Odor Quality and Chemical Structure - American Chemical Society

and has a relatively long retention time on this tissue (_1,2) . The same .... using the Spearman Rank Order Correlation (p). (A mean ... in Figure(1)...
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12 Structure-Activity Relations in Olfaction From Single Cell to Behavior—The Comparative Approach DAVID G. MOULTON

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Veterans Administration Medical Center and Department of Physiology, University of Pennsylvania, Philadephia, PA 19104

Odorants excite receptor cells presumably by interacting with the hypothetical receptor sites. But to reach these sites, odorants must first be transported from a point at which their concentration is known, across the liquid secretions (mucus) lining the surface nasal of nasal airways, through the mucus/air phase boundary and possible to the base of the mucociliary blanket. The mucus is rich in microproteins, Na ions and pigmented granules. Within the mucus, odorant molecules may partition between different liquid phases. Thus separate subsