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Induced waves in the olfactory bulb of the unrestrained cat Moore, Elizabeth Virginia 1971

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i INDUCED WAVES IN THE OLFACTORY BULB OP THE UNRESTRAINED CAT by ELIZABETH VIRGINIA MOORE B.Sc., University of London, 1966 A Thesis submitted i n p a r t i a l f u l f i l m e n t of the requirements f o r the degree of .Master of Science i n the Department of Physiology We accept t h i s thesis as conforming to the required standard 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 Brit 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 Physiology  The University of Brit ish Columbia Vancouver 8, Canada Date A p r i l , 1971. A B S T R A C T There are some discrepancies i n the literature regarding the response of the "induced waves" of the olfactory bulb to odorous stimuli. This work was designed to resolve the contro-versy by relating the different types of response to alertness of the animal and to concentration of the odour. The envelope of 40 Hz a c t i v i t y from the olfactory bulbs of unanaesthetised cats was recorded on a polygraph, and found to vary with respiration. The animal's nose projected into a stream of clean a i r to which odorant could be added at different fixed rates for about a minute at intervals. The amplitude of induced wave a c t i v i t y during the stimulus was compared to that shortly before i t . Odour concentrations were varied within a 5 x 10^-fold : range and the logarithm taken. The alertness of the cat was estimated on a 5-point scale. The data for the middle a l e r t -ness category were eliminated and those of the two extreme groups subjected to s t a t i s t i c a l analysis by multiple regression. The percentage change i n integrated induced wave a c t i v i t y during stimulus as compared to that during control i n a drowsy  cat was found to be independent of stimulus concentration and could be i n either direction but usually increased. In an aroused cat regression to a third order polynomial was s t a t i s -t i c a l l y significant (p < 0.02) and accounted for 0<>34 of the i i i v a r i a b i l i t y . T h i s r e s u l t appears s u r p r i s i n g l y good i n view of the enormous spontaneous v a r i a t i o n i n the s i g n a l and the unre-l i a b i l i t y of the s t i m u l u s , both as to i t s exact c o n c e n t r a t i o n and i n the resemblance of i t s p r e s e n t a t i o n parameters to a square wave. I t would be worth w h i l e to repeat t h i s study w i t h more animals, more odours and a. b e t t e r o l f a c t o m e t e r d e s i g n . The shape of the r e g r e s s i o n was p r e d i c t e d as f o l l o w s . At low c o n c e n t r a t i o n s an a l e r t c a t would show an o l f a c t o r y response i n the form of a d e p r e s s i o n of induced waves. At i n t e r m e d i a t e c o n c e n t r a t i o n s an alarm response would sometimes i n c r e a s e a l e r t -ness, augmenting the induced waves. At h i g h c o n c e n t r a t i o n s the t r i g e m i n a l - t o - a u t o n o m i c noxious vapour response would i n t e r v e n e , m e c h a n i c a l l y r e d u c i n g a c c e s s o f a i r to o l f a c t o r y r e c e p t o r s and/ or r e s p i r a t i o n . A drowsy c a t on the o t h e r hand might be s u b j e c t to a l e r t i n g by any s u p r a t h r e s h o l d c o n c e n t r a t i o n , or c o u l d i g n o r e the s t i m u l u s w i t h or without p e r c e i v i n g i t . E f f e c t s of n o n - o l f a c t o r y s t i m u l i and spontaneous v a r i a t i o n s were i n f a c t f a r more obvious than most of the " o l f a c t o r y r e s p o n s e s " . i v T A B L E O P C O N T E N T S I n t r o d u c t i o n 1 I Anatomy A. The nose 4 B . The o l f a c t o r y r e c e p t o r s 9 C. The o l f a c t o r y bulb 15 I I E l e c t r i c a l a c t i v i t y A. P e r i p h e r a l o l f a c t o r y system 25 B. C e n t r a l o l f a c t o r y system 31 - A r o u s a l 40 - C o n c e n t r a t i o n 47 - Present r e s e a r c h : the H y p o t h e s i s 49 Methods and M a t e r i a l s 51 I The p r e p a r a t i o n 52 I I The r e c o r d i n g system 57 I I I The o l f a c t o m e t e r 63 IV The odorants 67 V Procedure 69 VI P r e l i m i n a r y experiments: C and C 72 B and B 1 73 A 74 R e s u l t s 76 I P r e l i m i n a r y experiment A 77 I I P r e l i m i n a r y experiments B and B 1 79 V R e s u l t s (cont.) I I I P r e l i m i n a r y experiments C and G 1 8 1 I V The main Experiment 8 8 V Raw s i g n a l f r e q u e n c i e s 1 0 8 D i s c u s s i o n 1 1 2 O o n c l u s i o n s 1 2 6 B i b l i o g r a p h y 1 3 0 Appendices: I 1 3 8 I I 1 4 0 I I I 1 4 1 L I S T OP T A B L E S v i I Anatomical layers of the Bulb 18 II Alertness scale. 70 III Variables for regression 97 L I S T OP E Q U A T I O N S 1 Olfactory a c t i v i t y 83 2 Concentration of olfactory stimulus 83 v i i L I S T O P F I G U R E S 1 Sagittal section through the olfactory bulb 16 2 Neuronal connections of the olfactory bulb 19 3 Bipolar electrode construction 53 4 Gonnector plug 58 5 Recording system 59 6 F i l t e r characteristic 62 7 Olfactometer 65 8 Apparatus for experiment A 75 9. Record from experiment A 78 10 Results of experiment C 86 11 Sample records 89 12 Nasal obstruction - 92 13 Noxious vapour reflex 93 14 Results 98 15 Computer plot of D40 against SIZE 100 16 Computer plot of data from active cats 103 17 Non-olfactory responses 107 18 Autocorrelation of raw signals 109 19 Raw signals 111 v i i i A C K N O W L E D G E M E N T S I would l i k e to acknowledge the guidance of Dr H. McLennan i n a l l phases o f t h i s work, i n c l u d i n g p r i o r t r a i n i n g i n computer programming. I am v e r y g r a t e f u l to Mr. K. Henze f o r h i s m a s t e r l y p r e p a r a t i o n of the f i g u r e s . Mr. P. Graystone p r o v i d e d expert h e l p w i t h s e v e r a l a s p e c t s of the work, p a r t i c u l a r l y the i n s t r u m e n t a t i o n . Dr. P. L a r k i n v e r y k i n d l y a s s i s t e d w i t h the s t a t i s t i c a l a n a l y s i s . Dr. R. H. Wright gave a d v i c e on the o l f a c t o m e t e r and l i t e r a t u r e and ge n e r o u s l y loaned me some books and s u p p l i e d the methyl s a l i c y l a t e . Mrs. Y. Heap a s s i s t e d w i t h s e v e r a l i m p l a n t a t i o n s , read my t y p e s c r i p t and pr o v i d e d g e n e r a l encouragement. F i n a l l y , I owe to my mother the perseverance needed to complete t h i s t h e s i s d e s p i t e the i m p e r f e c t i o n s i n the experimental work. 1 I N T R O D U C T I O N 2 When odour laden a i r passes.; over the olfactory mucosa, the low amplitude, high frequency spontaneous e l e c t r i c a l a c t i v i t y recorded by a macro-electrode i n the olfactory bulb i s replaced by large waves of a lower frequency (Gerard & Young, 1937) . Adrian (1950) named these "induced waves", and used the term " i n t r i n s i c waves" for the more regular fast a c t i v i t y observed i n anaesthetized animals but usually desynchronised i n the conscious state. The frequency of the induced waves i s around 40 Hz i n most mammals, 55 - 60 Hz i n rabbits; i n poikilotherms i t i s naturally about 20 Hz but increases to 40 Hz when they are warmed to 37°G (Graystone et a l . , 1970). While room a i r appears odourless to man, both consciously and electrophysiologically (Sem-Jacobsen et a l . , 1956), i t i s an adequate stimulus for the rabbit, and extensive purification procedures (activated charcoal, etc.) are required to abolish the induced waves unless the animal i s breathing through a tracheal cannula. Waves apparently identical to the induced waves have been e l i c i t e d e l e c t r i c a l l y i n the strychninized bulb of curarized rabbits by Yamamoto & .  Yamamoto (1962). The induced waves generally occur during a DC s h i f t which i s seen by surface bulbar electrodes as a negative wave lasting through-out the duration of the passage of odour over the olfactory epithelium. This i s the i n i t i a l event i n the bulb and i s known as the Ottoson wave. Its properties were studied inten-sively by Ottoson i n 1956. (a, b, c). 3 The induced,waves, once present, have been variously reported to wax and wane i n response to a variety of para-meters. These include the nature, novelty and strength of the odour, alertness, emotion, stimulation of other senses, e l e c t r i c a l stimulation, anaesthetics and other drugs, lesions, etc. Some of these studies w i l l be reviewed below, with emphasis on chronic preparations, and found to show many discrepancies. These were suspected to be due mainly to reflex changes i n respiration and blood pressure and to different alertness levels and odour strengths. The present study set out to extend the range of odour strengths studied while also monitoring alertness i n the unrestrained cat. Before surveying i n d e t a i l the electrophysiology of the bulb, i t seems appropriate to give a brief synopsis of current knowledge and hypotheses of the anatomy and physiology of the olfactory sense i n general, with particular reference to those aspects which introduced d i f f i c u l t i e s i n the work to be reported or which may have contributed to the discrepancies i n the relevant l i t e r a t u r e . 4 I. A N A T O M Y A N D P H Y S I O L O G Y A. THE NOSE Noxious vapours such as ammonia, and danger signals such as smoke, can be detected by a l l the cranial nerves supplying the mucosa of the upper respiratory tract, mainly through free nerve endings. The olfactory epithelium i s specialised for the ide n t i f i c a t i o n of individual odours. Henkin (1967) studied patients whose tumour called for excision of the olfactory organ. A few weeks postoperatively they began to distinguish pure l i q u i d odorant from water by "smelling" i t but remained unable to name the substance. The sensation was f e l t i n the f i e l d of nerves V, IX and X. It has been calculated (de Vries & Stuiver, 1959) that i n humans only about 2% of the molecules i n inspired a i r strike the olfactory epithelium, which i s a delicate organ extending over the ethmoturbinals, b a f f l e - l i k e structures i n the upper nasal cavity. In order to smell e f f i c i e n t l y i t i s necessary to sn i f f , a procedure which may alter not only the velocity but also the flow pattern of the a i r . Mechelse & Lieuwens (1969) reported that after retraction of a nasal catheter a short distance away from the olfactory epithelium, a previously effective puff of room a i r was no longer able to generate e l e c t r i c a l burst a c t i v i t y i n the olfactory bulb. Thresholds 5 are very dependent on the v e l o c i t y of a i r f l o w . O l f a c t o r y nerve twigs sometimes show as great a response to an inc r e a s e i n f l o w r a t e as to an inc r e a s e of the same r a t i o i n c o n c e n t r a t i o n ; i . e . they appear to respond to the rat e of fl o w of odorant molecules r e g a r d l e s s of t h e i r d i l u t i o n , as reported by Tucker (1963) f o r the t o r t o i s e . One f a c t o r . c o n t r i b u t i n g to t h i s could be tha t the high e r v e l o c i t y and r e s u l t i n g turbulence would reduce the depth of the b a r r i e r of stagnant a i r i n contact w i t h the mucosa, which would enormously increase the r a t e of d i f f u s i o n to the air/mucus i n t e r f a c e . Another n a s a l chemoreceptor important i n some animals i s the vomeronasal organ, a l s o c a l l e d Jacobson's organ. I t i s bur i e d i n a pocket w i t h a narrow opening low i n the back of the n a s a l or bu c c a l c a v i t y , not apparently of easy access to a i r f l o w . I t was t h e r e f o r e w i t h some s u r p r i s e that Tucker (1963) found that the vomeronasal nerve twi g a c t i v i t y responded r e a d i l y to some odours, o f t e n but not c o n s i s t e n t l y w i t h even s h o r t e r l a t e n c y than the o l f a c t o r y nerve. However, he thought i t pos-s i b l e that some f i b r e s of t h i s nerve a r i s e from a more access-i b l e patch of e p i t h e l i u m which might be homologous w i t h the s e p t a l organ reported i n c e r t a i n s p e c i e s . I n any event t h i s nerve was found to respond w i t h a higher r a t e of f i r i n g than the o l f a c t o r y nerve to lower members of an homologous chemical s e r i e s . There are s t i l l other nerves which complicate the o l f a c t o r y p i c t u r e : the autonomic system. The main e f f e c t of parasympa-6 thetic activation seems to be an increased flow of mucus. This may be important i n the washout of noxious vapours. Washout may indeed represent the principal fate of any molecules which have excited the receptors, although Dravnieks (1964) visualizes the tissues as a sink. Wright & Burgess (1971), however, considered i t possible that the receptor-bearing c i l i a float on the surface of the mucus so that molecules can reach them without necessarily dissolving i n i t . Sympathet ic a l l y mediated changes i n the width of the lumen of the nose cause marked changes i n e x c i t a b i l i t y of olfactory receptors. This can be brought about by changes i n vascular engorgement of erectile tissue, e.g. nasal congestion during copulation. But the direction of change depends on species. In the rabbit the lumen expanded with cervical sympathetic nerve stimulation (Tucker, 1963) and some animals respond similarly to arousal stimuli; but the tortoise contracted i t s nasal passage on stimulation, with a corresponding reduction i n olfactory res-ponse (Tucker, 1963)• The afferent limb of this reflex has been shown to be trigeminal. In recording mucosal response to a series of concentrations of amyl acetate, Tucker noted a dis-placement of the olfactory epithelium relative to the electrode whenever a concentration of 10 "^'^  of saturation was reached. This he correlated with the trigeminal threshold for amyl acetate, between 10~^'^ and 10 "*'*<~>. It i s thus apparent that trigeminal-autonomic reflex arcs must be eliminated before any reli a b l e quantitative comparisons 7 o f o l f a c t o r y r e c e p t o r e f f e c t s can be made. U n f o r t u n a t e l y t h i s has been w i d e l y o v e r l o o k e d . The shape o f the n a s a l passages can a l s o be changed by m u s c u l a r a c t i v i t y i n the n o s e . Marevskaya (1961) s t u d i e d the l e v a t o r a l a e n a s i muscle i n the r a b b i t . He found t h a t i t p a r t i c i p a t e d i n the o r i e n t i n g r e f l e x o f the a n i m a l , i n w h i c h th e s e n s i t i v i t y o f the o l f a c t o r y a p p a r a t u s i s i n c r e a s e d . I t s a c t i v i t y was not a l w a y s a s s o c i a t e d w i t h t h a t o f the r e s p i r a t o r y a p p a r a t u s . O l f a c t o r y s t i m u l a n t s known t o a c t i v a t e the t r i -g e m i n a l nerve had a more l a s t i n g e f f e c t . There i s a l s o a d r a m a t i c n a s a l - g e n e r a l autonomic r e f l e x t o n o x i o u s v a p o u r s such as smoke, w h i c h i s s a i d t o have been f i r s t d e s c r i b e d i n 1870 by Kratchmer (quoted by A l l e n , 1 9 2 9 ) , and was s t u d i e d i n d e t a i l by A l l e n ( 1 9 2 9 ) . The a f f e r e n t l i m b seems to use e i t h e r o l f a c t o r y o r t r i g e m i n a l pathways, w h i c h e v e r i s i n t a c t , and the r e s u l t s a r e a s l o w i n g o f h e a r t r a t e , r i s e i n b l o o d p r e s s u r e and s l o w i n g o r i n t e r r u p t i o n o f r e s p i r a t i o n , d e p e n d i n g on t h e s e v e r i t y o f t h e s t i m u l u s . Such a w e l l s u p p l i e d r e f l e x would be h a r d t o e l i m i n a t e , and much o f the e l e c t r i c a l a c t i v i t y o f the b u l b i s s a i d t o be v e r y s e n s i t i v e t o b l o o d p r e s s u r e ( A d r i a n , 1950, G e s t e l a n d e t a l . , 1965, O t t o s o n , 1959b, e t c . ) . However, Yamamoto & Iwama (1961) s t a t e d t h a t a r i s e i n b l o o d p r e s s u r e was sometimes seen i n t h e i r p r e p a r a t i o n but a l w a y s o c c u r r e d l a t e r t h a n the change i n i n d u c e d waves. Never-t h e l e s s , i n a n i m a l s b r e a t h i n g s p o n t a n e o u s l y t h r o u g h the n o s e , the r e s p i r a t o r y component o f the r e f l e x must be t a k e n i n t o a c c o u n t . Some o f the e l e c t r i c a l s i l e n c e s seen by McLennan & G r a y s t o n e 8 ( 1 9 6 5 ) . i n the o l f a c t o r y bulb i n response to h i g h c o n c e n t r a t i o n s of smoke were probably due to the c a t ' s h o l d i n g i t s b r e a t h (see P i g . 13 )• Even such s m a l l changes i n depth and r a t e as occur on f a l l i n g a s l e e p have been shown by Mechelse & Lieuwens ( 1969) to reduce n a s a l a i r f l o w below the l e v e l a t which b u r s t s are generated. 9 B. THE OLFACTORY RECEPTORS The o l f a c t o r y e p i t h e l i u m c o n s i s t s of o l f a c t o r y r e c e p t o r c e l l s i n t e r s p e r s e d w i t h s u s t e n t a c u l a r c e l l s , p l u s a few mucus-s e c r e t i n g Bowman's g l a n d s . The s u s t e n t a c u l a r c e l l s have n u c l e i near the s u r f a c e , and m i c r o v i l l i . I t i s not known whether they p l a y more than j u s t a s t r u c t u r a l s u p p o r t i v e r o l e i n o l f a c t i o n , a l t h o u g h the presence of m i c r o v i l l i suggests some r e l a t i o n s h i p , perhaps n u t r i t i v e , w i t h the. mucus and/or the o l f a c t o r y c i l i a which i n t e r m i n g l e w i t h them. The s e c r e t o r y c e l l s are s t i m u l a t e d by the parasympathetic nerves, which are probably a c t i v a t e d when noxious, vapours s t r i k e the t r i g e m i n a l f r e e nerve endings. I t i s common to r e c o r d s e c r e t o r y . p o t e n t i a l s i n the r e g i o n o f a c t i v e e x o c r i n e c e l l s , and t h i s may account f o r some of the e l e c t r i c a l a c t i v i t y r e c o r d e d from the mucosa. The time taken f o r odorous a i r to fl o w through the narrow n a s a l passages c o u l d w e l l be o f the same or d e r of magnitude as the l a t e n c y i n a r e f l e x a r c from t r i g e m i n a l r e c e p t o r s a t the f r o n t of the n a s a l c a v i t y . If, so t h i s c o u l d be the e x p l a n a t i o n o f the f i n d i n g by K e r r ( i960) o f s u p p r e s s i o n by a s t r o n g odorous s t i m u l u s to the c o n t r a l a t e r a l n o s t r i l o f waves induced i n the bulb by weak odours. The o l f a c t o r y r e c e p t o r c e l l , the base of which g i v e s o f f an o l f a c t o r y nerve f i b r e , has i t s nu c l e u s f u r t h e r from the s u r f a c e than those of the s u s t e n t a c u l a r c e l l s . I t s mucosal end e n l a r g e s i n a v e s i c u l a t e d head, the o l f a c t o r y v e s i c l e , beyond the body of the s u s t e n t a c u l a r c e l l s , among the m i c r o -10 v i l l i . Andres (1969) found a variety of forms i n the cat and deduced that the olfactory vesicle i s capable of regeneration. These c e l l s are spaced about 3 urn apart. Lorenzo (1970) even observed contiguous receptor c e l l s i n the monkey, with what appeared to be a synapse between them. From the olfactory vesicle, emerge 3-10 c i l i a , about 10 -200 pm long and 0.1 - 0.5 M-m thick, as studied by Reese (1965) i n the frog, while Andres (1969) gave a probable maximum length of 80 u-m for the cat. In the frog they had the usual c i l i a r y structure of 2 inner and 9 outer filaments. They had a thick portion at the base, with double filaments, changing abruptly over a distance of 0.5 M-m to a long, thin t a i l i n which the f i l a -ments were single; Andres, who c a l l s the filaments microtubules, found that i n the t a i l of the cilium i n the cat their number decreased from 4.to only 2. The junctional region was very fr a g i l e and the t a i l s were easily broken off during histological procedures, which may account for the fact that the majority of diagrams i n books show the c i l i a looking much l i k e a bunch of thick radial m i c r o v i l l i crowning each olfactory c e l l ; whereas 1 i n fact the thick portions, as seen i n electron micrographs, are s t i f f and regular, sticking sideways among the m i c r o v i l l i as well as out, and the thin portions turn, to run p a r a l l e l to the mucus surface and reach across many dozens of other olfactory c e l l s . The c i l i a were stated to be non motile, but the stumps motile after the long.tail was lost; so i t i s probable that 11 the double filament section i s i n fact motile but the length of the :t a i l so damps the movement that i t cannot be seen. Respiratory mucosa normally has motile c i l i a which move the mucus and any entrapped particles and sloughed off epi-thelium, towards an o r i f i c e where they can be eliminated. Such motion as i s seen i n the short stumps of olfactory c i l i a i s not organised to perform this housekeeping function. Instead Reese (1965) attributed the movement of carbon particles that he observed, at less than 1 mm/sec and i n a pattern reproducible from frog to frog, to the flow of a very thin layer of watery mucus which was continuously secreted by the glands and sucked away by the current set i n motion by c i l i a i n adjacent areas of respiratory epithelium. Olfactory c i l i a with- intact t a i l s lined up p a r a l l e l to these flow lines on the surface of the subjacent layer of denser mucus about 25 - 35 thick. The t a i l of the cilium has been postulated, without direct evidence, to bear the specific olfactory receptor s i t e s . Some evidence may soon be, forthcoming since Gemne & D6ving (personal communication to Ottoson, 1970) have found that absorbent paper applied to the olfactory mucosa withdrew an almost pure prepara-tion of c i l i a r y t a i l s . Chemical tests of the effects of odorants on this substrate could be made, such as the u l t r a -v i o l e t absorbance studies of Ash & Skogen (1970), whose "olfac-tory scrapings" included the c e l l s of the epithelium above the lamina propria and showed a graded absorbance change at 267 nm 12 i n response to 7 out of 37 substances tested i n v i t r o . Meanwhile, the nature of the o l f a c t o r y receptor s i t e s and t h e i r mode of i n t e r a c t i o n with odorant molecules i s ground f o r much speculation. The bulwark of o l f a c t o r y thought i s Amoore's Stereochemical Theory (e.g. 1964), f i r s t published i n 1952, which stated that o l f a c t o r y d i s c r i m i n a t i o n depends on the r e l a -t i v e a b i l i t y of a molecule to f i t into seven d i f f e r e n t s i t e s on the receptors depending on i t s s i z e , shape and charge. Beets' (e.g. 1970a) Profile-Functional-G-roup theory i s s i m i l a r but stresses the.orientation of the molecule, which would depend on i n t e r a c t i o n of i t s f u n c t i o n a l groups with those of the receptor. Wright c|. Burgess (e.g. 1971), on the other hand, have developed the Low Frequency Molecular V i b r a t i o n Theory, i n which the receptor, which i s maintained i n a high energy state by an ac t i v e metabolic process, discharges when a molecule i s adsorbed which has a mode or difference mode of v i b r a t i o n perpendicular to the plane of adsorption at the c h a r a c t e r i s t i c frequency of the receptor; osmic i d e n t i t y would be established by the pres-ence or absence of a s p e c i f i c few out of about twenty-five such "primary" frequency bands. A fourth approach, to which Adrian (1963) lent some support, r e l i e s less on s p e c i f i c i t y of recep-tors and more on space-time e f f e c t s whereby the (electrophysio-l o g i c a l ^ and h i s t o l o g i c a l l y v e r i f i e d ) topographical projection of the epithelium on the bulb and the layering of a i r and mucus perform three-dimensional chromatography (e.g. Dravnieks, 1964). 13 There are many other theories in the literature, for example those founded on the principles of membrane puncturing or of enzyme activation. The support for these theories comes from work on different species, from insects to humans, and a l l workers hope that the fundamental mechanisms w i l l prove to be the same in a l l . The discovery by Reese & Brightman (1970) that elasmo-branchii (2 species) had v i l l i instead of c i l i a on their olfac-tory bipolar c e l l s need not be taken to rule out the assumption that the receptor sites are on the c i l i a i n other animals; the authors suggested on the contrary that these sites were always located on the c e l l membrane but that the c i l i a were an adapta-tion of the shape of the membrane which enabled the c e l l to span the mucous barrier i n air-breathing classes. Thus inter-species variations should be confined mainly to the numbers of primary odours that they are equipped to dis-tinguish. Gesteland et a l . (1963) i n electrophysiological studies i n the frog found evidence for eight "primary" groups of odours. The majority of studies aimed at elucidating olfac-tory receptor theory use ..panels of humans to report on d i f f e r -ent concentrations or on subjective s i m i l a r i t i e s of more or less carefully chosen series of chemicals. Beets (1970b) has very recently established an international consulting service to advise on the most f r u i t f u l assortment of. chemicals to use i n a particular investigation. 14 A refinement on the human panel approach i s the study of "specific anosmia", a condition i n which the patient has an abnormally high threshold for a particular substance. Chemi-cally related compounds are tested and the one for which the threshold ratio (patient : human norm) i s the greatest i s taken as exemplifying the physicochemical characteristics of one of the primary odorants of the human, e.g. isovaleric acid (Amoore, 1970). V/ith this technique Amoore now expects to find 20 - 30 of these, expanding on his original 7 (Amoore, 1964). It i s always worth hoping that nature has solved the problem of olfaction i n only one way; but i t should be borne i n mind that while i n t e r r e s t r i a l vertebrates the odorant mole-cules could either alight on receptors floating on the surface of the mucosa or dissolve i n i t to reach them, i n insects the process can surely only operate i n the gaseous phase and i n f i s h the aqueous. Tucker's (1963) successful stimulation of land tortoise olfactory nerves with both gaseous and aqueous odorants supports the suggestion that the fundamental mechanism of olfaction i s a universal one which i s independent of the physical state of the medium on which the stimulus i s borne. 15 C. THE OLFACTORY BULB The olfactory bulb i s an extension of the brain resting on the cribriform plate of the ethmoid bone, through which i t receives the olfactory nerves. It i s connected to the brain by the olfactory tract, which s p l i t s caudally into l a t e r a l and medial striae, and i n some animals also intermediate. Besides the bulb proper, the olfactory peduncle contains two other nuclei: the anterior olfactory nucleus, which i s behind the bulb and deep to the tract, and the accessory bulb on the posterior dorsal surface of the bulb (Fig. 1) . The accessory bulb receives the vomeronasal nerve and has a simpler structure than the regular bulb; i t therefore represents another whole area for study i n animals such as reptiles, i n which i t i s not v e s t i g i a l . The anterior olfactory nucleus (AON) appears to be an organ for coordination of the two bulbs and mediator of some of the centrifugal control exerted on the bulbs. The anterior bundle of the anterior commissure (AC) was long assumed to contain the axons of the tufted c e l l s of the bulb, but these are now known to go no farther i n this direction than the AON (e.g. Lohman & Lammers, 1963), while Valverde (1965) found no evidence that they leave the bulb at a l l . I t i s the.axons of the AON which make up the medial olfactory s t r i a and cross i n the anterior commissure. They synapse i n the contralateral AON and i n the i p s i l a t e r a l olfactory bulb on the peripheral processes of the F I G U R E 1 16 SAGITTAL SECTION THROUGH THE OLFACTORY BULB Cat, t o l u i d i n blue p r e p a r a t i o n x 5. From Fox (1940). bulb ...olf. b u l b . o l f . acc. c. f r o n t . com. ant. corp. c a l . f . r h i n . a r c . f x . i s l . G a l . 1. c e l . m i t . 1. glom. 1. gran 1. p l e x . e x t . 1. p l e x . i n t . n. acc. n. caud. n. d. b . B. n. o l f . ant. p. dor. n. o l f . ant. p. post. n. o l f . . ant.' p. vent. n. sept. l a t . . 1 tub. o l f . 1. p l e x . tub. o l f . 1. polym. v. l a t . o l f a c t o r y bulb accessory o l f a c t o r y bulb f r o n t a l c o r t e x a n t e r i o r commissure corpus callosum arcuate r h i n a l f i s s u r e f o r n i x i s l a n d of C a l l e j a m i t r a l c e l l l a y e r glomerular l a y e r g r a n u l a r l a y e r e x t e r n a l p l e x i f o r m l a y e r i n t e r n a l p l e x i f o r m l a y e r nucleus accumbens caudate nucleus nucleus of the diagonal band of Broca d o r s a l nucleus of the AON p o s t e r i o r nucleus of the AON v e n t r a l nucleus of the AON l a t e r a l s e p t a l nucleus p l e x i f o r m l a y e r of o l f a c t o r y t u b e r c l e polymorph l a y e r of o l f a c t o r y t u b e r c l e l a t e r a l v e n t r i c l e 1.7 internal granule c e l l s i(Pri'c.e -1969b) . As can be seen i n Fig. 1, the bulb i s made up of six layers as shown i n Table I. Some of the relations of these structures are diagrammed i n Fig. 2. The outer layer contains the primary olfactory nerves. The f i l a olfactoria of the receptor c e l l s come together i n bundles ensheathed by Schwann c e l l s , then .many bundles bunch together to pass through the holes i n the cribriform plate. On reaching the bulb the nerves spread out extensively i n the outer layer of white matter before turning i n to synapse i n the glomeruli, where for the f i r s t time they divide into many synapse-bearing branches. I t has been found by degeneration studies that there is.indeed a gross topographical, correspondence between the mucosa and the bulb, as seen electrophysiologically by Adrian (1963) •• I t i s not known precisely what purpose i s served by the interweaving seen i n this f i r s t layer. Besides the topo-graphical projection, there could be some degree of segregation by odour classes, and i t i s also possible that the meanderings of olfactory nerves represent delay lines adjusting the time of a r r i v a l of signals i n connection with a spatio-temporal theory such as that of Dravnieks (1964). Leveteau &MaeLeod (1966) studied the responses of 47 individual glomeruli ( i . e . the localised component of the Ottoson wave) to each of a set of 9 different odours. They found a wide range of differences i n T A B L E 18 A N A T O M I C A L L A Y E R S 0 F T H E B U L B . Name 1 Olfactory-fibre 2 Glomerular 3 External plexiform 4 M i t r a l 5 Internal plexiform 6 Internal granule Contents Olfactory nerves Glomeruli - axons of olfactory nerves - dentrites of mitral c e l l s - dentrites of tufted c e l l s - dentrites of periglom. c. Between glomeruli - external tufted c e l l s - periglom. granule c e l l s - short-axon c e l l s - centrifugal axons (from HDB) Secondary den. of mitral c. Secondary den. of tufted c. Axon collaterals of tufted c. Periph. processes of gran. c. Tufted c e l l s M i t r a l c e l l s Axon collaterals of mitral c. Axon collaterals of tufted c. Dendrites of granule c e l l s Centrifugal axons (from AON) Granule c e l l s Short axon c e l l s + same as layer 5 Synaptic effect + + + + + + + + + none + Quantity (in rabbit) 7 5 x 10 1,900 150,000 45,000 Abbreviations: den. dendrites c. c e l l s gran. granule HDB horizontal nucleus of diagonal band periglom. periglomerular periph. peripheral Prom A l l i s o n & Warwick (1949), Moulton & Tucker (1964), Pinching'(1970), Price (1969a, b), Valverde (1965), etc. 19 F I G U R E 2 H E XI F O R M . G L O M E R U L U S 1.900 * I l U H f D C ( U I i 0.00 0 R E C E P T O R S 3 0 . 0 0 0 . 0 0 0 NEURONAL CONNECTIONS OF THE OLFACTORY BULB Structure of the ol f a c t o r y bulb (in the r a b b i t ) , showing r e l a t i o n s h i p to the o l f a c t o r y nerves and mucosa, d i f f e r -ent layers and c e l l types i n the bulb, numbers1 of each. c e l l type as found by A l l i s o n & Warwick (1949),.and some of the interconnections within the bulb. For a more accurate diagram of the cen t r a l connections, defer-to F i g . 2B. From Moulton & Tucker (1964). Central connections of the o l f a c t o r y bulb ( i n the r a t ) . The "cells shown i n the bulbs are m i t r a l only, the dots glomeruli. From Price (1969a). 20 their "response spectra"; a quarter of them reacted to a l l odorants tested, while only 3 pairs responded i n a similar way to each. This result i s comparable to that of studies on the discriminating a b i l i t y of individual receptors, and therefore does not rule out the p o s s i b i l i t y that each glomerulus receives input from a class of similar receptors, so that glomeruli and receptors both have a similar degree of s p e c i f i c i t y . The glomeruli are large spherical structures containing a mass of bare nerve processes synapsing on the primary dendrites of mitral c e l l s . Dendrites of tufted c e l l s and periglomerular c e l l s also participate i n this conglomeration, while the short-axon interglomerular c e l l s do not contact olfactory nerves but serve as second order interneurons within this layer (Pinching, 1970) . Centrifugal fibres sometimes reach this far, affecting either the interneurons or the glomeruli directly, although the majority act via the granule c e l l s . In addition to the longer-established relationships, the recently discovered reciprocal synapse has been identified i n several layers of the bulb by various workers, e.g. Andres (1969). Reciprocal synapses between the process of a granule c e l l and the soma or secondary dendrite of a mitral or tufted c e l l are frequently seen. The synapse from mitral to granule c e l l has round vesicles and i s asymmetric, a morphology which i s now thought to characterize excitatory synapses, while the synapse from granule to mitral c e l l i s symmetrical and has some oval vesicles and i s taken to be inhibitory. 22 i n the bulb. Ferrer (1969) made a detailed study of the d i s -t r i b u t i o n of axons from the r o s t r a l nucleus of the AON i n the hamster. He implicated the ol f a c t o r y tubercle, pyriform cortex, corticomedial nucleus of the amygdala, dorsomedial nucleus of the thalamus, l a t e r a l habenular nucleus, l a t e r a l hypothalamus and supraoptic nucleus. I t i s therefore possible that axons of m i t r a l c e l l s i n f a c t reach only the AON and the prepyriform cortex. A l e s i o n r e s t r i c t e d to the cortex of the bulb would be needed to v e r i f y t h i s since the LOT includes AON f i b r e s (Ferrer, 1969). . From the r e l a t i v e numbers counted by A l l i s o n & Warwick (1949), i t can be said that each glomerulus receives input from about 26,000 receptors and relays i t through 24 m i t r a l c e l l s and 68 tufted c e l l s i n the rabb i t . However, experimental evidence that the d i s t r i b u t i o n of actual connections shows any such r e g u l a r i t y has not been found. Nevertheless both V/right & Burgess (1971) and Hainer et a l . (1954) have concluded that ••' there are 24 - 25 primary odours which make up a l l o l f a c t o r y sensations. They supported t h i s by a c a l c u l a t i o n of the number of primary odours supposedly necessary to give enough permuta-tions to account f o r the t o t a l number of odours p o t e n t i a l l y distinguishable by a trained person such as a perfumier ( i t i s strange that Amoore, working d i r e c t l y with the perfumery indus-t r y , should have considered 7 s u f f i c i e n t , but see Amoore, 1970,.. i n which t h i s figure was increased to 25). 23 Below the m i t r a l c e l l l a y e r i s the i n t e r n a l p l e x i f o r m l a y e r , f o l l o w e d by the granule c e l l l a y e r . W i t h i n t h i s , i n some s p e c i e s , i s the o l f a c t o r y v e n t r i c l e , an e x t e n s i o n of the v e n t r i c u l a r system of the b r a i n , and the r o s t r a l nucleus o f the AON c a u d a l l y . In many s p e c i e s the granule c e l l s are d i s -seminated throughout the r e g i o n i n t e r n a l to the m i t r a l c e l l s so t h a t separate i n 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 cannot u s e f u l l y be d i s t i n g u i s h e d . The plexus i s made up of axons and axon c o l l a t e r a l s of m i t r a l and t u f t e d c e l l s , c e n t r i f u g a l axons and processes of granule c e l l s . The g r a n u l e c e l l s , as mentioned above, are the main r e c i p i e n t s o f c e n t r i f u g a l i n f l u e n c e s on the b u l b . The l a t t e r come from two immediate s o u r c e s . The f i r s t , the AON, r e c e i v e s f i b r e s from the c o n t r a l a t e r a l AON and from the p y r i f o r m c o r t e x v i a the medial f o r e b r a i n bundle and l o n g i t u d i n a l a s s o c i a t i o n bundle, presumably through the amygdala and o l f a c t o r y t r a c t . The pyramidal c e l l s o f the AON send axons v i a the AC to synapse on the body and secondary d e n d r i t e s of the gr a n u l e c e l l s w i t h i n the c o n t r a l a t e r a l granule c e l l l a y e r , w h ile axon c o l l a t e r a l s r e t u r n to the i p s i l a t e r a l bulb where they terminate 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 on the s p i n e s r a t h e r than the gemmules of the p e r i p h e r a l p r o c e s s e s of granule c e l l s ( P r i c e , 1969b). The other focus i m p l i c a t e d i n o l f a c t o r y c o n t r o l i s the nucleus o f the h o r i z o n t a l limb of the d i a g o n a l band ( P r i c e , 1969a), which r e c e i v e s f i b r e s from the m i d b r a i n and l a t e r a l hypothalamus 24 and has a c o n t r a l a t e r a l association loop v i a the habenular commissure. The f i b r e s from t h i s nucleus synapse on the gemmules of the i n t e r n a l granule c e l l s i n the external p l e x i -form layer (Price, 1969b), where Valverde (1965) suggested presynaptic i n h i b i t i o n might occur. 25 I I . E L E C T R I C A L A C T I V I T Y A. PERIPHERAL OLFACTORY SYSTEM The anatomical relations of the olfactory bulb have been outlined above. In order to form a hypothesis regarding the behaviour of i t s e l e c t r i c a l a c t i v i t y i t was important to establish, as far as i s known, the type of signals passing i n and out along these pathways under various conditions. The i n i t i a l event i n the excitation of a sense organ i s generally expected to be a generator potential. A slow negative potential which answers this description was indeed found in-the mucosa. Ottoson (1956b) named i t the Electro-olfactogram or EOG. There i s now some doubt as to whether i t i s indeed a generator potential. Shibuya (1964) found that placing a small piece of absorbent paper on the tortoise olfactory mucosa for a few, seconds abolished the EOG but f a i l e d to influence the response of the primary olfactory neurons recorded simultaneously, and concluded that the EOG i s not important i n olfaction. However the model proposed by Gesteland et a l . (1965) appears to offer a simple explanation. Moreover, Shibuya may simply have been recording from the processes of neurons from unaffected epithelium passing by; the loc a l receptors were probably badly damaged by his proced-ure -(Ottoson, 1970). , 26 Gesteland et a l . (1965) made a very careful study of the mucosal potential and the unit a c t i v i t y of the axons of local olfactory receptor neurons i n the frog. They point out the dangers of recording from decorporate or otherwise poorly vascularised preparations; the receptors become hypersensitive and therefore lose their s e l e c t i v i t y . They also studied the state of the mucus microscopically and reported that changes i n the constitution of the secretions that seriously interfered with responsiveness could be produced either by the secretory response to rough handling or bythe drying effect of stimulating with inadequately humidified gases. Gesteland et a l . (1965) found that the shape of the slow potential was characteristic for different odours and that the positive-potentials and off responses sometimes seen were not art i f a c t s but reproducible and important components of the response. Using paired odour stimulations with increasing latency and measuring impedance changes i n the tissue they derived the following hypothetical model of the olfactory transduction process. Gesteland likened the receptor sites on the olfactory c i l i a to those on postsynaptic c e l l s responding to the chemical mediators of synaptic transmission, with some extra property giving them a considerable variety i n the sets of chemicals to which any one responds. The olfactory environment would there-27 fore be represented holographically on the mucosa. There were two types of ionic permeability changes e l i c i t e d i n these receptor sites, one a hyperpolarisation giving rise to the regular negative EOG and to a depolarisation of the membrane on the soma tending to generate an action potential; the other a depolarisation towards an equilibrium potential a l i t t l e more positive than the resting potential, producing the positive slow potential and a hyperpolarisation at the soma inhibiting spike generation. The currents involved would flow down the core of the cilium and dendrite and back through the mucus. Lorente de No (1947) has shown that the external resis-tance i n e l e c t r i c a l transmission down a fibre makes surprising-ly l i t t l e difference, so Shibuya's (1964) absorbent paper (see above) would have l i t t l e effect on olfactory transduction i n any c i l i a i t happened to spare but would greatly alter the spread of the slow potential to his mucosal electrode. Gesteland et a l . (1963) saw the potential o s c i l l a t i o n described by Ottoson (1959b) and studied by Takagi & Shibuya (1961) in decapitated toad, only after deterioration of the preparation. After Gesteland et a l . (196 5) had stimulated the mucosa e l e c t r i c a l l y , nearby neurons were seen to be activated electrotonically. Comparison of this effect with the reported behaviour of other overstimulated or poisoned nervous tissue led them to believe that the potential o s c i l l a -tions reported as sometimes occurring i n mucosa and olfactory 28 nerve were art i f a c t s of overstimulation or ischaemia. They noted with interest that i n olfactory epithelium there can be no appeal to neural feedback loops such as are cited to explain this effect elsewhere. The a b i l i t y of Gesteland to change the shape of the slow potentials by changing the odour without moving the recording electrode seems to rule out the slow potential generating mechanism proposed by Moulton & Tucker (1964). They applied Lorente de No's (1947) experimentally verified calculations of the potential waveform seen i n a volume conductor when a volley passes along a nerve within i t , to the model of olfactory epithelium with f i l a olfactoria passing under i t and turning up into the holes of the cribriform plate. They were thereby able to account for the relative preponderance of negative and positive potentials and their shape. However a clearcut s p e c i f i c i t y of large groups of neurons, which i s not i n fact seen, would be required to explain Gesteland's findings i f the model of Moulton & Tucker were to be accepted. There i s some discussion i n the literature as to whether olfactory receptors are mechanosensitive. Most workers have eventually achieved the required purity i n the stimulating gas to abolish a l l olfactory response. Ueki & Domino (1961), however, were able i n one half of their twelve acute dogs to find areas i n the bulb which responded to the flow of pure odourless gases. They stated that these only appeared after 29 a diligent search, so there was time for an injury to develop from their experimental procedure. Their apparatus lacked a humidifier and their stimulus was administered through a nasal catheter bypassing some of the respiratory mucosa which normally supplies water vapour. It i s therefore possible that some neurons were injured by desiccation and became hyper-sensitive. The p o s s i b i l i t y that some so-called odourless gases are osmically active i n other species should also be borne i n mind; for example, water vapour receptors and CO^ receptors can be found i n insects. Whatever may be i t s generating mechanism, the a c t i v i t y i n the olfactory nerves i s what ultimately affects the olfactory bulb. The f i l a olfactoria were found by Gesteland et a l . (1965) to be tonically active. Each fibre was inhibited by some odours, excited by others, unaffected by yet others, and sometimes the noisy baseline a c t i v i t y became more rhythmical. For every pair of compounds that.affected one fibre both i n the same way, they could easily find another fibre whose response discriminated between the two. . A similar variety of response spectra was. seen i-n.the several types of bulbar a c t i v i t y , the glomerular response recor-ded by Leveteau & MacLeod (1966), the secondary olfactory neuron a c t i v i t y studied by Moulton & Tucker (1964), and the induced waves seen i n the human by Sem-Jacobsen et a l . (1956). The 30 slow p o t e n t i a l i n the bulb, known as the Ottoson wave, i s s i m i l a r to those of the e p i t h e l i u m (EOG) and o l f a c t o r y nerves, but i s a p p a r e n t l y not merely d e r i v e d from the l a t t e r by e l e c t r o t o n i c c o n d u c t i o n s i n c e i t i s more r a p i d l y a f f e c t e d by a s p h y x i a . Moreover, i t f a i l s to show the a d a p t a t i o n found i n the EOG and p e r s i s t s i n the f a c e of a n t i d r o m i c a c t i v a t i o n o f the d e n d r i t e s of m i t r a l c a l l s i n the g l o m e r u l i by LOT s t i m u l a -t i o n . I t can t h e r e f o r e be a t t r i b u t e d to the p r e s y n a p t i c p o t e n t i a l i n the g l o m e r u l i . Ottoson (1959c) f u r t h e r showed t h a t a p o s t s y n a p t i c component can be separated out of i t . At t h i s p o i n t (the glomerulus) the p i c t u r e becomes more obscure: feedback l o o p s and c e n t r i f u g a l i n f l u e n c e s b e g i n to modify the o l f a c t o r y s i g n a l . 31 B. CENTRAL OLFACTORY SYSTEM V/hen only a part of the bulb i s stimulated (e.g. i n mapping the topographical projection either of primary or secondary neurons (Shepherd & Haberly, 1970), a positive counterpart of the negative slow potential (Ottoson v/ave) appears on the other side of the bulb. MacLeod & Leveteau (1963) noted that the sign of the slow potential changed as the olfactory stimulant was--, altered from a hydrophilic to a l i p o p h i l i c compound. Mozell (1958) studied bulbar spike responses to different odours and concentrations with anteroposteriorly spaced electrodes and doubted that the topographical projection of the mucosa on the bulb i s exploited i n physiological olfactory discrimination, since he did not find enough reliable differences. MacLeod & Leveteau (1963) stated that i n the guinea pig the Ottoson wave occurred 50 msec after the EOG and the induced waves appeared superimposed on i t 20 - 30 msec la t e r . However, i n their later paper on the rabbit (Leveteau & MacLeod, 1966) they stated that the induced waves began 200 msec after the EOG and cited their 1963 paper as reference. Is this a typographi-cal error or a species difference? In any case whether the delay between EOG and induced waves i s 20 or 150 msec, i t allows time for several synaptic delays; the induced waves could therefore represent summed a c t i v i t y from many polyneuronal feed-back loops. 32 It was orig i n a l l y thought that the mitral c e l l s f i r e d in synchrony with the induced waves and formed a part of this loop (Adrian, 1950), but attempts to substantiate this by simultaneous recording of induced waves and unit a c t i v i t y have been unsuccessful (Sakai, 1969). In fact, epinephrine, while enhancing wave a c t i v i t y (Penaloza-Rojas & Alcocer-Cuaron, 1967), depressed mitral c e l l s (Salmoiraghi et a l . , 1964).. The only work i n which the locking of mitral c e l l s to induced waves was sometimes seen, Baumgarten et a l . (1962a), i s open to certain criticisms. They stated that the mitral c e l l s were consider-ably less well locked than other types of c e l l s (granule and . tufted) and that no locking was obtained u n t i l they used max-imal stimuli (saturated vapours) and in j acted strichnine. The warnings of Gesteland et a l . (1965) should be borne i n mind, concerning the deterioration of the preparation both on over-stimulation with odour and on any insult to the circulation. Baumgarten sometimes used animals whose olfactory peduncles had been cut 3 - 4 weeks prior to the acute experiment. They thus eliminated by axonal degeneration any centrifugal fibres which could interfere with the pure antidromic stimulation of the LOT. While this seems an excellent aid to the study of evoked potentials and other unphysiological properties of bulbar c i r c u i t r y , the dangers of vascular damage to the bulb make this preparation of doubtful s u i t a b i l i t y for the observa-33 tion of physiological processes. It i s generally held that i n a deteriorated preparation, c e l l s that would not normally f i r e are l i k e l y to be activated. It i s interesting to note that induced and/or i n t r i n s i c waves are found to be enhanced after a variety of surgical man-oeuvres, including section of the LOT, AC and/or olfactory nerves(Callens & Boisacq-Schepens, 1963). Appropriate stim-ulation of these pathways i s widely reported to produce a depression of various types of bulbar a c t i v i t y . It i s not entirely clear, however, whether these effects imply a degree of tonic inhibitory bulbopetal a c t i v i t y , or whether degenera-tive hyperexcitability and recurrent axonal antidromic i n h i b i -tion i s the true explanation (for the enhancement and the depression respectively), or both. Baumgarten et a l . (1962b) showed that purely orthodromic commissural a c t i v i t y (by stim-ulation of the contralateral LOT with the i p s i l a t e r a l LOT severed) as well as purely antidromic stimulation of either commissural or LOT fibres (in the chronically isolated bulb) could inhibit bulbar neurons (some unidentified bulbar c e l l s , however, v/ere excited by orthodromic commissural stimulation) . They were surprised at their i n a b i l i t y to f i r e tufted c e l l s antidroinically by stimulation of commissural fibres. It apparently never occurred to them that these could be other than tufted c e l l axons, but Valverde (1965) found c e l l s in 34 the AON which sent axons to the AG with collaterals back to the i p s i l a t e r a l bulb. Mechelse & Lieuwens (1969), on the other hand, found no difference i n the induced waves after isolation of a bulb in chronic cats; the response of both Ottoson waves and induced wave bursts to airflow, both awake and on going to sleep, was the same as i n the intact bulb. Baumgarten et a l . (1962a) also studied changes in the amplitude of the induced waves and the evoked potentials as the electrode was moved down through the layers of the bulb. Both the waves and the second component of the potential evoked by LOT stimulation changed sign i n or.'just below the mitral layer. The resultant voltage profile led them to infer two time-locked v i r t u a l generators, one i n the external plexiform layer and the other i n the granule layer, where the voltage was greatest. Since the induced waves are 180° out of phase i n these layers, and both types of c e l l were locked to the negative-going phase, tufted and granule c e l l s were f i r i n g alternately. This would suggest that they are involved i n a feedback loop which generates the waves. There i s anatomical evidence for this loop, the peripheral processes of granule c e l l s being found i n inhibitory synaptic contact with dendrites of tufted c e l l s i n the external plexiform layer while axon collaterals of tufted c e l l s excite secondary dendrites of granule c e l l s in the granular layer (Price, 1969b). 35 I t i s interesting to note the role of strychnine. Baum-garten et a l . (1962b) found that intravenous strychnine did not affect the inhibition of c e l l f i r i n g produced by AG antidromic stimulation. However, topical application of strychnine to the bulb enabled Yamamoto & Yamamoto (1962) to e l i c i t waves identical to the induced waves by e l e c t r i c a l stimulation of the olfactory mucosa. This i s the only report of successful wave generation by pure e l e c t r i c a l stimulation of any structure i n the absence of airflow across the mucosa. This might be explained as follows. E l e c t r i c a l stimulation as applied i n this case was only able to excite olfactory neurons, whereas Geste-land et a l . (1965) and others have shown that olfactory stimula-tion inhibits a proportion of neurons. This inhibition must be important i n "authenticating" the olfactory stimulus, perhaps by dis i n h i b i t i o n > o f some c e l l such as a peri glomerular c e l l , which could be sensitive to topical strychnine. This could explain why we do not suffer an olfactory hallucination when we get a punch on the nose or a whiff of steam, for example. While induced waves are d i f f i c u l t to suppress in a macr-osmatic animal breathing through i t s nose, for which room a i r i s an adequate olfactory stimulus, they are not always obtained even with odorants i n the human (Sem-Jacobsen et a l . , 1956). Different electrodes were found to record a response to d i f f e r -ent chemicals. Hughes et a l . (1970), measuring the frequency spectrum of induced waves with a heterodyne wave analyser i n 36 the human, found that a l l the same frequencies were present whether the odour was perceived or not, but that the amplitude was lower when the patient did not report an odour. These workers have devised the Frequency Component Hypothesis of the olfactory coding mechanism, which maintains that an odour i s identified by the admixture of certain frequencies (Hughes & Hendrix, 1967). In their 1970 study a different repertoire of frequencies could be recorded from each electrode, which they adduce as support for their theory. The presence of different frequencies simultaneously i n one bulb casts doubt on the prediction: of Hainer et a l . (1954) that the induced waves would be found to serve as a coincidence sampling mechanism for extracting the complete picture of the olfactory environment from numerous component memory units, and summing i t over a number of cycles to reduce noise. This would presumably require waves of the same frequency to be present i n a l l areas projecting to stimulated parts of the epithelium. The difference between induced and i n t r i n s i c waves should be emphasized. The i n t r i n s i c waves described by Adrian (1950) and i n certain earlier studies appear to represent synchrony of the normal resting multiunit discharge. They are consider-ably more sensitive to anaesthetics and anoxia than the induced waves and reports of them vary greatly with species and recording techniques. In the more physiological preparations of several species their frequency i s often about double that 37 of the induced waves. They disappear with the advent of an induced wave. I t i s thus permissible to v i s u a l i s e the i n t r i n -s i c waves as representing a c t i v i t y i n a smaller loop within the bulb, perhaps r e s t r i c t e d to the i n t e r n a l granule c e l l layer, with some overlap between t h i s loop and the one generating induced waves, so that the two types of a c t i v i t y are mutually exclusive. There does not appear to be evidence f o r a r e a l f u n c t i o n a l difference between the condition of " i n t r i n s i c waves" and asynchronous " i n t r i n s i c a c t i v i t y " . The bulbar counterpart of the c o r t i c a l desynchronization on arousal appears to enhance the induced waves (only Yamamoto & Iwama (1961) found them to be depressed), and w i l l be discussed below. The type of i n t r i n s i c a c t i v i t y seen seems to depend mainly on the e l e c t r i -c a l c h a r a c t e r i s t i c s of the recording system and the nature and depth of anaesthesia. Urethane seems to be the anaesthetic of choice, at l e a s t i n the rabbit; . i t does not a l t e r the wave frequencies and i f l i g h t enough enables induced waves to be e l i c i t e d i n those instances when they would be expected from chronic studies. Barbiturates (nembutal and d i a l ) a l t e r the frequency of induced waves (Stone et a l . , 1968); i t i s much reduced and may decline further during a burst (Adrian, 1950). At intermediate l e v e l s of anaesthesia (Adrian, 1950) or with halothane (Graystone, personal communication) wave a c t i v i t y i s continuous, even i f the o l f a c t o r y t r a c t i s severed. 38 Electrodes and recording techniques have now been devel-oped to the point that a particular mode of a c t i v i t y - wave, multiunit, single c e l l , D.C. potential shi f t - can be isolated without resorting to anaesthesia to eliminate i n t r i n s i c noise as Adrian was obliged to do i n 1950. It i s important to wait a few days after preparation of chronic animals under nembutal. Unfortunately Perialoza-Ro jas & Alco.cer-Cuaron (1967) only waited' 24 hours; they found the i n t r i n s i c waves reduced to 30 - 40 Hz, although their induced waves at 44 Hz are within normal li m i t s for the cat. Acute experiments present a more serious problem. Most workers have stopped using nembutal, but a popular alternative i s ether. While apparently an hour or two i s sufficient for effects on the central nervous system to "blow off",. and.:.many workers report having duly waited for this time, they have then proceeded with their experiments apparent-ly unaware of the fact that the receptors i n the epithelium have been seriously damaged (Mozell, 1958). This explains why Baumgarten et a l . (1962a, b) found they had to stimulate a macrosmatic animal with saturated vapours! Trigeminal effects on olfaction i n general were discussed towards the beginning of this introduction. One group of workers (Stone et a l . , 1966, & 1968)','.'however, has studied their effect s p e c i f i c a l l y on the induced waves. They used rabbits with cannulas chronically implanted i n the Gasserian ganglia. They obtained a reversible trigeminal block by injecting 39 xylocaine, and compared responses of the organism to olfactory stimulation i n the two conditions. Xylocaine reduced the con-t r o l respiratory rate to less than half and abolished the tachy-pneuic response to odour. The same could be said of the heart rate i l l u s t r a t e d i n the preliminary paper of 1966, but no i n -crease i n heart rate on odour presentation pre-xylocaine was reported i n 1968. The bulbar response and the c o r t i c a l desyn-chronisation on odour presentation were l i t t l e affected by xylo-caine i n the 1966 account. In the s t a t i s t i c a l analysis of 1968, however, the frequency and amplitude of the bulbar induced wave response were increased by the block while the c o r t i c a l arousal was usually abolished. The change in frequency i s most unex-pected and does not seem to be consistent with the bulk of the literature (e.g. Graystone et a l . , 1970). The i n i t i a l decrease i n bulbar a c t i v i t y noted by Stone et a l . on presentation of the olfactory stimulus pre-xylocaine, particularly at high concentrations, i s consistent with the noxious vapour reflex of Allen (1929), as are the other auto-nomic changes. This i s to be expected since they deliberately chose i r r i t a t i n g odours. Their concentrations may be compared to those used i n the work to be reported below. They used one of the same odorants, propionic acid, at an "olfactory a c t i v i t y " (see equation 1) of the order of 14, which w i l l be shown to be within the range where the noxious vapour reflex intervenes. 40 Arousal Olfactory induced a c t i v i t y i s closely linked to arousal and emotion. Hernandez-Peon and coworkers, after finding that second order neurons of various sensory modalities were affected by arousal through another sensory modality mediated by the mesencephalic reticular formation, turned their atten-tion to olfaction (Lavin et a l . , 1959) i n chronic cats. They found that bursts of 34 - 48 Hz a c t i v i t y i n the bulb were e l i c i t e d or enhanced by alerting the animal through any sensory modality or by 50 Hz e l e c t r i c a l stimulation of the retic u l a r formation. They named these "arousal discharges", thinking that they differed from Adrian's induced waves (and i n t r i n s i c waves) i n requiring integrity of centrifugal pathways. In the li g h t of later work, however, i t would appear that i n their prestimulus a c t i v i t y , as in Adrian's (1950) unanaesthetised preparation, the induced waves were simply buried i n spontane-ous a c t i v i t y as a result of recording techniques. •-The means of measuring respiration i s not stated, so i t i s d i f f i c u l t to explain Lavxn et a l . (1959)'s i n a b i l i t y to correlate burst a c t i v i t y with respiration; they probably used the thoracic measure, which most workers have now replaced by a thermistor to distinguish nasal from buccal airflow. They noted that the bursts occurred at 20 - 25/min, the range of respiratory rate in the cat, and pointed out that neither inspiration nor blowing at the no s t r i l s guarantees airflow over 4 1 the olfactory part of the nasal passages. They suggested that the reticular influence i s the neural mechanism for the focussing of attention, enhancing the sensi-t i v i t y of the modality whose information promises to be the most pertinent. The cat, being a macrosmatic animal, would be anxi-ous to obtain olfactory corroboration of other exteroceptive data. McLennan & Graystone (1965) suggested that the burst a c t i v i t y constitutes a "gated scanning system" such as i s used i n radar for improving sensitivity (noise rejection). On the other hand, i f an olfactory stimulus was delivered i n the pres-ence of "arousal discharges", the l a t t e r were suppressed (Hernandez-Peon et a l . , I960). McLennan & Graystone confirmed that, when this a c t i v i t y i s present in an alert cat, i t i s inhibited by odorous stimulation; this may infer that maximum information has immediately been extracted from this sense, so attention passes to other modes of perception. On the other hand, when signals are enhanced i n the inattentive cat alerted by olfactory stimuli (also confirmed by McLennan & Graystone), thi s response seems biologically appropriate too, since there may well be additional information i n the a i r besides the particular cause for the alarm. It would thus appear that the process of olfactory dis-crimination as opposed to olfactory alerting i s accompanied by a diminution i n bulbar a c t i v i t y . But much of the li t e r a t u r e . : i s quite unsure about this. Most people assume u n t i l proven 42 otherwise that sensory stimulation entails an augmentation of e l e c t r i c a l a c t i v i t y of a l l sorts. This i s not necessarily so, even i n f i r s t order neurons; for example, Hodgson & Steinhardt (1967) studied the inhi b i t i o n of sugar receptors by certain hydrocarbons: they found i t to play a physiological role. .How-ever, many common experimental procedures tend to reinforce this prejudice, such as the suppression of resting discharge by anaesthetics and the frequent practise of giving odours without a background of nasal airflow (tracheal cannula). I t i s also often unclear to one author what type of e l e c t r i c a l a c t i v i t y another was talking about and on what model of anatomical con-nections his reasoning was based. Takagi & Shibuya (I960) found induced waves increasing with intensity of odour, but they used the doubly unphysiological preparation of a toad decapitated under ether. Gault & Leaton (1963) found an enhancement of 40 Hz a c t i v i t y i n the bulb and amygdala during olfaction and attributed the suppression seen by others to changes i n respiratory parameters. It has been sug-gested i n turn that they were using inattentive animals, but i n the records referred to the cat was actively sniffing and clear-l y attentive; the true cause seems to be the improved velocity and pathway of airflow associated with sniffing (Tucker, 1963)• The increase i n a c t i v i t y with snif f i n g would actually be great enough (de Vries & Stuiver, 1959) to mask the depressions found i n response to an olfactory stimulus in the present work (Pig. 11B) . 43 I t should also be borne in mind that EEG alertness has been shown to para l l e l autonomic tone as measured by that important parameter, peripheral a r t e r i a l pressure (Bonvallet et a l . , 1954). Thus i t i s possible, despite Yamamoto & Iwama's (1961) denial, that the changes i n wave a c t i v i t y i n the bulb are mediated i n part by the vascular system, particularly since the changes i n c o r t i c a l arousal accompanying sympathetic or sympathomimetic a c t i v i t y were found by Bonvallet to be mediated by the caudal mesencephalic reticular formation, Yamamoto & Iwama (1961) stated that i n the rabbit breath-ing through a tracheal cannula the i n t r i n s i c waves of the bulb were depressed by sensory or reticular stimulation i n a way reminiscent of c o r t i c a l desynchronization, but with a different time course. These workers, however, also found the induced waves to be depressed; they attribute the enhancement seen by Hernandez-Peon et a l . (i960) to respiratory changes accompany-ing arousal (as do Gault & Leaton, 1963). They differed from Kerr & Hagbarth (1955) i n finding the suppression to be mediated by a pathway other than the AC. Their acute animals were atropinised and chronic experiments performed from 1 day to a week after pentobarbital anaesthesia. Harada & Takagi (1961) have studied the effects on bulbar "induced a c t i v i t y " of stimulation of each of the senses with a l l others excised, i n the frog. Their "induced a c t i v i t y " was usually an e l e c t r i c a l evoked potential, but a puff of saturated 44 ether vapour was used occasionally to. confirm that the same effect would be obtained on an induced wave. By this strange procedure they found that induced a c t i v i t y was inhibited by auditory, photic and noxious and warming cutaneous stimuli, while cutaneous touch and cold augmented i t . They suggest that the difference i s due to the significance of the particular ;types of stimuli i n the natural habitat of the animal; the augmentation sometimes seen might represent the arousal of curiosity by a non-threatening sensation; species differences i n the response spectra would therefore be expected. Wave a c t i v i t y of the same frequency as that i n the olfac-tory bulb has been recorded i n other rhinencephalic structures, the amygdala (Gault & Leaton, 1963), amygdala and pyriform cortex (McLennan.- et • a.l.; 1967) and various rhinencephalic and non-rhinencephalic structures (Mechelse & Lieuwens, 1963); Freeman (I960) studied what appears to be the same phenomenon i n the pyriform cortex but without monitoring the olfactory bulb; a l l these studies were done i n the cat. Freeman found pyriform c o r t i c a l wave a c t i v i t y which varied with respiration. I t was increased by food odours i n proportion to the degree of starvation and did not show adaptation to food odours i n a hungry cat, whereas adaptation was seen to chemical odours. In a satiated cat no response was seen except to such biologi-c a l l y but non-alimentarily interesting odours as urine and catnip. I t i s noticeable that the conditions for augmentation 45 are a l l s i t u a t i o n s i n which the animal i s motivated to i n t e n s -i f y i t s o l f a c t o r y i n v e s t i g a t i o n ; i n f a c t h i s r e c o r d s show an i n c r e a s e i n r e s p i r a t o r y r a t e which would d o u b t l e s s be accom-panied by i n c r e a s e d n a s a l a i r f l o w so t h a t a s i m i l a r augmenta-t i o n would have been found i n the o l f a c t o r y b u l b . The t e m p o r a r i l y augmented response to chemical odours i n the hungry c a t (48 hours d e p r i v a t i o n ) i s more com p l i c a t e d to e x p l a i n . H i s s u b j e c t s were t r a i n e d to work f o r food when hungry, so a s l i g h t l y d i f f e r e n t response might be expected from them than, from animals not l i k e l y to i n t e r p r e t the odour as a c o n d i t i o n e d s t i m u l u s . The r e l a t i o n s h i p of p y r i f o r m c o r t i c a l b u r s t s to those i n the bulb has a l s o been the s u b j e c t of c o n t r o v e r s y . Gault & Leaton (1963) s t a t e d t h a t b u l b a r b u r s t a c i t v i t y was a n e c e s s a r y but not a s u f f i c i e n t c o n d i t i o n f o r b u r s t s i n the amygdala, and Mechel.se & Lieuwens (1963, 1969) always found t h a t 40 Hz a c t i v - ' i t y d i s a p p e a r s from o t h e r areas e a r l i e r than from the bulb on f a l l i n g a s l e e p . Evoked p o t e n t i a l s t u d i e s , on the o t h e r hand, l e d McLennan & Graystone (1965) to b e l i e v e t h a t the b u r s t s -were . i n i t i a t e d i n the amygdala. However, phase r e l a t i o n s h i p s c a l c u l a t e d i n 1967 w i t h the p y r i f o r m c o r t e x i n c l u d e d and b i -l a t e r a l r e c o r d i n g suggested t h a t waves began i n the p y r i f o r m c o r t e x and were t r a n s m i t t e d to the c o n t r a l a t e r a l p y r i f o r m c o r t e x b e f o r e a p p e a r i n g i n the o l f a c t o r y bulb or amygdala of e i t h e r s i d e . I t has been w i d e l y r e p o r t e d , however, t h a t the 46 induced waves s t i l l appear i n the bulb a f t e r n e u r a l c o n n e c t i o n s w i t h the b r a i n have been severed, and Mechelse & Lieuvens (1969) found.no d i f f e r e n c e i n the responses of an i n t a c t and a c h r o n i -c a l l y i s o l a t e d bulb under s t i m u l a t i o n by p u f f s of gas or under the n a t u r a l r e d u c t i o n of a i r f l o w over the o l f a c t o r y organ a s s o c i a t e d v/ith f a l l i n g a s l e e p . The o s c i l l a t o r y evoked poten-t i a l seen by McLennan & Graystone (1965) on s t i m u l a t i o n o f the amygdala may simply be due to the h i g h n a t u r a l p r o p e n s i t y of the b u l b a r n e u r o n a l c i r c u i t r y f o r t h i s behaviour ( c . f . i t s enhancement i n e a r l y d e g e n e r a t i o n of acute p r e p a r a t i o n s ) . McLennan et a l . ( p e r s o n a l communication) now suggest t h a t phase r e l a t i o n s t u d i e s should be repeated w i t h one n o s t r i l plugged to e l i m i n a t e c o n f u s i o n due to a p o s s i b l e simultaneous g e n e r a t i o n of waves i n both b u l b s . The phase l a g of 6 msec found by McLennan et a l : f o r appearance of p y r i f o r m c o r t i c a l waves i n the o l f a c t o r y bulb, i f i t r e f l e c t s t r a n s m i s s i o n of s i g n a l s i n a n e u r a l pathway, suggests t h a t t h e r e i s time f o r the second o r d e r o l f a c t o r y neurons to a c t i v a t e the h y p o t h e t i c a l wave g e n e r a t o r i n the p y r i f o r m c o r t e x w i t h i n the l a t e n c y found by MacLeod & Leveteau (1963) f o r induced waves i n the o l f a c t o r y b u l b . A d d i t i o n a l study i s c l e a r l y needed. 47 Concentration Another possible source of the discrepancies i n the l i t e r -ature i s the concentration of the odour used as stimulant. McLennan & Graystone (1965) used extremely strong smells (dense smoke of burning rags, pipe, etc.) and the responses e l i c i t e d may not have been purely olfactory, but complicated by noxious vapour reflexes, watering of the eyes, etc. Hughes et a l . (1970) reported electrophysiological changes at concentrations (absolute values unspecified) at which his patients were unable to detect the odour. It i s a very curious fact that, while the human olfactory threshold i s said to be of the order of 10""^ molar, no electro-physiological reports have been found in which macrosmatic animals have been presented with such a weak stimulus. Yet most people (except Adrian, 1963) say that olfaction at well below human threshold i s highly e f f i c i e n t i n many animals. No exten-sive concentration studies on induced waves have been found at a l l . Mozell (1958) studied spike a c t i v i t y i n the deeply anaes-thetized bulb over anrange of 2 log units; the a c t i v i t y rose from zero to a plateau. Tucker (1963) measured the compound action potential of olfactory nerve twigs over a 5 log unit variation i n odour intensity; it increased (not from zero) to a peak and declined over the last log unit below saturated vapour. Both these authors used amyl acetate as one of-their stimuli, and expressed their concentration in (different) 48 a b s o l u t e u n i t s . T h i s p a r t i c u l a r odorant was t h e r e f o r e used i n the present study f o r ease of comparison. Other chemicals were a l s o used to a s s i s t i n extending the range and reduce o l f a c t o r y a d a p t a t i o n ; i f a more e l a b o r a t e o l f a c t o m e t e r c o u l d have been c o n s t r u c t e d the whole range c o u l d have been covered w i t h each odorant. 49 P r e s e n t Research: The Hypothesis Prom an assessment of the l i t e r a t u r e the outcome of the p r e s e n t i n v e s t i g a t i o n was p r e d i c t e d as f o l l o w s : In a drowsy  animal an o l f a c t o r y s t i m u l u s was expected to. produce an a l e r t i n g response (augmentation of induced waves) sometimes, w h i l e a t o t h e r s the odour might not be p e r c e i v e d or might e l i c i t anc o l f a c t o r y response ( d e p r e s s i o n ) unmasked by a r o u s a l . The lowest c o n c e n t r a t i o n a v a i l a b l e from the o l f a c t o m e t e r was the human d e t e c t i o n t h r e s h o l d , which might be h i g h enough to s t i m u l a t e . t h e r e c e p t o r s of a c a t not a c t u a l l y a s l e e p . N e g l i g i b l e responses . would t h e r e f o r e not n e c e s s a r i l y be expected to appear at t h i s end of the s c a l e . The changes seen i n an aroused c a t , on the o t h e r hand, were expected to v a r y i n a c o m p l i c a t e d f a s h i o n w i t h s t i m u l u s i n t e n s i t y . In the r e g i o n of the f e l i n e t h r e s h o l d , i f t h i s c o u l d be reached w i t h the apparatus a v a i l a b l e , random r e s u l t s would be expected, c o r r e s p o n d i n g td the f r e q u e n t spontaneous v a r i a t i o n s i n " a r o u s a l d i s c h a r g e s " . I n the next range, where, s t i m u l u s i n t e n s i t y would not suggest to the animal t h a t the source of odour was c l o s e a t hand, a pure o l f a c t o r y response ( d e p r e s s i o n ) v/as expected. F u r t h e r up the s c a l e , the odour might seem to r e p r e s e n t more imminent changes i n the e n v i r o n -ment and prompt a r e d i r e c t i o n of a t t e n t i o n from s e l f - c e n t r e d a c t i v i t y such as washing and p l a y i n g to e x t e r o c e p t i o n , so a p r o p o r t i o n of responses would be i n the d i r e c t i o n of enhance-50 merit. Fina l l y , a concentration was expected to be reached at i which nasal discomfort would e l i c i t reflex and/or conscious physical processes tending to reduce contact of the mucosa with the offending vapour. It was not possible to predict the absolute values of concentration over which each of these effects would occur (except for the range of 10r^"^ - 10"^ **^  of saturation of amyl acetate quoted by Tucker•' (1963) for appearance of the reflex change i n physical dimensions of the nasal passages) nor how much they would overlap. But the general hypothesis was clear enough to prompt analysis of the results by regression of the independent variable, percentage change of integrated 40 Hz ac t i v i t y , on fourth order polynomials of the logarithm of concentration relative to human threshold. The results for different odorants were pooled after converting the data into this form ("olfactory activity'^, on the assumption that while species differences in sen s i t i v i t y i n general might be large, the d i f f e r e n t i a l response would probably depend largely on physicochemical constants and was l i k e l y to be i n proportion for any randomly chosen pair of odorants. 51 M E T H O D S A N D M A T E R I A L S 52 I . T H E P R E P A R A T I O N Experiments were performed on f o u r l a r g e a d u l t c a t s (3 male and 1 female, c a t I I ) , a l l c h r o n i c a l l y p r e p ared. The g r o s s anatomy of the n a s a l passages and o l f a c t o r y apparatus were f i r s t observed i n a d i s s e c t i o n of f r e s h c a t cadaver.. A l l c a t s were implanted b i l a t e r a l l y w i t h b i p o l a r s t a i n l e s s s t e e l e l e c t r o d e s i n the o l f a c t o r y bulb and w i t h s i l v e r p l a t e d screws i n the f r o n t a l and p a r i e t a l s k u l l bones; c a t IV a l s o had b i p o l a r e l e c t r o d e s i n the b a s o l a t e r a l amygdalae, and c a t I had 4 e l e c t r o d e s i n o t h e r r h i n e n c e p h a l i c s t r u c t u r e s which were not made use of i n t h i s study. For c a t s I - I I I , e l e c t r o d e s were made from 25 gauge s t a i n l e s s s t e e l tubes (manufactured by Becton D i c k i n s o n & Go.,. R u t h e r f o r d , N . J . ) . These were cut and ground to 5 . 9 cm and a ho l e ground i n the s i d e to about h a l f the diameter o f the tube ( P i g . 3A) a t a s u i t a b l e l e n g t h from one end. The b u r r was removed from t h i s h o l e and the ends w i t h a 24 gauge needle c l e a n e r . E l e c t r i c a l c o n n e c t i o n to t h i s e l e c t r o d e was made w i t h 32 gauge enamelled copper wire c a b l e c o r d (from C o n s o l i d a t e d Wire and A s s o c i a t e d Companies, Chicago, 1 1 1 . ) bared f o r 2 mm, looped around the tube j u s t below the hole and s o l d e r e d i n p l a c e ( P i g .3?).''The tube served as the o u t e r p a r t o f the concen-t r i c b i p o l a r e l e c t r o d e . The i n n e r c o n s i s t e d of 30 gauge t e f l o n - c o a t e d s t a i n l e s s s t e e l wire ( o b t a i n e d from R.A.E., 53 F I G U R E B. C. D. Ground with Dremel Tool (plan) 2 5 gauge Steel Tube ^ Solder Joint 7v ~—' bared Epoxy 0.5 mm enamelled Cu Wire 'Tetlon coated Steel Wire Shaft cut here after Electrode cemented in Place Beldenamel Varnish Insulation Cut, bared and soldered to Cannon Plug BIPOLAR ELECTRODE CONSTRUCTION A. A hole i s ground i n the outer tube. B. Copper wire i s soldered to the tube. C. Teflon-coated wire i s threaded through the hole. D. The inner wire i s cut and glued in place. E. The electrode i s insulated and the t i p s bared ready f o r implantation. 54 Vancouver) threaded through the hole and down inside the tube (Pig. 3C). A f t e r t e s t i n g f o r short c i r c u i t s caused by s t r i p -ping of the t e f l o n by burrs, the inner wire was cut o f f to project 0.5 mm beyond the outer and fix e d i n place with a small blob of epoxy r e s i n which also covered the solder j o i n t (Pig. 3D). The electrodes were insulated with several coats of Beldenamel varnish each baked f o r 4 hours at 250°P. The ti p s were bared under a binocular microscope at 40x magnification u n t i l the resistance of the inner was down to a few hundred Kfl measured at 10 - 100 Hz and a patch at the end of the outer bared to match t h i s resistance (Pig. 3E). When the i n s u l a t i o n broke down at the jo i n t i t was sealed with beldenamel or Q-dope. Cat IV was implanted with concentric electrodes as above but bared down to a few tens of Kil resistance at 60 Hz, with the outer bared a l l round the end, on the l e f t side. On the r i g h t side he received pairs of monopolar electrodes made from smaller (28 gauge) s t a i n l e s s s t e e l tubes and bared to a r e s i s -tance s i m i l a r to those on the l e f t . The two electrodes were implanted to the same depth about 1 mm apart i n the antero-posterior d i r e c t i o n , the anterior being treated l i k e the "inner" of the bipo l a r electrodes f o r recording purposes. The electrodes were implanted i n an aseptic operation 55 under Nembutal anaesthesia, 35 mg/kg intraperitoneally. The animal's head was held i n a stereotaxic instrument from La Precision Cinematographique, Asnieres, Prance. The scalp was opened with a 6 cm midline incision and the periostium elevated. The frontal sinus was opened with a trefine and a 2 mm diameter hole d r i l l e d i n the bone overlying the olfactory bulb at i t s saddle point. The dura was pricked with a needle and when bleeding subsided the electrode was lowered v e r t i c a l l y to 7 mm below the surface of the bone. The sinus was f i l l e d with Gelfoam and the electrode cemented i n place with dental cement (Yates Manufacturing Co., Chicago, 111., Plash A c r y l i c ) . When the cement was dry the shaft of the electrode (the part of the tube above the blob of epoxy) was cut off. In the case of cat IV the basolateral amygdala.electrode coordinates were , determined from the Merck Stereotaxic Atlas and electrodes made so that the epoxy blob was just at the level of the top of the s k u l l . The c o r t i c a l screws were fixed i n the s k u l l i n small d r i l l e d starter holes 1 - 2 cm behind the suture, two on the right and one on the l e f t . Enamelled copper wire was soldered to them. The wires were then soldered to. a female 15 pin Can-non plug with the metal casing removed and the two halves melted together, except for cat IV which retained the casing. The plug was then bu i l t up smoothly with dental cement, separ-ating a l l wires, and the wound closed with a suture i f neces-56 s a r y . Animals were allowed a t l e a s t a week to r e c o v e r from the o p e r a t i o n ; c a t I I took two months' m a t e r n i t y l e a v e . Cat I d i e d of a pulmonary i n f e c t i o n a f t e r c o n t r i b u t i n g to p r e l i m i n a r y s t u d i e s , and h i s b r a i n was p e r f u s e d w i t h 10% f o r m a l i n . The ot h e r c a t s were not s a c r i f i c e d i n view of the s h o r t supply, t h e r e being l i t t l e room f o r doubt as to the presence of a macroelectrode somewhere i n the o l f a c t o r y b u l b . 57 I I . T H E R E C O R D I N G S Y S T E M Con n e c t i o n t o the c a t ' s p l u g was made w i t h a male cannon p l u g from which, r a n a c a b l e made from 2 s t r a n d s o f 3 x 28 gauge conductor s h i e l d e d audio c a b l e (from Western Ag e n c i e s , Vancouver) wound t o g e t h e r w i t h one wire removed from each. T h i s c a b l e was v e r y f l e x i b l e and had no outer sheath so the c a t c o u l d move f r e e l y . A " f u s e " was made i n the c a b l e , i n case the c a t should p u l l i t too hard, by t e r m i n a t i n g the cable i n an a r r a y o f cannon p l u g p i n s h e l d t o g e t h e r w i t h d e n t a l cement, to be i n s e r t e d i n t o another s e t a t t a c h e d to the male p l u g ( P i g . 4). Wires were a t t a c h e d i n the l a t t e r to each p i n corr e s p o n d -i n g to an e l e c t r o d e , so t h a t s e l e c t e d e l e c t r o d e s c o u l d be plugged i n t o the r e c o r d i n g system. S t r o n g e r w i r e s were used' f o r t h i s to a v o i d breakage. A l l these w i r e s were kept i n s u l -a t e d from one another w i t h M u e l l e r # 32 v i n y l i n s u l a t o r s l e e v e s . The s h i e l d was connected to a c o r t i c a l screw. T h i s whole assembly was covered w i t h a sheet of aluminum f o i l a f t e r the e l e c t r o d e s e l e c t i o n had been made. The c a b l e from the two b i p o l a r e l e c t r o d e s s e l e c t e d was f e d i n t o the d i f f e r e n t i a l i n p u t of a type 502 d u a l beam T e k t r o n i x O s c i l l o s c o p e ( P i g . 5) which was grounded, a l l o t h e r apparatus being i s o l a t e d from the l i n e and grounded to t h i s o s c i l l o s c o p e . S e n s i t i v i t y was s e t a t 200 u-V/cm, timebase a t . 20 msec/cm, so t h a t 40 Hz a c t i v i t y c o u l d be seen c l e a r l y and 58 F I G U R E 4 Connector Plug To Oscilloscope Input /Olfactometer 2x2-Core bore-shielded Wires wound together Mechanical "Fuse" 6 Cannon Plug Pins Dental Cement Hook-up Wire (x6) Threaded through Holes in Cannon Plug Casing for Strength Connection to chosen Electrode (x5) Vinyl Sleeve One Representative non-chosen Electrode (x5) (eth Pin connects- fo Cannon Plug Casing) One Representative non-chosen Electrode stowed in Vinyl Sleeve (x6) 15 pin Cannon Plug Aluminum Foil Shielding Odor Tube, attached to Plug by Rubber Bands, guided by groove crimped in Foil to send Jet straight over Cat's Nose CONNECTOR PLUG The male cannon pl u g shown here i s plugged i n t o the femal on the c a t ' s head f o r r e c o r d i n g , w i t h the odour tube d i r e c t i n g a j e t of a i r over i t s nose. The mechanical " f u s e " and the method of c o n n e c t i n g to s e l e c t e d e l e c t r o d e s are i l l u s t r a t e d . 59 F I G U R E 5 Recording System Plug ROB LOB Cort c Screw Oscilloscope Cat or Reduction Box Oscillator 40 Hz Filters Vokam Power Supply Poly-graph RECORDING SYSTEM 60 distinguished from hum. The oscilloscope stand was f i t t e d with a Grass Kymograph camera which was occasionally used, with f i l m moving at 100 mm/sec, to photograph the amplified raw signals with the horizontal sweep disabled. Calibration was provided by a Hewlett Packard model 200CD wide range 4 o s c i l l a t o r set at 2 volts fed through a 10 : 1 reduction box to a female cannon plug wired to produce a single-ended version of the arrangement of the cats' electrodes, giving a 200 u,V signal. The frequencies of the sinusoidal a c t i v i t y on the developed f i l m were measured using a Gerber Sc i e n t i f i c Instru-ment Go. (Hartford, Conn.) Variable Scale, to determine the characteristic frequencies of each cat, i f any. Signals from cat I, i n addition to being filmed on photo-graphic paper as above i n single-ended mode, were fed into the analog-to-digital converter of Graystone (1970) and written on binary magnetic tape i n blocks (;Pig;. 18A).: . The tape was run on an autocorrelation program (Moore, 1968) on the IBM 360/67 computer, which computed the frequency from the zero-crossing intervals of the autocorrelation function of the f i r s t 250 msec of each block. This function was converted back from the computer tape and displayed on the oscilloscope for photography as above so that the v a l i d i t y of the frequency print-out could be checked; i n cases where there was no real correlation, a frequency print-out was s t i l l obtained i f there were sufficient zero-crossings to compute i t , , but i t was quite meaningless: i t 61 was necessary" to inspect the shape of the function to make sure that a l l significant fluctuations, and no others, actually did cross the zero l i n e , as i n Pig. 18B. The oscilloscope output was fed into 40 Hz bandpass f i l t e r s powered by a (Shandon Scie n t i f i c Co., Ltd., London) . Vokam constant voltage power supply. The f i l t e r s had 3 db cut off points at 34 and 42 Hz (peak 38 Hz) for channel ROB (led from the right olfactory bulb) as shown i n Pig. 6, 33i and 45 Hz (peak 39 Hz) for channel LOB. The f i l t e r e d signals were re c t i f i e d and integrated and displayed at 5 mm/sec on long pen chopper channels of a G-ilson polygraph, on which the calibration was made. The area under the curve (Pig. 11) was measured with a K & E compensating polar planimeter. Results were run on ... the IBM 360/67 computer on UBC library program TRIP to obtain orthogonal polynomial generation, correlation matrices (Appendix I - I I I ) , multiple regression analyses and plotting (Pig. 15 and 16). 62 too 4 Amplitude 40 20 10 2 -15 F I G U R E ; o i o I o d I o o \ o o \ % \ 9 o 3db cut off i i i i i i i l l ) 25 40 60 100 Freqency Hz F I L T E R C H A R A C T E R I S T I C The o r d i n a t e i s mm d e f l e c t i o n of the polygraph pen i n response to 200 u,V o s c i l l a t o r s i g n a l s f e d through the f i l t e r n o r m a l l y connected to the e l e c t r o d e i n the r i g h t b u l b . The othe r f i l t e r had a s i m i l a r c h a r a c t e r i s t i c . 6 3 I I I . T H E O L F A C T O M E T E R The cat was placed i n a sound-damped cubical wooden box, 320 l i t r e s i n volume, provided with a one-way mirror through which behaviour was monitored. The laboratory was darkened and the box l i t by 5 bulbs around the mirror powered by a 6 volt heavy duty car battery. The box had a few holes for ventila-tion; the recording cable was passed, through one i n the centre of the roof closer to the rear. Elastic thread was tied to the cable so that i t would retract a l i t t l e i f the cat moved closer to this hole, instead of tripping i t ; this reduced the amount of a r t i f a c t due to the cats playing with the cable ; .. or..: biting through i t . A piece of elastic thread was hung from the roof by a thumbtack to provide a harmless plaything. A i r was introduced through a 10 foot length of f l e x i b l e l/8" I.D. tygon tubing which ran up through the l i d of the box and down along the recording cable to the connector plug to. which i t was firmly attached with rubber bands in such a way that the stream of a i r passed the cat's nose'. The flow rate was set at 4 litres/minute so that-the supply was always adequate to the cat's respiratory demands and box a i r , with i t s traces of past odorants and occupants, would never be breathed. Ueki & Domino (1961) have calculated the maximum inspiratory flow for dogs at 1.5 - 4 l/min. 64 Clean a i r was obtained from the compressed a i r supply of the building. Prom the a i r line, a i r was fed continuously into an Airco oxygen therapy regulator adjusted to 4 - 5 l/min (Pig. 7). l/4" I.D. tygon tubing led to a glass Y-piece from which two similar lengths of such tubing led to another Y-piece whose stem was adapted to f i t into the 1/8" tygon tubing going into the catbox. One of the par a l l e l paths was the bypass tube which remained clamped with a Day Pinchcock Clamp during most of the experiment. The bypass tube could not be used between stimuli during a run since movement of the clamp produced a change i n the airstream over the cat's nose which would have acted as a conditioned stimulus. The other path contained.a T-piece and then a Y-piece. The Y-piece introduced a side,tube which was closed with a screw clamp and could be opened to sample the a i r or blow off excess odour. The side arm of the T-piece was closed with a Rubber Cap Stopper through which passed a Yale Luerlok hypodermic needle, sometimes 21 gauge, with the o r i f i c e i n the middle of the airstream. Odorant i n the form of a pure l i q u i d was delivered down such a hypodermic needle from a disposable 1 ml tuberculin syringe or by a (Hamilton Co., Inc., Whittier, C a l i f . ) 25 M-l syringe which has i t s own needle as an integral part. A 2.5 ml syringe was used i n some preliminary experiments. Sometimes the syringe contained saturated vapour with a l i t t l e l i q u i d i n the bottom below the needle o r i f i c e ( i t was held horizontally). 65 F I G U R E Olfactometer Air Regulator \ I.D. Line Tygon ' Flowmeter Tygon Infusion Pump Hypodermic Needle Sample Tube I.D Tygon x 3' Cat Box N Cat's Plug Tygon x10 ' Bypass Line OLFACTOMETER I n experiment C, the i " t u bing l e d d i r e c t l y from the hypo-dermic needle to the o u t l e t i n the roo f of the'catbox. 66 The s y r i n g e was h e l d i n one b a r r e l o f a Harvard model 940 d u a l i n f u s i o n / w i t h d r a w a l pump. The g l a s s T-piece was supported by a wire frame from the pump so t h a t i t d i d not r e s t on the needle and touch i t when jogged, which was found to cause l i q u i d to be sucked out by c a p i l l a r i t y . T h i s r e s u l t e d i n an u n d e s i r a b l y l o n g l e n g t h of t u b i n g so t h a t the pre s s u r e a t the needle r e q u i r e d to ac h i e v e the d e s i r e d f l o w through the down-stream t u b i n g was 9 mm Hg. The g l a s s s y r i n g e s were unable t o wi t h s t a n d t h i s p r e s s u r e ; u n f o r t u n a t e l y g a s - t i g h t s y r i n g e s were not a v a i l a b l e . The pump had 12 gears p r o v i d i n g speeds i n l o g a r i t h m i c increments o f 3 g e a r s / l o g c y c l e ( i . e . gear n g i v e s twice the speed of gear n + 1). Nine of the gears c o u l d be used without s t o p p i n g the motor to s h i f t . The motor was kept r u n n i n g and the gears engaged f o r p e r i o d s o f 4 - 1 minute so t h a t odorant would be s u p p l i e d • t o the end o f the needle f o r v a p o r i s a t i o n i n the a i r stream without the c a t ' s b e i n g aware through any oth e r sense t h a t a s t i m u l u s was be i n g p r e s e n t e d . The motor provided a h i g h background n o i s e l e v e l . U n f o r t u n a t e -l y the o p e r a t i o n of s h i f t i n g was sometimes accompanied by a "tok" n o i s e ; t h i s v/as marked on the r e c o r d when i t o c c u r r e d . A t y p i c a l i n f u s i o n r a t e , w i t h a 1 ml s y r i n g e a t gear 10, was .1 nl/min. The amount of t r a v e l i n v o l v e d was so s m a l l (e.g. 50 u.m/min) t h a t i t was c o n s i d e r e d i m p r a c t i c a l to attempt to i n s e r t and withdraw the needle between s t i m u l i without c a u s i n g s p u r i o u s s t i m u l i . 67 I, V.. T H E 0 D 0. R A N T S Odorants were chosen from the Laffort (1963) l i s t of 192 "pure" compounds for which the human olfactory threshold had. been assessed. While i t was thought to be unlikely that the feline threshold i s i n fact the same as the human, i t was con-sidered probable that for the majority of odorants i t varies lin e a r l y with i t . I t i s to be expected that olfactory recep-tor: mechanisms at the molecular level are qualitatively uniform at least among mammals, except for possible variations i n the set of "primary" receptors. It was a r b i t r a r i l y assumed that the odorants used would not be affected by any such differences. Laffort's results were expressed i n "p.ol." (the negative logarithm of molarity) and i n moles/litre of inspired pure a i r , and represented the detection threshold (the subject's opinion.that the sample presented i s something other than pure water/air), which i s considerably below the recognition threshold. A series of tests was carried out i n the v i c i n i t y of this human threshold value. Other experiments were perfor-med at much greater concentrations to allow for impurity of the baseline a i r , adsorption by the tygon apparatus, olfactory adaptation, the fact that the subject i s not actively sniffing so that i t s nasal airflow pattern i s not at i t s most favourable for olfaction, etc. 6 8 The main s e r i e s of experiments was performed u s i n g com-m e r c i a l l y "pure" u n d i l u t e d samples of F i s h e r ' s amyl a c e t a t e (a.a.) or Baker's methyl s a l i c y l a t e ( o i l of wintergreen) (m. s . ) . P r e l i m i n a r y experiments a l s o employed F i s h e r ' s p y r i d i n e and p r o p i o n i c a c i d . Some e a r l i e r n o n - q u a n t i t a t i v e runs used pipe tobacco smoke, ammonia and a sample of peppermint o i l from U n i v e r s i t y Pharmacy which c o n s i s t e d mainly of 5% menthol i n a l c o h o l . Allowance was made f o r d i f f e r e n c e s i n v o l a t i l i t y of the d i f f e r e n t odorants (see R e s u l t s f o r c a l c u l a t i o n s ) . The volume of deadspace i n the apparatus when the bypass tube was i n use, between experiments, was such t h a t 1.2 u,l of a.a. would f i l l i t w i t h s a t u r a t e d vapour. T h i s i s the o r d e r of magnitude of the f i r s t s t i m u l u s of a r u n . 69 V . P R O C E D U R E A f t e r the e l e c t r o n i c s had warmed up f o r an hour, the o s c i l l o s c o p e i n p u t was balanced, a s e r i e s of c a l i b r a t i o n f r e -quencies from the o s c i l l a t o r r e c o r d e d and the o s c i l l a t o r and b a t t e r y c h a r g e r unplugged. The f i r s t c a t was then placed i n the box and the r e c o r d i n g c a b l e plugged i n t o i t s head. The behaviour of the c a t was monitored throughout the experiment and l a t e r scored on a f i v e - p o i n t a l e r t n e s s s c a l e (Table I I ) . The occurrence of movement was u s u a l l y apparent from the p a t t e r n of the r e c o r d , p a r t i c u l a r l y i n the case of washing. V/hen the c a t ' s behaviour and s i g n a l s seemed r e l a t i v e l y . s t a b l e , the i n f u s i o n pump was engaged a t a time and i n a gear r e c o r d e d on the p o l y g r a p h . A f t e r 2 - 1 minute the s t i m u l u s was d i s c o n t i n u e d (sooner i f the response was c o m p l e t e l y b u r i e d i n movement a r t i f a c t ) . Some minutes l a t e r another t r i a l was made a t another gear s e t t i n g , u s u a l l y one p r o d u c i n g a h i g h e r c o n c e n t r a t i o n of odorant, up to a t o t a l o f 3 - 6 t r i a l s , u s u a l l y 4. Where more than one c a t was t e s t e d i n a day, the t e s t s were w e l l spaced to a l l o w f o r d i s s i p a t i o n of adsorbed odour t r a c e s , e t c . , and the o s c i l l a t o r c a l i b r a t i o n was not repeated.. The nature of any extraneous s t i m u l i t h a t might have reached the c a t were noted. The animal was not r e l e a s e d While i t s behaviour appeared to be d i r e c t e d towards escape, except 70 T A B L E I I ' A L E R T N E S S S C A L E . 1 very active 50 - 100% continuous a c t i v i t y such as walking, scratching, washing, playing, attacking the door or cable 2 moderately active less than 50% of the time spent i n not-too-violent a c t i v i t y 3 quietly alert usually s i t t i n g staring at the one-way mirror on the door 4 sleepy lying down or s i t t i n g with eyes half closed 5 asleep lying motionless with eyes shut. 71 i n cases of detachment of the r e c o r d i n g p l u g from the c a t ' s head, or i t s a t t a c k i n g or g e t t i n g t a n g l e d up i n the c a b l e , when immediate a t t e n t i o n was r e q u i r e d . 72 V I . P R E L I M I N A R Y E X P E R I M E N T S EXPERIMENT C Some of the main s e r i e s of experiments were done without the s i d e arm and by-pass path. In a d d i t i o n , p r e l i m i n a r y experiments were done without the t h i n odour tube, the a i r supp l y b e i n g d e l i v e r e d a t 16 l/min by t " I.D. tygon t u b i n g p r o j e c t i n g a few i n c h e s below the h o l e i n the r o o f of the box. Amyl a c e t a t e , p r o p i o n i c a c i d (p.a.) and p y r i d i n e (pyr.) were used i n t h i s s e r i e s . EXPERIMENT C In a subset o f the above s e r i e s r e c o r d s were taken from the b a s a l amygdala of c a t IV i n p l a c e o f one of the two channels of o l f a c t o r y s i g n a l s . The 40 Hz f i l t e r s , . e t c . , were employed aa u s u a l . The o l f a c t o r y s t i m u l a n t was p y r i d i n e . 73 EXPERIMENT B An earlier series was conducted i n another room where the apparatus could not be l a i d out so that the cat could be observed at a l l times; an assistant was seldom available to perform this service. Instead of two channels of 40 Hz, one was recorded, the other channel being led from the c o r t i c a l screws (d i f f e r e n t i a l l y ) via a 5 Hz bandpass f i l t e r of similar Q. Both barrels of the infusion pump were used with different odours, which were presented alternately to each cat. The drive head of the pump had to be positioned manually in contact with plunger of the second syringe, so these runs were not quantitative. Amyl acetate and peppermint were used. Gats II and I I I were studied, cat IV being not yet implanted. EXPERIMENT B' In a subset of this series the oscilloscope output from the c o r t i c a l channel was potted down to 10% (voltage) and led directly onto the EEG channel of the polygraph. The polygraph was sometimes run at 25 mm/sec i n this case. 74 EXPERIMENT A The earliest preliminary experiments were done on cat I using radically different methods and materials (Pig. 8). The cat was held on the experimenter's knee with his head kept close to the funnel out of which the air/odour flowed. Nasal respira-tion was recorded using a thermistor probe which had to be held manually i n the nasal o r i f i c e without touching i t , which would i r r i t a t e the subject. This occupied the f u l l attention of the experimenter, as any movement of the cat must be followed, but with assistants to operate the olfactometer and polygraph experiments could be done. The thermistor signal was taken to one arm of the wheatstone bridge c i r c u i t of a chopper channel on the polygraph, the next arm being f i t t e d with a 2.5 K Jl poten-tiometer to set the le v e l . Signals were led from one bipolar olfactory bulb electrode only, with the indifferent from a c o r t i c a l screw. Amplification was performed by battery operated Grass P15 AC Preamplifiers instead of an oscilloscope. The two P15s were connected i n p a r a l l e l . One, with the cutoff set at 10 - 100 Hz and amplifi-cation at 100, fed to the usual 40 Hz f i l t e r for display of the induced waves on the polygraph. The other, on minimum low and high cutoff and gain 1000, sent i t s output straight to a poly-graph chopper channel to record the DC shif t or "Ottoson waves". Odour stimulation was by ammonia via the syringe or by blowing a puff of Erinmore tobacco smoke at the cat. 75 F I G U R E 8 Preliminary Experiment A P 15 s Vokam n n n Polygraph 40 Hz Filter D.C. Level Induced Waves Ottoson Waves Respiration Hypo. — < — Charcoal — Respirator Needle Filter Pump Infusion Pump Outdoor Air APPARATUS FOR EXPERIMENT A In the experiment shown i n F i g . 9 , the o l f a c t o m e t e r shown a t the f o o t o f t h i s f i g u r e was r e p l a c e d by a b r e a t h of smoke blown a t the c a t . 76 R E S U L T S 77 I . P R E L I M I N A R Y E X P E R I M E N T A . Nasal a i r f l o w was d i s p l a y e d on the polygraph t o g e t h e r w i t h d i f f e r e n t i a l l y a m p l i f i e d s i g n a l s from one o l f a c t o r y b u l b , r e c o r d i n g both the DC component and the 34 - 44 Hz f i l t e r e d i n t e g r a t e d a c t i v i t y . These parameters were found to v a r y syn-c h r o n o u s l y w i t h i n the accurac y o f the 5 mm/sec paper speed. Both components of the o l f a c t o r y s i g n a l rose s h a r p l y to a peak d u r i n g the i n s p i r a t o r y phase and d e c l i n e d to b a s e l i n e by the end o f e x p i r a t i o n , remaining there d u r i n g the b r i e f pause bef o r e the next b r e a t h . A t y p i c a l p o r t i o n o f the r e c o r d i s shown i n P i g . 9. When a p u f f . o f tobacco smoke was blown a t the c a t ' s f a c e , the r e s p i r a t o r y r a t e d i d not change except f o r two s h o r t s n i f f s , but the p a t t e r n of a i r f l o w showed a d i s t i n c t s t e e p e n i n g of the r i s i n g edge i n d i c a t i n g t h a t the flow a c r o s s the o l f a c t o r y mucosa had been improved i n speed and volume. A f t e r h a l f a minute t h i s e f f e c t began to d e c l i n e towards normal. Both p a r a -meters of the o l f a c t o r y s i g n a l were a t l e a s t doubled i n amplitude. - _ The s m e l l of ammonia had much l e s s e f f e c t on the o l f a c t o r y s i g n a l s and appeared to have an i n h i b i t o r y e f f e c t on i n s p i r a -t i o n , which was s h a l l o w e r than e x p i r a t i o n or sometimes d e l a y e d . 78 F I G U R E . 9 C c | t : 1 I sec. 4 0 H z Respiration Blow Smoke RECORD FROM EXPERIMENT A The upper t r a c e was obtained w i t h the f i l t e r c h a r a c t e r i s e d i n F i g . 6. The bottom t r a c e was l e d from a t h e r m i s t o r h e l d i n the r i g h t n a r i s . 79 I I . P R E L I M I N A R Y E X P E R I M E N T S B & B f The b u l b a r e l e c t r i c a l a c t i v i t y f o r s e r i e s B and B' i s d i s -c u s s e d t o g e t h e r here s i n c e the o n l y d i f f e r e n c e was i n the treatment o f the c o r t i c a l s i g n a l s . No s i g n i f i c a n t changes i n the c o r t i c a l s i g n a l were seen, i n e i t h e r s e r i e s , which c o u l d be c o r r e l a t e d w i t h changes i n a l e r t n e s s . In f a c t both the c a t s used i n t h i s s e r i e s remained throughout t h e i r time i n the box a t h i g h l e v e l s of a l e r t n e s s ; o n l y one r e c o r d was obtained a t l e v e l 3 ( a l e r t but not moving, see Table I I ) from c a t I I , wh i l e c a t I I I seldom dropped.-below l e v e l 1 ( a c t i v e > 50% of the t i m e ) . S i n c e the c a t co u l d not be watched d u r i n g the o p e r a t i o n of the odour i n j e c t i o n apparatus used i n t h i s s e r i e s , i t s behaviour had to be deduced from the shape of the t r a c e s on the po l y g r a p h . The two pens were r e c o r d i n g d i f f e r e n t parameters so they would not n o r m a l l y move t o g e t h e r except d u r i n g movement a r t i f a c t s , which c o u l d t h e r e f o r e e a s i l y be r e c o g n i z e d . Washing produced a t y p i c a l o s c i l l a t i o n , p a r t i c u l a r l y on the EEG t r a c e , a t two per second. Cat T I d i d a g r e a t d e a l o f washing and o f t e n played w i t h the c a b l e . Cat I I I spent much of i t s time pawing a t the window or the c o r n e r iwhere the door opened. The r e s u l t i n g e l e c t r i c a l a r t i f a c t s may have been conducted to the c a t ' s b r a i n as w e l l as to the r e c o r d i n g apparatus; the animals became pro-g r e s s i v e l y calmer as the connector p l u g d e s i g n was improved i n s e r i e s G, and c a t IV who was never s u b j e c t e d to the o l d connec-80 t o r was even more r e l a x e d . S i n c e the e l e c t r i c a l a c t i v i t y i n the o l f a c t o r y bulb which was under study had been shown to appear on i n s p i r a t i o n , r e c o r d i n which the l e v e l of t h i s parameter d i d not var y a t the r e s p i r a t o r y r a t e were r e j e c t e d . Responses are d e s c r i b e d as p o s i t i v e i f the are a under the curve of i n t e g r a t e d 40 Hz a c t i v i t y appeared to i n c r e a s e , n e g a t i v e i f i t decreased, zero i f no s i g n i f i c a n t change was seen. When the r e s u l t s were examined they appeared to be q u i t e random. When they were p l o t t e d as a f u n c t i o n of odorant c o n c e n t r a t i o n , a s l i g h t t r e n d was seen, i n or d e r of i n c r e a s i n g c o n c e n t r a t i o n : - p o s i t i v e , random, p o s i t i v e , n e g a t i v e . 81 I I I . P R E L I M I N A R Y E X P E R I M E N T S C & C1 Experiment C:' differs from G i n that one of the channels of 40 Hz a c t i v i t y was recorded from the amygdala, i n which an electrode was implanted in cat IV, i n the hope of using this as an index of arousal (Pagano & Gault,1964).Pyridine was used as the stimulus in this subset. A small reversible change i n the level of amygdaloid a c t i v i t y was seen with alerting produced by odour. The accom-panying change i n olfactory signals was much larger, however, and with the improved arrangement, of the apparatus i t was also possible to monitor the cat's overt behaviour and the raw signals on the oscilloscope at a l l times. Indeed i t was found necessary to do so as the improved connector plug design reduced the movement art i f a c t s to the same order of magnitude as the signals. The amygdaloid recording was therefore held to be redundant and was not resumed after cat IV recovered from i t s cold which had abolished signals in both structures. To make the different t r i a l s comparable for analysis, various adjustments were made. If the stimulus was delivered for one minute{ as i t usually was except i n the case of very high concentrations when there was danger of the evaporation rate being exceeded so that drips accumulated i n the T-piece), the odour i f perfectly mixed would be diluted twenty times by the volume of the box. In computing the results this protocol was treated as the norm and other durations were prorated and 82 p l o t t e d as i f they had been d e l i v e r e d a t another gear s e t t i n g f o r one minute. However, the a c t u a l c o n c e n t r a t i o n a t the c a t ' s nose would depend on i t s whereabouts i n r e l a t i o n to the a i r f l o w p a t t e r n s i n the box, which were not determined. Two p r o p e r t i e s of the chemicals were assumed l i k e l y to a f f e c t t h e i r o l f a c t o r y a c t i v i t y : vapour p r e s s u r e , which might be expected to a f f e c t the r a t e of e v a p o r a t i o n of the l i q u i d from the needle, and the r e l a t i o n s h i p of the c o n c e n t r a t i o n d e l i v e r e d to the o l f a c t o r y t h r e s h o l d . The vapour p r e s s u r e of p y r i d i n e i s f o u r times t h a t o f amyl a c e t a t e and of p r o p i o n i c a c i d , which are s i m i l a r . I f p y r i d i n e i s j u s t a b l e to v a p o r i s e as f a s t as i t i s extruded, then the same q u a n t i t y of amyl a c e t a t e would be expected to take r o u g h l y f o u r times as l o n g , so i t would behave as i f d e l i v e r e d a t two gears slower. In i f a c t the d i f f e r e n c e would not be as g r e a t s i n c e the i n c r e a s e d s u r f a c e a r e a .and reduced s u r f a c e t e n s i o n of the drop of accum-u l a t i n g l i q u i d would f a v o u r f a s t e r e v a p o r a t i o n . Moreover, i f a l l the compounds used were w e l l a b l e to evaporate a t a l l i n f u s i o n r a t e s used there would be no d i f f e r e n c e a t a l l ; how-ever, d r i p s i n the T- p i e c e and r e s i d u a l odour emerging from the a i r l i n e were o c c a s i o n a l l y apparent a f t e r h i g h c o n c e n t r a -t i o n s were a d m i n i s t e r e d . I t was t h e r e f o r e d e c i d e d as a f i r s t a p p r o x i m a t i o n to assume that e v a p o r a t i o n v a r i e d l i n e a r l y w i t h vapour p r e s s u r e , so the p y r i d i n e data were s l i d the a p p r o p r i a t e d i s t a n c e a l o n g the a b s c i s s a . 83 In accordance with the rationale set forth under Methods and Materials, the concentration of the stimulus was then expressed as a multiple of the human olfactory threshold for that substance, to give a measure of "olfactory a c t i v i t y " . For the purposes of this study," the quantity "olfactory a c t i v i t y " has been defined as follows. ( concentration i n moles/litre a i r ) Olfactory (SQT) _ -z lo_ ( ) ""activity ^ ' g10( concentration at human olfactory ).' ( threshold ) equation 1 Since an increase of three gear units produces a tenfold reduction i n rate of extrusion of odorous l i q u i d into the a i r -stream, this w i l l correspond to a decrease of three AQL units. In other words, one AOL unit change i s the negative of one gear unit change for each substance ( i t i s also one third of one unit change i n Laffort's (1963) "p.ol."). The data could therefore be plotted i n terms of gear units, traced and the page of tracing paper reversed; the equivalent gear unit to give threshold concentration was calculated and labelled as zero on the AOL scale. The calculations involved were as follows. infusion rate /^-\ x density , +. (mole) ( m i n ) ( m l ) Concentration ^ ^  ^ box dil u t i o n v a i r ( 1 ) v molecular (gm weight (mol ... equation 2 factor flow (min) weight (mol 84 At gear 10 and 16 l/min airflow, from equation 2:-* i n 10' 3 x 0.876 p . c • = 2.1 x 10"° mole/1 a .a. C 1 0 pyr. 20 x 16 x 130 10" 3 x 0.98 20 x 16 x 79 = 3.9 x 10 8 mole/l , n 10 3 x 0.99 • o G ± v = = 4.2 x 10~° mole/1 p , a * 20 x 16 x 74 At threshold, from Laffort (1963), **C t h = 3.72 x 10" 1 1 mole/1 a .a. G t h = 5.13 x 10~ 1 0mole/l pyr. C t h = 7.08 x 10" 1 0 mole/1 p.a. The olfactory a c t i v i t y of each of the three substances at gear 10 can be found from equation 1, giving to the nearest AOL unit:-*** in 2.1x 10 A 0 L a a = 3 l oS H I = 8 a- * 3.72 x 10 u AOL 1 0 = 6 AOL 1 0 = 5 p.a. * read "concentration i n box a i r of amyl acetate after infusion for one minute at gear 10" ** read "concentration of amyl acetate at human olfactory threshold" *** read "olfactory a c t i v i t y of amyl acetate i n box after delivery at gear 10 for one minute 85 The p r o p i o n i c a c i d and p y r i d i n e data must t h e r e f o r e be s h i f t e d three and two u n i t s to the l e f t r e s p e c t i v e l y to convert from, , c o n c e n t r a t i o n to o l f a c t o r y a c t i v i t y . The net e f f e c t of a l l these s h i f t s i s that p r o p i o n i c a c i d i s s h i f t e d three u n i t s to the l e f t from amyl a c e t a t e , p y r i d i n e one u n i t to the r i g h t ; ; i . e . gear 7 w i t h p r o p i o n i c a c i d produces a stimulus of the same o l f a c t o r y a c t i v i t y as gear 10 w i t h amyl a c e t a t e . The r e s u l t s ( P i g - 10.) are p l o t t e d w i t h zero (no change i n induced wave amplitude) on the centre h o r i z o n t a l l i n e , i n c r e a s e s upwards and decreases downwards, by a d i s t a n c e p r o p o r t i o n a l to the " c r e d i b i l i t y " of the response estimated on a t e n - p o i n t s c a l e by s u b j e c t i v e c r i t e r i a such as the a u t h e n t i c i t y of the s i g n a l s (judged from t h e i r v a r y i n g at the r e s p i r a t o r y r a t e r a t h e r than the r a t e of washing or other movement), the l a t e n c y of the response and the absence of extraneous s t i m u l i (such as n o i s e s or the animal's g e t t i n g tangled up i n the r e c o r d i n g c a b l e ) . Prom the graph i t can be seen that responses from the drowsy cat ( a l . 4) are upwards. Those from the a l e r t cat depend 2 on the s t i m u l u s s t r e n g t h : f o r weak s t i m u l i ( t h r e s h o l d x 10 ) they are down but f o r stronger ( t h r e s h o l d x 10 ) they are up; 7 3 f o r one enormous stimulus ( t h r e s h o l d x 10 ) i t was a g a i n down, but as the subject seemed unduly a g i t a t e d t h i s concentra-t i o n was not repeated. U n f o r t u n a t e l y l i t t l e o v e r l ap occurs between t r i a l s w i t h p r o p i o n i c a c i d and w i t h the other odours; 86 F I G U R E 1 0 A = Propionic Acid • = Pyridine O = Amyl Acetate Numbers = Alertness + i.o -+ 0.8 -+ 0.6 -+ 0.4 -+0.2 o -0.2 -0.4 --0.6 --0.8 --1.0 -A A A i t A' A 3 A 3 A 0 3 A 3 4 4 • • O i ^ 0 3 fl' 0 3 2 O 3 D l 4 2 2 | | O C J J D ' — D O C D O I 3 O i 0 3 8 A O L 10 12 22 RESULTS OF EXPERIMENT G 87 u s a b l e c o n c e n t r a t i o n s were l i m i t e d by the r e l i a b i l i t y of the i n f u s i o n pump (good down to gear 10) and the production' of d r i p s due to the l i q u i d flow exceeding the a b i l i t y of the a i r s t r e a m to v a p o r i s e i t , which was apt to occur a t h i g h i n f u s i o n r a t e s (gear 6 and above). Responses to n o n - o l f a c t o r y s t i m u l i t h a t o c c u r r e d d u r i n g the p e r i o d r e p o r t e d above w i l l be c o n s i d e r e d t o g e t h e r w i t h those of the main experiment. 8 8 I V . T H E M A I N E X P E R I M E N T The 40 Hz a c t i v i t y , when p r e s e n t , i s l o c k e d to r e s p i r a t i o n (as proved by p r e l i m i n a r y experiment A) and the r e s p i r a t o r y r a t e c o u l d be measured e a s i l y from a good r e c o r d ; i t was found to be about 22/min, a normal v a l u e f o r c a t s . In the a i r f l o w -f r e e pause f o l l o w i n g e x p i r a t i o n ( at the " t u r n of the t i d e " , as i t were), 40 Hz a c t i v i t y was c o m p l e t e l y absent, and a b a s e l i n e c o u l d be v i s u a l i s e d through these p o i n t s . A measure of a c t i v i t y c o u l d t h e r e f o r e be obtained by computing the a r e a under the curve w i t h a p l a n i m e t e r over a standar d time p e r i o d , chosen as 20 seconds. The e r r o r i n t r o d u c e d by v a r i a t i o n s i n r e s p i r a t o r y r a t e was found to be n e g l i g i b l e , and t h a t a s s o c i a t e d w i t h the f a c t t h a t the time p e r i o d may not i n c l u d e an exact number of br e a t h s was found to be about + 2%. The moment when the i n f u s i o n pump engaged was marked on the r e c o r d by an arrow ( P i g . 11) and the time p e r i o d s over which a c t i v i t y was measured always bore a s e t r e l a t i o n s h i p to t h i s time (to the n e a r e s t second), which was chosen as f o l l o w s . The c o n t r o l p e r i o d was s e t back to end 6 seconds before the s t i m u l u s to a l l o w f o r f a i l u r e o f the one-way m i r r o r to hide the move-ments of the experimenter. On one o c c a s i o n when s t r o n g s u n l i g h t overcame attempts to darken the l a b o r a t o r y and the b a t t e r y used to l i g h t the c'atbox was p a r t i c u l a r l y weak, c a t I I was c l e a r l y a b l e to see when the experimenter reached over to operate the i n f u s i o n pump. A l l the responses on t h i s run o c c u r r e d 5 seconds 89 F I G U R E 1 1 A. Cat: 3 , ROB "SIZE" - 102 700 MV "ABS " - 142 Cat: 3 , LOB "SIZE" - 88 Signals 300 / J V 1 A B S " - 67 SAMPLE RECORDS A. I n c r e a s e ( a l e r t i n g response) B. D e p r e s s i o n ( o l f a c t o r y response) The areas measured by p l a n i m e t e r are shaded. The l i n e marks the p e r i o d of odour p r e s e n t a t i o n , b e g i n n i n g 10 seconds a f t e r the pump was switched on. 90 before the pump was engaged. The other cats under similar conditions demonstrated no such a b i l i t y . The responses to such non-blfactory stimuli w i l l be discussed lat e r . The time period for measurement of the response was started 12 seconds after the pump was engaged. The time taken for a i r to travel down the 3 feet of i""tubing beyond the odour injec-tion point (see Pig 7) was calculated from the airflow rate and the cross-sectional area (assuming a uniform velocity profile as a f i r s t approximation) to be 5 seconds and that . for the 10 feet of "^8" tubing to be also 5 seconds, giving a total transit time of 10 seconds. At some point i n the next 0 - 2 seconds inspiration w i l l occur and the response should be expected. A 12 second latency i s therefore appropriate; however this choice was made before the above calculations were done, simply from observation of the most common time (to the nearest 0.05 minute) at which an obvious response was noted. This was taken as confirmation that a genuine olfactory response had been obtained. Another advantage of the 12 second delay was that i t allowed time for the "tok" response, i f any, to subside completely. The measurement periods are shown i n Fig. I I . The calculation of stimulus concentration i s s l i g h t l y different from that already described for experiment G; the catbox dilution factor i s no longer applicable since the odourr-laden a i r i s directed straight past the cat's nose, eliminating the. uncertainty due to a i r currents and mixing i n the box. The airflow was also reduced from 16 to 4 l/min. There was a disadvantage to the new arrangement: the draught sometimes caused the cats' eyes to water and a n o s t r i l to be-come congested. Signals were then completely absent on the side of the obstruction. They were sometimes seen to disappear gradually and then suddenly return to f u l l amplitude when the cat shook i t s head (Fig. 12; compare Fig. 13 which shows a b i l a t e r a l disappearance of signals indicating an abrupt arrest of respiration). Only two odorants were used i n this series, amyl acetate and methyl salicylate ( o i l of wintergreen). The concentrations at gear 10 ( l u,l/min) (from a modified equation 2) were:-G 1 0 = 1.68 x 10"6 mole/l a .a. i n 10 3 x 1.18 , c = = 1.73 x 10"D mole/l m , s * 4 x 152 The concentration at threshold, from Laffort (1963), was:-G t h = 3.72 x 10" 1 1 mole/l, C m o t h + 3.16 x 10" 1 0 mole/l The olfactory a c t i v i t y at gear 10 was therefore, from equation AOL 1 0 = 14, AOL 1 0 = H i = 11 to the nearest AOL unit, £L # 3, • III • S • so methyl salicylate data must be shifted 3 units to the l e f t . The vapour pressure of methyl salicylate i s f i f t y times lower than that of amyl acetate, so the results for methyl salicylate must be shifted another 5 units to the l e f t , making a total of 8. 92 F I G U R E 1 2 NASAL OBSTRUCTION The r e c o r d shows r e a p p e a r a n c e o f b u r s t s o f 40 Hz a c t i v i t y a t d i f f e r e n t t i m e s i n the two b u l b s , i n d i c a t i n g c l e a r i n g o f n a s a l passages b l o c k e d by mucus when the a n i m a l made a sudden movement. 93 P.I G U R E 1 3 Cat: 3 10 sec. ROB t Pyridine (approx. saturated = "AOL 19") J NOXIOUS VAPOUR REPLEX B i l a t e r a l i n h i b i t i o n of induced wave a c t i v i t y indicates arrest of r e s p i r a t i o n i n response to a strong unpleasant odour. 94 The s a t u r a t e d vapour of amyl a c e t a t e was a l s o i n j e c t e d . I t s ; : c o n c e n t r a t i o n was found from the i d e a l gas law. s a t u r a t e d vapour p r e s s u r e a t room / -i \ temperature / \ 273 „ 1 c s v mple = P mm x ( o R ) x ( U t r e s ) v x ' 760 ^ n s ; room 22.4 temp. 5 273 1 ' C S V = x x = 2.7 x 10" 4 m o l e / l (so AOL s v •=• 21) a , a " 760 295 22.4 a , a ' —6 (A s i m i l a r c a l c u l a t i o n f o r methyl s a l i c y l a t e y i e l d s 5.6 x 10 m o l e / l f o r s a t u r a t e d vapour, o n l y three times the C ; f o r -t u n a t e l y the c o n c e n t r a t i o n s a c h i e v e d by i n j e c t i n g l i q u i d methyl s a l i c y l a t e were not l i m i t e d a t gear "8^" s i n c e the data were a l r e a d y s h i f t e d down because v a p o r i s a t i o n c o u l d not keep pace w i t h i n j e c t i o n ) . F o r i n j e c t i o n of amyl a c e t a t e vapour from a 1 ml s y r i n g e : -IO" 6 G 1 0 = 2.7 x 10 4 x = 6.7 x I O - 1 1 a.a.sv ^ t h e r e f o r e AOL = 1. a. a. sv A 25 M-l s y r i n g e was a l s o used w i t h methyl s a l i c y l a t e . By com-p a r i s o n of the c a l i b r a t i o n s on i t s b a r r e l i t was found to i n j e c t a volume f o r t y times s m a l l e r than the 1 ml s y r i n g e , so these data were p l o t t e d another 5 u n i t s to the l e f t . As a matter of i n t e r e s t , the c o n c e n t r a t i o n s of the pure l i q u i d s were c a l c u l a t e d to be:-C a . a . l i q * = 6 ' 7 4 m o l e / 1 = 54 AOL, V ^ 1 1 ^ = 7.76 m o l e / l . 95 The r e s u l t s of a l l t r i a l s where the planimeter could be used were plotted together i n a s i m i l a r fashion to those of ex-periment C, except that there was no need to take account of the duration of s t i m u l i . The planimeter was not used i f there were no f l u c t u a t i o n s at the respiratory rate or i f movement a r t i f a c t s had c l e a r l y d i s t o r t e d the sign a l over an appreciable portion of the period. In cases where the signals from both bulbs, both before and a f t e r stimulus, could be measured, credence was given to the bulb which apparently showed an ol f a c t o r y response as opposed to a n e g l i g i b l e or mis-timed one, or to that which had the largest signals as noted from observa-t i o n of the oscilloscope or deduced from the gain settings of the polygraph. The " i n f e r i o r " bulb was l i k e l y to be associated with a case of the progressive nasal obstruction which appeared to occur as a r e s u l t of the draught over the subject's face (Pig. 12.'0 • The planimeter reading f o r the standard period following the stimulus was divided by the con t r o l value and expressed as a percentage. Augmentations (Pig. 11A) are thus, above 100% and depressions (Pig. 11B) below. The r e s u l t s are plotted i n Pig. 14, The human threshold l e v e l appears as zero on the ordinate ( t h i s applies to both odorants since t h e i r concentrations have been adjusted to bear a constant ..relationship to i t i n converting to " o l f a c t o r y a c t i v i t y " ) . Small arrows are drawn to show the d i r e c t i o n s of trends appearing f o r st i m u l i of the same o l f a c t o r y a c t i v i t y i n 96 preliminary experiment G . Unfortunately there i s a dearth of data over the range of positive response of series C. Another odorant or another syringe size would be required to f i l l the gap, but time did not permit t h i s . The data were punched on IBM cards for correlation and multiple regression analysis. The variables are described i n table I I I . In addition, orthogonal polynomials were generated i n AOL up to the 4th power so the "non-linear" regressions could be computed. Orthogonal polynomials were also sometimes genera-ted i n several other variables. The program eliminated from the regression equation any variable whose inclusion did not reduce significantly (at the probability level p < 0.05) the sum of squares of deviations of actual points from those predicted by the equation. In cases where only one variable or powers there-of was l e f t i n the equation, a two-dimensional plot could be obtained. In the correlation matrix (e.g. Appendix I for a l l data) a variable name followed by a colon, or two stars, and a numeral indicates the variable raised to that power. The prog-ram required that a l l variables i n which orthogonal polynomials were generated be raised to the same maximum.power. Fi r s t of a l l i t was important to see i f the size of the control signals was stable. The regression equation was SIZE = 118.9823 - 31-0031 AL + 19.5047 GAT The negative coefficient of AL i s highly significant (p< .0001). 97 T A B L E I I I V A R I A B L E S F O R R E G R E S S I O N DESCRIPTION VALUES % change i n 40 Hz a c t i v i t y 100 = no change Planimeter reading for control (adjusted for polygraph gain) Notes n^*1 stimulus of one run 100 - 300 uV 1 - 6 Olfactory a c t i v i t y of stimulus Alertness of cat, see Table II Month day, e.g. 1103 = Nov. 3rd Cat number 0- 19 1 - 5 1021 - 1115 2- 4 1 = 4 = a.a., 2 = m.s., 3 = 25 u.1 1 - 5 syringe of m.s., 5= a.a. vapour 1= a.a., 2 = m.s. 1 or 2 1 = f i r s t presentation of new syringe to this cat (ORDER = 1) Adjusted planimeter reading after stimulus 1 or 0 SIZE x D40 / 100 F I G U R E 1 4 98 120 -8 0 4 0 • • • • • • - i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i i6o ^ 5 s 120 ° 8 0 4 0 B. o «o • o J— i—i—i—i—i—i—i—i—i— i—i—j—i—i— i—i—i— i—i—i—i—i 160 -120 -8 0 -4 0 0 o o o G % o ° Amyl Acetate • Methyl Salicylate -i—i—i—i—r-4 - i — i—i—i— i — i—i—i—i—i— i—i— i — i — i — i 8 12 AOL 16 20 22 RESULTS A. From c a t s at a l e r t n e s s l e v e l s 1 and 2. The arrows below i n d i c a t e the d i r e c t i o n of changes (from 100%) seen i n experiment C. B. AL = 3. G . AL = 4 or 5• 99 The positive coefficient of CAT reflects the fact that larger signals were obtained from cat I I I than from cat I I . Those of cat IV were smaller but this i s compensated i n the equation by i t s habitually higher alertness index. The date, however, i s not significant. The negative correlation v/ith date in the correlation matrix (Appendix I) merely reflects the fact that during October cat IV, which had small signals, was recovering from a cold so a lesser proportion of the results were obtained from this cat than v/as the case i n November (the fact that "i. . there are 31 days i n a month instead of 100 gives this d i f f e r -ence undue weight). Next, regression equations for the response to odour were sought, both for the percentage change, D40, and for the absolute value of the signals during presentation of odour, ABS. A l l other variables were included as "potential indepen-dent variables", including orthogonal polynomials of AOL up to the fourth power. After a l l insignificant variables were eliminated, the equations were as follows. D40 = 110.9717 - 0.16007 SIZE (Pig. 15) ABS = 28.6125 + 0.75 SIZE - 22.606 ODOR + 5-5381 AOL -0.32137 AOL2 I t should be borne i n mind that SIZE i s a linear combination of AL and CAT. Thus an alert animal i s more l i k e l y to show a decrease i n signal amplitude, a relaxed one an augmentation. 100 F I G U R E 1 5 150 o o o o O 1001 Q 50 10.0 60.0 110.0 160.0 210.0 260.0 SIZE COMPUTER PLOT OF D40 AGAINST SIZE Double c i r c l e s indicate data points on the regression l i n e . 101 The positive dependence of ABS on SIZE probably reflects mainly the characteristics of the individual electrodes such as resistance, local necrosis and exact location i n the bulb. The data were not divided into l e f t and right bulb, but at least i n cats II and IV most of the data were obtained from one electrode. I t i s interesting that less a c t i v i t y i s seen i n the presence of wintergreen than with amyl acetate (p<.0l). When the results of preliminary experiment C were divided into different odours an even clearer difference was seen: propionic acid better than 50% of responses negative, amyl acetate nearly 50% "zero" (otherwise very slight trend to positive), pyridine 70% .positive, no negatives at a l l (Fig,'lO) . 2 The dependence of ABS on AOL i s only just significant and disappears i f the other variables are not already included i n the equation. Taking powers of AL, up to 3, and of CAT, up to 2, each alone, the following equations were obtained.. D40 = 74.2839 + 7.6147 AL SIZE = 151.8008 - 21.356 AL ABS = 121.5786 - 13.387 AL and D40 not significant SIZE = -449.2922 + 378.13 CAT - 62.081 CAT2 ABS = -412.6116 + 347.65 CAT - 57.076 CAT2 102 I t i s important to note that while absolute values of signal size are highly variable from cat to cat, the percentage response to odorants i s independent of the individual. The dependence of signal size on alertness i s as expected. The difference between the equations for SIZE and ABS partly reflects the reduction in the alertness index which occurred i n the cases when the animal was aroused by the olfactory stimulus but was not recorded i n the data, AL being always the control alertness l e v e l . Multiple regressions were then performed on two separate groups taken from the data, the active, composed of those with alertness index 1 or 2, and the drowsy, with AL = 4, against a l l variables,including the 4th power of AOL. The correlation matrices are shown i n Appendices II and I I I . The equations were as follows. ( D40 = 101.7941 - 20.564 AOL + 3-4786 AOL2 - 0.14386 ( 3 ( AOL (Pig. 16) Active ( ( ABS = 52.1595 + 0.75987 SIZE - 30.021 AOL + 4.6659 ( AOL2 - 0.19296 AOL3 ( D40 - not significant Drowsy ( ( ABS = 14.6080 + 0.82716 SIZE Segregation by alertness has thus removed a l l SIZE dependence from D40. ABS naturally continues to depend on SIZE. 103 COMPUTER PLOT OF DATA FROM ACTIVE CATS 104 The big difference here, however, i s in the contribution.of AOL. In a drowsy cat the strength of olfactory stimulus had no effect on either the change i n bulbar induced waves or the even-tual amplitude. In active animals, on the contrary, both meas-ures show a cubic relationship with AOL. The equation for D40 . could conveniently be plotted by the computer (Pig. 16) because, i t was not complicated by the third dimension SIZE (the three-dimensional predicted surface would be projected onto the AOL/ ABS plane to give a meaningless duplication of "predicted points"). The equation intersects the "zero l i n e " ( D 4 0 = 1 0 0 ) at approximately AOL = 0 , 12, 1 3 2 . The finding of a zero-cross-ing at AOL = 0 i s most interesting, since i t implies that the feline olfactory threshold for these compounds i s identical to the human. However, there i s no reason to suppose that the equation i s followed outside the range covered by data points, even i f i t i s valid within. It would be more reasonable to ex-pect the response curve to level off towards 100% on approaching threshold, as the chances of odour detection diminish; this suggests that the feline threshold might be a factor of 4 - 100 lower than the human. Bearing in mind the deficiencies of the olfactometer used i n this study as compared to the types commonly employed in human panel studies and the fact that the cats were not motivated to s n i f f and detect odours, the real threshold i s probably several orders of magnitude lower s t i l l . 105 It i s interesting to note the relationship of this "olfactory a c t i v i t y " scale to the saturated vapour of the odorants at room temperature, since some workers have describe the concentration of their odours i n submultiples of that of the saturated vapour. For example, Tucker (1963) located the threshold for the trigeminal-to-autonomic reflex at a concen-tration of amyl acetate equivalent to AOL 17. The vapour of wintergreen, on the other hand, becomes saturated at AOL = 13. A preliminary linear regression analysis on the O l i v e t t i Programma 101 desk computer showed that the response of cat IV at AL = 3 was very similar to that of cats II and I I I at AL = 2. The subsequent inclusion of these data with those of the aroused cats hardly altered the results of multiple regression Gats II and I I I at AL = 3, on the other hand, reacted quite differently from any other subset: their linear regression of D40 on AOL was negative. There was, however, a detectable difference between their apparent behavioral state and that of cat IV when both were cl a s s i f i e d as A l ^ 3; cat IV appeared genuinely alert while the others could best be described as mesmerized, 'i'heir results were therefore not used in further analysis since the implications of this unusual psychological state were not clear. For the sake of symmetry the data from cat IV at AL = 3 were also excluded from the "active" category although i t was suspected that they genuinely ( i . e . by EEG c r i teria) belonged there and that this cat simply had an undemon-strative personality. 106 It remains to be considered just how important i s the olfactory environment, as compared to other factors, i n regulating the level of 40 Hz a c t i v i t y i n the olfactory bulb. Some of the changes found by planimetry were not obvious to the eye. However, changes that were obvious often occurred several times i n a minute while olfactory stimuli were only presented once i n several minutes (avoiding i f possible such times of frequent spontaneous changes) and produced no significant (let alone obvious) change much of the time. The majority of changes occurred spontaneously for no apparent reason, or were accompanied by alterations i n the a c t i v i t y of the cat. For example, lying down produced a marked increase i n 40 Hz signals lasting about 5 sec (Fig. 17A), probably attributable to changes i n respiratory pattern. Noises such as the "tok" of the infusion pump (Fig. 17B), a knock on the box (Fig. 17G) or a jangling of keys also caused a transitory increase. Likewise a change in the airflow over the cat's nose such as was pro-duced by opening or closing the side arm of the olfactometer often resulted in an increase (Fig. 17D). 107 F I G U R E 1 7 A . Cat: 4 ROB 10 sec. i 1 Cat lies down Cat: 4 ROB Knock on Door oT Box 3. Cat: 4 ROB t Noise made by switching off Pump Cot: 4 ROB Open "Side Arm" ( Air Flow past Nose greatly reduced ) • NON-OLFACTORY RESPONSES A B, C D Respiration enhanced during movement. Arousal by auditory stimulus. Arousal by cutaneous stimulus. 108 V . RAW S I G N A L F R E Q U E N C I E S Since the f i l t e r in the> recording system passes a band of frequencies, i t was desirable to obtain from the raw amplified signal a more accurate sample of the frequencies actually exhibited by the individual cats. Raw signals from cat I were analysed by the 8 channel computer program (Moore, 1968) as i n Fig. 18A, B, with signals led from both poles of the electrodes i n monopolar recording mode. A considerable degree of independence both i n occurrence and frequency of bursts was seen not only between the two bulbs but also between the inner and outer of the concentric' electrode i n the one bulb. When bursts were seen i n the pyriform cortex they were always present at the same time some-where i n the i p s i l a t e r a l bulb. Bursts i n the bulbs occurred approximately every second, that i s twice i n the respiratory cycle; alternate interburst intervals tended to show a more complete disappearance which by analogy with the polygraph records from this cat probably represenfed.ithe pre-inspiratory phase. The frequency of bursts i n the bulbs of cat I varied from 40.5 - 45 Hz and occasionally higher; a typical value was 43 Hz. With only two channels of data from cats II - IV, the oscilloscope trace could be photographed at a high enough gain for rough measurement by hand without additional enlargement. 109 F I G U R E 1 8 A. r-. A V 5 0 msec. 3 vv v v v v J Olfactory bulb AUTOCORRELATION OF RAW SIGNALS A . B . 8 channel r e c o r d i n g i n b l o c k s of 250 msec (experiment A) A u t o c o r r e l a t i o n of top channel o f A. 110 Records were interspersed with calibrate frequencies from the os c i l l a t o r so that uneven stretching of the film could be allowed for. Cat IV had no signals during the two weeks when i t was sought, to photograph them; he was probably suffering from a cold again. Cat II's bursts were mainly 36 - 39 Hz, occasionally 44 Hz (Pig. T9A) . Between bursts there were a lot of quite regular 56 - 60 Hz and 115 - 120 Hz waves. These were inter-preted as hum and re c t i f i e d hum respectively. Cat III (Pig. 19B) had frequent bursts of 36 Hz, some of 44 Hz, and when washing picked up hum of 58.5 and 128 Hz. Bursts were sometimes of constant frequency, sometimes gradually increasing, particularly at the beginning. F I G U R E 1 9 A. Cat: 2 L O B ROB 3 8 HZ 116 HZ B. Cat: 3 L O B ROB 37.4 Hz 5 7 Hz 2 0 0 / J V 4 0 Hz 0.5 sec. RAW SIGNALS 112 D I S C U S S I O N 113 There i s some disagreement i n the. literature about the exact relationship of the burst a c t i v i t y known as olfactory induced waves to the phases of respiration. This i s due in part to species differences, as i s clear from Ueki & Domino (1961). They studied both the monkey and the dog by similar methods and found that the bursts were synchronised with i n -spiration i n both but also occurred during expiration only in the dog. In the cat Hernandez-Pe6n et a l . (i960) found no significant correlation with any particular phase of respira-tion whereas Mechelse & Lieuwens (1969) found that burst a c t i v i t y commenced during inspiration and declined during ex-piration, frequently with a short period of silence between the two. It was therefore important to check the relationship of the bursts to respiration as recorded with the apparatus described above. The results of preliminary experiment A exactly confirm those of Mechelse & Lieuwens. The disappearance of the a c t i v i t y when the animal was suffering from a cold which blocked access of a i r to the olfactory mucosa, noted generally in the literature, was also observed i n cat IV during experiment G. However, the loss of induced waves persisted long after the gross manifestations of laboured breathing associated with the i l l n e s s had cleared, up. This i s consistent with the careful observations of Gesteland et a l . (1965) in the frog. They showed by microscopic obser-vation of the olfactory epithelium that the mucosal response 114 (upon which bulbar a c t i v i t y depends) i s drastically inhibited by any change i n mucus secretion, either thickening or water-ing. This explanation also accounts for the observation that the draught of a i r blowing across the cat's face i n the main experiment occasionally produced a watering of the eye and a reversible unilateral block of burst a c t i v i t y (Fig. 12). The clearing of this block was usually accompanied by a shaking of the head. Since scrupulously odourless a i r was never used i n these experiments, they shed no light on the controversy as to whether there are receptors producing bursts in response to mechanical stimulation alone. Bursts were present i n a l l cases except those suspected of nasal or mucosal obstruction and i n extreme drowsiness. This was satisfactory since the main intent was the study of changes in the level of pre-existing burst a c t i v i t y . Experiment A also provided confirmation of the association of the induced waves with the DC s h i f t or Ottoson wave (e.g. Ottoson 1959a, b). The waveform was not identical, the Ottoson waves tending to have a f l a t t e r peak than the induced wave envelope, but both showed a similar response to a l l stimuli presented, olfactory, auditory, t a c t i l e and emotional, and a similar change during movement, a l l of which appeared to depend upon changes i n respiration, as predicted by Gault & Leaton (1963). 115 The association of these three parameters, inspiration, Ottoson waves and induced waves, being thus established, the induced waves were chosen for further -study. The direction of response of the induced waves having been found to vary with alertness by McLennan & Graystone (1965), i t was clearly necessary to have some measure of the arousal state of the animal. Pagano & Gault (1964) showed that fast a c t i v i t y i n the amygdala could be correlated with c o r t i c a l and behavioural arousal with a high degree of r e l i a b i l i t y . V/hile Gault & Leaton (1963) have shown that amygdaloid fast a c t i v i t y i s only seen i n the presence of similar bursts i n the i p s i -l a t e r a l olfactory bulb, the l a t t e r do not provide an index of arousal as suggested by Lavin et a l . (1959); Pagano (1966) was able to stimulate a c t i v i t y i n the bulb without producing c o r t i c a l arousal. The reduction i n bulbar a c t i v i t y found by several workers on olfactory stimulation was also not normally accompanied by diminished alertness, except when an anaesthetic gas was used as the stimulus. Experiments B and C were therefore concerned with estab-lishin g which of these three parameters would provide the best alertness index. The co r t i c a l measure tested i n experiment B was abandoned since i t i s re l a t i v e l y insensitive at the "highly aroused" end of the spectrum (Pagano & Gault, 1964) and the animals in use at the time varied mainly over this range. In 116 experiment C the apparatus was rearranged to a l l o w f o r con-t i n u o u s o b s e r v a t i o n of the s u b j e c t , and a c a t was implanted w i t h e l e c t r o d e s i n the b a s o l a t e r a l amygdala. A d e f i n i t e change i n amygdaloid a c t i v i t y w i t h b e h a v i o u r a l a r o u s a l was seen, p r o v i d i n g s u g g e s t i v e c o n f i r m a t i o n of the work of Pagano & G a u l t . However, i t was not a ve r y marked change, and nor was i t p a r t i c u l a r l y r e l i a b l e , p robably on account of the d i f f e r e n c e i n measuring t e c h n i q u e s . A b e h a v i o u r a l estimate of a l e r t n e s s was t h e r e f o r e s e l e c t e d , and a f i v e - p o i n t s c a l e comparable to t h a t o f Pagano & Ga u l t was e s t a b l i s h e d (Table I I ) . U n f o r t u n a t e l y i t was not p o s s i b l e to o b t a i n d a t a a t a l l a l e r t n e s s l e v e l s from each c a t . Cats I I and I I I never r e a l l y r e l a x e d even when an exp e r i m e n t a l run l a s t e d three q u a r t e r s of an hour. Cat IV c o u l d seldom be a l e r t e d f o r l o n g enough to o b t a i n a complete t r i a l ; i t s resumption of drowsiness would g i v e a s p u r i o u s d e p r e s s i o n . However, the response to odour as measured by percentage change, D40, was not s i g n i f i -c a n t l y c o r r e l a t e d w i t h powers of CAT even when oth e r v a r i a b l e s which might have masked the r e l a t i o n s h i p were excluded from the r e g r e s s i o n . The a b s o l u t e s i z e o f s i g n a l s n a t u r a l l y was s i g n i f i c a n t l y d i f f e r e n t between c a t s , but the r e g r e s s i o n e q u a t i o n was second order, i n d i c a t i n g t h a t i t was not c a t IV but the a p p a r e n t l y s i m i l a r c a t I I I which was out of l i n e 117 with cat I I . Pour possible explanations may be offered for the small size of signals from cat IV. Electrode location and electrode resistance are probably the two most important. Thirdly, the distance between the two poles of each pair may play a part; i t was only of the order of the thickness of the teflon coat on the inner wire of the concentric electrode on the l e f t where the signals were minute; on the right, where the two leads were independent, the separa-tion was considerably greater. The fourth possible factor i s the thermoelectric effect. The electrodes i n contact with the cat's brain were a l l stainless steel (the s i l v e r plated c o r t i c a l screws were supposedly extradural) as recommended by Fisher et a l . (1961) to avoid the local necrosis produced by copper or s i l v e r . The lack of a sig-nificant change i n signal size with time suggests that this attempt to minimise necrosis around the electrode t i p was successful. Gat IV had steel tubes with copper connecting wires for both poles of the electrode on one side but only the outer on the side with small signals. This.is the reverse of what would be expected i f a thermoelectric current were stimulating e l e c t r i c a l a c t i v i t y . However, calculations showed that the current, even when the animal was f i r s t plugged in to a cold connector, would be only 118 o f the o r d e r o f picoarnperes and t h e r e f o r e u n l i k e l y even to aggravate n e c r o s i s . The o l f a c t o r y response d a t a from experiment G were v e r y encouraging;, drowsy c a t s showed enhancement, a l e r t c a t s showed the p r e d i c t e d down-up-down v a r i a t i o n w i t h s t i m u l u s i n t e n s i t y (the v i c i n i t y of t h r e s h o l d was not r e a c h e d ) . However n e i t h e r v a r i a b l e was a c c u r a t e l y Measured and t h e r e was l i t t l e o v e r l a p between odorants on the c o n c e n t r a t i o n s c a l e , no one odour c o v e r i n g the f u l l range. ( P i g . 10). The main experiment produced q u a n t i t a t i v e measures of both s t i m u l u s and response as w e l l as a c o n s i d e r a b l y g r e a t e r number of t r i a l s . To e l i m i n a t e c o n f u s i o n , d a t a f o r the middle c a t e g o r y of a l e r t n e s s (A.L\= 3) were omitted from the d i f f e r e n t i a l a n a l y s i s o f a l e r t and drowsy c a t s ; i t was not c l e a r to which subset they should belong, i f e i t h e r . A t e n t a t i v e i n t e r p r e t a t i o n of these d a t a has been d i s c u s s e d under R e s u l t s . I n view of the f r e q u e n t f l u c t u a t i o n s i n the s i g n a l i t was q u e s t i o n e d whether the " c o n t r o l " used was a p p r o p r i a t e . If, the d e f i c i e n c i e s i n the apparatus which d i c t a t e d , the gap between the p o r t i o n o f r e c o r d used as c o n t r o l and the a r r i v a l of the s t i m u l u s c o u l d have been removed, there would have been l e s s time f o r spontaneous changes to i n t e r v e n e and g i v e s p u r i o u s o l f a c t o r y r e s p o n s e s . Furthermore i t was important to know whether i t was a p p r o p r i a t e to compare the response to a c o n t r o l p e r i o d or whether each c a t had a s t e r e o t y p e d a c t i v i t y l e v e l i n 119 a specific olfactory environment. The absolute size of the response was computed from the data already compiled and named ABS. A l l regression equations were computed for both forms of independent variable, ABS and the percentage change over control, D40. ABS varied principally with i n i t i a l size whenever i t was included i n the regression. Size i n turn i s dependent on alertness and the individual cat; when tested against powers of AL or of GAT, both SIZE and ABS were found to vary linearly with AL, parabolically with CAT (owing to the order i n which the animals were numbered). The percentage change, on the other hand was independent of the individual cat and depended only on alertness. This shows that the l a t t e r was indeed a , suitable measure of olfactory response. The inclusion of ODOR i n the equation for ABS (and pre-sumably for i t s correlate SIZE) i s interesting; the negative coefficient shows that, at a l l times during recent application of wintergreen (or during continued desorption of trace amounts from the air l i n e s ) induced waves i n the bulb were lower than ' with amyl acetate. Now the l a t t e r i s a pleasant odour at reasonable concentration, whereas wintergreen i s l i s t e d by Allen (1929) as one of the i r r i t a n t vapours producing the noxious vapour reflex discussed above. This confirms that i t was appropriate to consider this reflex i n formulating a hypo-thesis of olfactory induced wave response. 120 ABS also varied parabolically with AOL provided i t had already been corrected for the variation due to the above variables; when run against powers of AOL alone, regression was not even significant at p < 0.1. It was concluded that i f ABS were'physiologically important, this would only be brought out after segregation of the data by alertness. The separate analysis of data from drowsy and active cats showed spectacular differences. In the case of the drowsy cat, ABS s t i l l depended on SIZE i n spite of the fact that GAT and AL no longer varied; this implies a real size dependence. There was no significant dominance of any particular type of reaction to odour; arousal, olfactory response and lack of " response a l l seemed to be randomly distributed over the whole concentration range (Pig., 140). Active cats, on the other hand, react quite differently to various odour intensities. Regardless of whether the inde-pendent variable i s D40 or ABS adjusted for SIZE, the induced waves vary very significantly with the cube of AOL (p < 0.02 for D40, p < 0.0002 for ABS). In this subset AL and CAT both vary so SIZE i s a mixed parameter. The curve plotted for D40 cuts the zero-change axis at AOL = 0, the lowest stimulus used. I f i t i s assumed that this represents the transition point from a concentration so close to threshold that response i s random to one where the odour e l i c i t s a reproducible olfac-tory response, then the entire hypothesis can be supported. 121 I t cannot be s a i d to be proved, s i n c e r e g r e s s i o n should never be e x t r a p o l a t e d from c o r r e l a t i o n to c a u s a t i o n ; i t i s always c o n c e i v a b l e t h a t o t h e r f a c t o r s to which the v a r i a b l e s are a l s o r e l a t e d r e p r e s e n t the tr u e cause, o r t h a t the r e l a t i o n s h i p d i d i n f a c t occur by chance, u n l i k e l y though t h a t may seem. I n view of the g r e a t spontaneous v a r i a t i o n i n the s i g n a l and the u s u a l l y c o n s i d e r a b l e responses seen on s t i m u l a t i o n of ot h e r senses, i t i s amazing t h a t the i n t e n s i t y of o l f a c t o r y s t i m u l a t i o n accounts f o r as much as 34% of the v a r i a b i l i t y i n response. The data can t h e r e f o r e be taken as c o n f i r m i n g the t h e o r y . I t i s hoped t h a t the s t a t i s t i c a l techniques employed s u c c e s s f u l l y averaged out not o n l y the spontaneous v a r i a t i o n s i n the s i g n a l s , but a l s o c e r t a i n s y s t e m a t i c d e f e c t s i n the experiments and t h e i r a n a l y s i s . The a n a l y s i s o f the polygraph r e c o r d s shows a few minor f l a w s such as the e a r l y time p e r i o d chosen as the c o n t r o l and the o c c a s i o n a l doubt as to the a p p r o p r i a t e c h o i c e of a b a s e l i n e f o r p l a n i m e t r y . The r e c o r d i n g system has a few shortcomings such as the r a t h e r wide bandpass of the f i l t e r , the f a i l u r e to make permanent r e c o r d s o f the raw s i g n a l , and above a l l the l a c k o f c o r t i c a l EEG, which might a t l e a s t have been u s e f u l f o r c a t IV. The o l f a c t o m e t e r , however, l e a v e s much to be d e s i r e d . I t would be n i c e to be a b l e to monitor the a c t u a l composi-t i o n of the gas r e a c h i n g the.animal, but i n the range of 122 t h r e s h o l d c o n c e n t r a t i o n s t h i s i s uneconomic i f not i m p o s s i b l e . The l i t e r a t u r e has y i e l d e d no r e p o r t s whatever of such a measurement; i t i s common p r a c t i s e to deduce what must be com-i n g out of the o l f a c t o m e t e r from what went.in. In t h i s case there i s room f o r doubt as to the p r e c i s e t i m i n g and r a t e of d e l i v e r y of the s t i m u l u s . The v e l o c i t y of movement of the d r i v i n g p l u n g e r o f the i n f u s i o n pump was o f t e n as low as 50 urn/min. At such speeds t h i s machine i s h i g h l y r e l i a b l e over l o n g periods, but under the c o n d i t i o n s of the ex-periment i t would be nece s s a r y to e s t a b l i s h s t a b l e v e l o c i t y i n t o an exhaust l i n e b efore s w i t c h i n g the o d o r i s e d a i r to the c a t ' s tube, to take up the s t r e s s e s i n the system ( p a r t i c u l a r l y when u s i n g deformable p l a s t i c s y r i n g e s ) . T h i s would have the advantage e i t h e r of e s t a b l i s h i n g a steady s t a t e e v a p o r a t i o n r a t e o r of e l i m i n a t i n g the use o f the p a r t i c u l a r gear s e t t i n g f o r t h a t l i q u i d on account of d r i p a ccumulation. However, i t would d e s t r o y the p o s s i b i l i t y of a pure o l f a c t o r y s t i m u l u s , s i n c e pressure waves would be bound to occur d u r i n g s w i t c h i n g (see F i g . 17D); the r e d u c t i o n of these waves by adding a fl o w r e s i s t a n c e to the system ( p l a s t i c sponge) would aggravate the problem due to h i g h p r e s s u r e i n the system. The h i g h p r e s s u r e i n the T-piece due to the l e n g t h of downstream t u b i n g r e s u l t e d i n leakage o f odorant past the plu n g e r i n the s y r i n g e , p a r t i c u l a r l y w i t h g l a s s s y r i n g e s . 123 Gas s l o w l y r e p l a c e d the l i q u i d i n the s y r i n g e ; i t f i r s t appeared from the d i r e c t i o n o f the needle, as c o u l d be seen i n the narrow-bored 25 p i s y r i n g e i n which the g a s / l i q u i d i n t e r - , f a c e was v e r t i c a l i n s t e a d o f h o r i z o n t a l . A l t e r n a t i v e l y t h i s phenomenon c o u l d have been due to the substance b e i n g sucked out o f the needle by the V e n t u r i e f f e c t e x e r t e d by the f a s t stream o f a i r . I n any event i t i s c e r t a i n that the d e p l e t i o n of the l i q u i d l e v e l i n the s y r i n g e sometimes r e s u l t e d i n the i n f u s i o n o f vapour i n the pl a c e of l i q u i d , and e q u a l l y c e r t a i n t h a t t h i s was not always the case; both c o n d i t i o n s were u t i l i s e d . The problem was to decide a t what moment the t r a n -s i t i o n o c c u r r e d ! However, the two descending p o r t i o n s o f the c u b i c curve bear almost i d e n t i c a l r e l a t i o n s to the gear s e t -t i n g s , one f o r vapour and the oth e r f o r l i q u i d i n f u s i o n , s u g g e s t i n g that t h i s q u e s t i o n was i m m a t e r i a l . A f u r t h e r problem was the use of tygon t u b i n g . I t i s m a l l e a b l e , durable and o d o u r l e s s , but has a g r e a t p r o p e n s i t y f o r odour a d s o r p t i o n . T h e r e f o r e the s t i m u l u s was probably not d e l i v e r e d as>a step f u n c t i o n over a p r e d i c t a b l e time a t the i n d i c a t e d r a t e , but r a t h e r was smeared out over a much l o n g e r time as. the adsorbed molecules g r a d u a l l y r e v a p o r i s e d . I t was noted that, w h e n , i n a d v e r t e n t l y contaminated w i t h l i q u i d odorant, a p i e c e o f t u b i n g remained so i n d e f i n i t e l y and had to be d i s c a r d e d ; t h i s was o b v i o u s l y happening to some extent even a f t e r an i n f u s i o n s u c c e s s f u l l y evaporated w i t h i n the T - p i e c e . 124 The recommended c o n s t r u c t i o n m a t e r i a l f o r o l f a c t o m e t e r s i s g l a s s . The a d s o r p t i o n on a g l a s s s u r f a c e i s much l e s s than on p l a s t i c , and i t can e f f e c t i v e l y be c l e a n e d w i t h superheated steam. But g l a s s i s f r a g i l e ; i t would be d i f f i c u l t to con-s t r u c t apparatus r o b u s t enough to use w i t h u n r e s t r a i n e d c a t s . R e s t r a i n t would help but would i n t e r f e r e w i t h the o b s e r v a t i o n of b e h a v i o u r a l a l e r t n e s s ; moreover, when r e s t r a i n t was attempted, i t met w i t h v i g o r o u s r e s i s t a n c e from the i n d i v i d u a l concerned, which i n t e r f e r e d w i t h e l e c t r i c a l r e c o r d i n g . I f the o p t i o n tb s t i m u l a t e s e v e r a l times i n one r u n were s a c r i f i c e d , s e v e r a l of these problems would be more amenable t o s o l u t i o n , but the e x p e r i m e n t a l work would be . . d r a s t i c a l l y slowed. An a l t e r n a t i v e more i n tune w i t h the o b j e c t s of t h i s work would be m u l t i p l i c a t i o n o f the o l f a c t o m e t e r to a l l o w f o r p r e s e n t a t i o n of s e v e r a l d i f f e r e n t odours. G a s t i g h t s y r i n g e s of v a r y i n g s i z e should be used, each on i t s own i n f u s i o n pump, and the temperature o f the entire, o l f a c t o m e t e r c o n t r o l l e d . • S i n c e the s i g n i f i c a n t s t i m u l u s i s a n o v e l odour, i t would not-be n e c e s s a r y to c l e a n the apparatus more thor o u g h l y than would n a t u r a l l y occur i n the pause between p r e s e n t a t i o n s , u n t i l a l l the d i f f e r e n t odorants had been used. The o r d e r o f p r e s e n t a -t i o n would have to be c o n t r o l l e d c a r e f u l l y s i n c e i t might w e l l be s i g n i f i c a n t . I t i s p o s s i b l e t h a t w i t h . i n c r e a s i n g concen-t r a t i o n s , as i n the p r e s e n t study, a second round o f a l l the odorants might be p e r m i s s i b l e . At h i g h e r c o n c e n t r a t i o n s the 125 lesser e v i l would have to be determined as between vapour flow rates significant compared to the diluting airflow and stimulus a r t i f a c t s due to switching i n a l i q u i d infusion line previously-brought to dynamic equilibrium. The cat would probably have to be restrained i n such a way that i t s struggles could not damage the olfactometer, and alertness judged mainly from an EEG. A thermistor should be implanted for measuring the airflow inside the nasal cavity i n front of the olfactory part of the epithelium, probably by the frontal sinus approach of Mechelse '&. Lieuwens (1969). The recordings should be subjected to spectral analysis and the variation with time of the power within different bands of frequencies plotted by a computer program such as that of. Moore et a l . (1968). The apparatus suggested above might be i suitable for extending the scope of the study to d i f f e r e n t i a l adaptation. As concerns the non-olfactory stimuli presented i n the course of these experiments, i t should be noted that while the majority usually caused a brief (e.g. 5 - 10 sec, see Pig. 17) enhancement of induced wave a c t i v i t y , the only visual stimulus produced a depression. A possible inference might be that the cat i s at a point on the evolutionary scale where vision i s beginning to gain ascendency over olfaction. 126 C O N C L U S I O N 127 This study has confirmed the findings of many others who have investigated the induced wave a c t i v i t y of the o l f a c t o r y bulb.. Data were c o l l e c t e d over a wide range of conditions and i f v a l i d , provide a basis f o r i n t e g r a t i n g the apparently d i s -crepant r e s u l t s obtained under more r e s t r i c t e d conditions by others into a coherent scheme. Bothbthe main experiment and the preliminary experiment C reported here are open to severe c r i t i c i s m . However, the p a r t i c u l a r shortcomings of each are d i f f e r e n t i n many respects, yet both give good s t a t i s t i c a l v e r i f i c a t i o n of the hypothesis being tested; moreover the predictions were formulated before e i t h e r set of experiments was performed. I t therefore seems j u s t i f i a b l e to suppose that most of the d e f i c i e n c i e s have been eliminated by the s t a t i s -t i c a l methods employed. U n t i l more rigourous studies are done the following scheme should serve as a u s e f u l working hypothesis. The induced waves i n the o l f a c t o r y bulb are bursts of s i n u s o i d a l e l e c t r i c a l a c t i v i t y seen by a macroelectrode. In the cat they vary i n frequency betwen about 36 - 45 Hz, but each burst has a r e l a t i v e l y constant frequency except some-times at the beginning. They occur during i n s p i r a t i o n and at a lower amplitude during expiration (unless scrupulously p u r i f i e d a i r i s presented). They are very s e n s i t i v e to flow rate and are depressed during nasal obstruction or when sleep, 128 v o l i t i o n or certain reflexes reduce respiratory movements and reroute the flow of a i r so that the olfactory organ i s bypassed more than usual; they are also very susceptible to pathological conditions of the mucosa. On the other hand they are greatly enhanced by a l l factors increasing or improving the airflow, such as arousal, physical movement and sn i f f i n g . As. the common . expression "cat nap" suggests, conscious cats are subject to periodic spontaneous changes of alertness, which are reflected i n sizeable fluctuations i n the amplitude of induced waves, of the order of 25 - 125%. External stimuli, such as sudden noises or changes i n wind speed, .tgenerally arouse the animal, with a corresponding enhancement of respiration and induced wave a c t i v i t y . Olfactory stimuli frequently serve as arousal stimuli i n this way, particularly i f the animal i s drowsy or i f the concentration i s "alarmingly" high ( i n absolute value this range i s quite low compared to the stimuli used by most investigators). A true olfactory response on the other hand i s accompanied by a depression of induced waves without necessarily causing any change i n alertness. This response may occur under any condi-tions but predominates i n an aroused animal at low stimulus intensity. At very high concentrations of odour, of the order of those used by most workers (particularly on acute, r a d i c a l l y dissected preparations) depression also predominates, but i n this case i t appears to be attributable principally to another 129 mechanism, which has been referred to as the noxious vapour reflex. Depending on the degree of i r r i t a t i o n produced by the particular substance, the reflex response may include total or p a r t i a l i n h i b i t i o n of respiration, bradycardia or cardiac Larhythmias, increased blood pressure, aversive behaviour, increased mucous flow and a change i n physical dimensions of the nasal passages probably achieved by vascular engorgement. This reduces the contact of the animal i n general and the olfac tory epithelium i n particular with the contaminated a i r . 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Physiol. 12, 14 -24. 1 i 138 A P P E N D I X POLYNOMIALS IN TERMS OF ORIGINAL VARIABLES AOL AOL **2 AOL **3 AOL **4 AOL :2 -17.23 1.000 AOL :3 195. 1 -26. 85 1.000 AOL :4 -1882 . 455 .9 -37 .70 1.000 AL AL **2 AL **3 . AL **4 AL -5 .595 1 .000 AL :3 21 .30 -8.365 1.000 AL : 4 -78.40 47. 3 5 - 11.63 1.000 CAT CAT **2 CAT **3 CAT **4 CAT .:_2. -_6_._09.1 .1.0.00 CAT :3 26.00 -9.000 1.000 CAT :4 -102.0 53.00 -12 .00 1.000 84 OBSERVATIONS 83 DEGREES OF FREEDOM NAME MEAN S.0. 1 NAME MEAN L t d * D40 96.77 29.55 ABS 82.05 46. 82 SIZE 88.75 49.17 AOL :2 -43.98 20.99 ORDER 2. 738 1. 432 AOL :3 290.7 115.9 ACL 9.107 5.512 AOL :4 -1813. 534.4 AL 2.952 0.8630 AL :2 -7.065 0.9700 .DATE. 1090_. 34. 45 AL :_3 _ 15.84 1_.07.8 . CAT 3.143 0.7784 AL :4 -42.95 1.351 MODE 2.881 1. 675 CAT :2 -8 .667 0.4834 ODOR 1.214 0.4128 CAT :3 24.00 0.1491D-04 NEW 0.9524D-01 0.2953 CAT : 4 -72.01 0.7252D-06 y 133 _C.0RRELAU.0J4 V A R I A B L E 0 4 0 S I Z E M A T R I X D 4 0 1 . 0 0 0 0 - 0 . 2 6 6 4 S I Z E 1 . 0 0 0 0 O R D E R A O L AL OAT E CAT M O D E ODOR NEW A B S O R D E R A O L _A.L D A T E C A T MODE 0 . 1 1 2 9 0 . 0 8 6 2 ..0 ..2 2.2 4. 0 . 0 0 6 6 0 . 0 9 8 5 - 0 . 0 9 3 6 • 0 . 13 56 0 . 0 3 0 6 : 0 , 3 1 4 . 8 . 0 . 1 7 C 2 0 . 0 1 0 4 0 . 0 4 9 8 1 . 0 0 0 0 0 . 3 2 7 1 O.087-3_ 0 . 1 9 4 3 0 . 0 3 4 0 - 0 . 2 7 4 4 1 . 0 0 0 0 - 0 . 0 4 9 6 0 . 0 6 83 -0 . 0 6 8 2 - 0 . 6 0 5 4 .1 .ooop_ 0 . 0 4 0 4 0 . 5 4 8 4 0 . 1 0 4 4 1 . 0 0 0 0 0 . 1 3 1 6 - 0 . 0 3 3 6 1 . 0 0 0 0 0 . 1 7 0 3 1 . 0 0 0 0 ODOR NEW A B S . _ A O L A O L AOL 2 3 4 -0 . 1 3 9 4 - 0 . 1 4 3 1 0 . 2 8 1 4 • 0 . 1 7 0 6 ' 0 . 1 4 1 0 0 . 8 1 7 0 -0 . 0 3 0 5 • 0 . 0 3 4 8 0 . 1 5 3 9 0 . 0 4 9 3 - 0 . 1 0 8 2 • 0 . 0 3 9 7 •0 . 0 8 7 3 -0 . 3 9 6 1 ___J6_42_ -0 . 0 3 1 6 0 . 2 7 6 8 0 . 0 7 7 0 - 0 . 3 0 1 4 -0 . 0 8 0 4 _o_._o.azi_ 0. 0 0 0 0 0 . 0 0 0 0 - 0 . 0 0 0 0 - 0 . 1 0 6 3 - 0 . 3 6 0 2 - 0 . 2 4 6 8 0 . 0 3 5 3 -0 . 0 5 4 0 0 . 0 5 7 2 - 0 . 5 9 8 8 • 0 . 2 2 9 1 _0_.J_9„I6_ 0 . 3 4 2 1 - 0 . 2 8 3 7 - 0 . 0 7 0 3 -0 . 2 4 6 4 - 0 . 1 1 2 3 0 .0.2 59.. 0 . 2 2 8 6 • 0 . 1 2 8 4 0 . 1 0 1 3 - 0 . 1 7 1 8 - 0 . 0 7 4 2 - 0 , 0 1 9 6 0 . 3 7 0 0 0 . 0 3 9 2 -0 . 2 3 5 8 1 . 0 0 0 0 0 . 2 2 5 9 •0. .2 6 5 8 - 0 . 4 7 4 7 0 . 3 9 6 3 0 . 1 7 5 7 1 . 0 0 0 0 _a.-03.7.0_ - 0 . 0 9 4 5 0 . 0 3 8 7 • 0 . 0 5 3 6 _L..Q_Q00_ - 0 . 0 1 0 5 - 0 . 1 7 6 5 0 . 0 4 9 5 A L A L _ _ _ . _ C A T C A T C A T :2 : 3 :4 :2 : 3 :4 0 . 0 3 1 8 - 0 . 1 6 2 4 -_0._.03_79_ 0 . 0 3 7 9 0 . 0 5 1 7 -0 . 0 0 0 0 - 0 . 0 9 0 5 0 . 1 4 0 8 : 0 _ 0 J 3 L 5 . - 0 . 6 1 0 3 • 0 . 6 0 2 2 •0 . 0 0 0 0 -0 . 1 0 5 2 0 . 0 4 4 9 .J_.___i.___ -0 . 0 1 1 6 • 0 . 0 0 6 6 -0 . 0 0 0 0 - 0 . 1 9 0 6 - 0 . 0 9 4 7 0 . 0 4 4 1 - 0 . 2 8 20 - 0 . 2 8 8 8 • 0 . 0 0 0 0 • 0 . 0 0 0 0 0 . 0 0 0 0 -O.J30LOO 0 . 2 8 0 1 0 . 3 5 6 5 - 0 . 0 0 0 0 - 0 . 0 1 9 2 0 . 1 2 8 6 - 0 . 0 9 3 1 • 0 . 0 0 6 5 0 . 0 1 2 7 - 0 . 0 0 0 0 0 . 2 1 5 9 0 . 1 0 0 1 - 0 . 3 6 7 7 - 0 . 0 0 0 0 0 . 1 4 4 8 - 0 . 0 0 0 0 0 . 1 1 4 6 0 . 1 9 0 8 -0 . 1 3 3 A - 0 . 0 3 7 0 - 0 . 0 1 1 9 - 0 . 0 0 0 0 - 0 . 0 7 7 0 - 0 . 1 3 2 1 _Q_..l.83_L 0 . 2 0 8 6 0 . 1 7 0 7 -0 . 0 0 0 0 0 . 2 9 4 8 - 0 . 0 9 5 8 - 0 . 0 2 7 9 0 . 0 2 0 5 0 . 0 0 4 0 -0 . 0 0 0 0 -0 . 0 8 4 2 0 . 0 7 6 5 • 0 . 0 4 4 4 - 0 . 5 8 9 3 - 0 . 5 7 9 2 • 0 . 0 0 0 0 C O R R E L A T I O N MA TRI X V A R ! AB.L.E A O L :.2 AOL :.3_ A O L U L A O L :2 1 . 0 0 0 0 AOL : 3 0 . 0 0 0 0 1 . 0 0 0 0 A O L :4 0 . 0 0 0 0 - 0 . 0 0 0 0 1 . 0 0 0 0 J L L _ A L . A L . : 4 C A T :2. C A T : 3 CAT .: 4. . . A L AL .At.. C A T C A T C A T :2 : 3 :4_ •2 : 3 : 4 - 0 . 0 1 7 5 0 . 0 8 9 1 0 . 0 1 9 9 - 0 . 0 6 0 1 - 0 . 0 2 6 3 0 . 0 0 0 0 - 0 . 0 6 0 4 - 0 . 0 6 9 2 0 .17.2.2 0 . 0 7 4 0 0 . 0 5 . 4 7 - 0 . 0 0 0 0 - 0 . 1 2 9 4 - 0 . 0 6 2 9 . P . . 00 3.6. 0 . 0 8 0 9 0 . 0 9 4 7 0 . 0 0 0 0 1 . 0 0 0 0 - 0 . 0 0 0 0 . . . 0 ,000 .0 . 0 . 2 9 4 1 0 . 3 2 2 2 0 . 0 0 0 0 1 . 0 0 0 0 0 . 0 0 0 0 - 0 . 2 3 2 0 - 0 . 2 1 5 0 - 0 . 0 0 0 0 1 . 0 0 0 0 0 . 0 2 7 5 - 0 . 0261 0 . 0 0 0 0 1 . 0 0 0 0 0 . 9 8 9 3 0 . 0 0 0 0 1 . 0 0 0 0 - 0 . 0 3 2 4 1 . 0 0 0 0 140 A P P E N D I X I I DATA FOR THIS ARRAY IS FILTERED SO THAT* AL MUST BE IN THE RANGE 1.000 TO 2.000 POLYNOMIALS IN TERMS OF ORIGINAL VARIABLES AOL AOL **2 AOL **3 AOL **4 AOL :2 -16.55 1.000 AOL : 3 155.6 -24. 18 1. OOP  AOL :4 -1376. 368.6 -33.85 1.000 22. Oj3.SE.R_VAJIGN S 21 DEGREES OF FREEDOM NAME MEAN S.D. NAME MEAN S.D. D40 86.41 25.85 MODE 3.045 1.731 SIZE ORDER AOL 116.5 2.500 8.545 40 .42 1. 62 6 5. 587 ODOR NEW ABS 1.182 0.2727 99. 17 0.3948 0.4558 _._44_._8.6_ AL DATE CAT 1 .773 1092. 2.864 0.4289 33. 54 0.6396 AOL AOL AOL :2 -38.59 :3 215.0 :4 -1278. 17. 05 77.62 271.2 CORRELATION VARXAB.LE MATRIX D40 _ S I ZJE ORDER AOL. AL . _ D_ATE CAT MODE ODOR NEW ABS D40 SIZE ORDER 1 .0000 -0.1459 -0 .1897 1. 0000 0.0652 1 .0000 AOL AL DATE 0.3930 -0.1145 -0.18 57 -0.1435 0.1518 -0 .2223 0 .3618 0.1707 0.1637 1.0000 0.0343 0. 12 74 1.0000 0. 2968 1•0000 CAT MODE ODOR -0.4254 -0.2505 -0 .2979 0.3893 -0.1112 0.2960 0 .0687 -0.1776 -0 .1484 -0.1781 -0. 6180 -0.2846 0.5759 0.2710 -0.3068 0.0564 0.0417 -0. 7104 1.0000 0.3069 0. 1029 1 .0000 -0.2217 1 .0000 NEW ABS AOL :2 0.0644 0.5655 -0.1471 -0.1156 0.7148 -0.4253 -0.5783 0.0015 0 .0279 -0.1734 0.1443 0.0000 -0 .398 5 0. 1151 0.1726 -0.4977 -0.2224 0.4711 -0.1930 0.0505 -0. 0149 -0.0768 -0 .2231 .. .0.3511 0.5052 1.0000 -0.0425 -0.1216 -0.5245 -0.1683 1.0000 -0 .3987 AOL : 3 AOL :4 -0. 4320 0 .0 592 -0. 0903 0.1813 0.4140 0.2449 0.0000 -0. 0000 -0.1805 0. 0500 -0.2374 -0.2356 0.1769 -0.0144 0.0121 -0.2949 0.3744 0. 0162 0.1860 -0.0616 -0.3 957 0.2298 CORRELATION VARIABLE AOL :2 MA TRI X AOL : 2 1 ._QO00„ AOL : 3 AOL : 4 AOL : 3 AOL : 4 -0.0000 0 .0000 1. 0000 0.0000 1.0000 141 < A P P E N D I X I I I DATA FOR THIS ARRAY IS FILTERED SO THAT AL MUST HAVE THE VALUE 4.000 POLYNOMIALS IN TERMS OF ORIGINAL VARIABLES AOL AOL **2 AOL **3 AOL **4 AOL :2 -15.77 1.000 AOL :3 16 0. 3 -24.44 1 .000 AOL :4 -1232. 336.9 -32.28 1.000 2.1 QB.S.ERVAT IONS 20 DEGREES OF FREEDOM NAME MEAN S.D. NAME MEAN S.D. D40 108.5 33 .46 MODE 3.2 86 1.821 SIZE 61.52 24.36 ODOR 1 .095 0.3008 ORDER 2.714 1.347 NEW 0.4762D-01 0.2182 AOL . 8..048 5_._5_54 ABS 65 .50 28.83 AL 4.000 0.5331D-07 AOL tZ -32.76 16.85 DATE 1092. 32.9 1 AOL :3 ' 210.0 64.91 CAT 4. OOP 0. 5331D-07 AOL :4 -1159. 229.7 CORRELATION MATRIX . VARI ABLE DAO_ SLZ.E ORDER AOL _AJ___ JD.ATJE CAT. M.O.D.E OD.OR NE W ABS D40 1.0000 SIZE -0.1648 1.0000 ORDER 0.3367 -0. 1232 1 .0000 AOL -0.1484 -0.2441 0.3896 1.0000 AL 0.0000 0.0000 O.DOOO 0.0000 1.0000 DATE .0.3368 0. 34.89 0.42 02 ... -0.2 3 50. 0 .00.0 0 1 .0000 . CAT 0 .0000 0.0000 0.0000 0.0000 1. 0000 0.0000 1 .0000 MODE 0.0262 0.1814 -0.4748 -0.7530 0.0000 -0.1707 0.0000 1.0000 ODOR -0.2934 -0.0754 -0.2997 -0.0328 0.0000 -0.2889 Q.QOOO -0.1435 1 .0000 NEW -0 .2865 -0.2965 -0.2916 -0.0845 0.0000 -0. 4950 0. 0000 -0.1618 0.6892 1.0000 ABS 0.5493 0.6987 0.1883 -0.3625 0.0000 0.5374 0. 0000 0. 1588 -0.2082 -0. 3615 1.0000 ... AOL :2 0.0960 -0. 1403 . -0.2341 0.0000 -0.0000 . -0.0052 -0.0000 0.2063 -0.5330 "0.3516 -0.1672 AOL :3 -0.0976 -0.1572 0 .1395 0.0000 0.0000 -0.2118 0. 0000 0.1666 0. 1788 0.3102 -0.1772 AOL :4 0. 1796 -0.2563 -0.0343 -0.0000 -0.0000 0.0066 -0.0000 -0.099.3 0.4482 0. 2219 -0.1259 CORRELATION MATRIX VARIABLE AOL :2 AOL :3 AOL :4 AOL. 1.2 1...0.000 AOL :3 -0.0000 1.0000 AOL :4 0.0000 0.0000 I.0000 

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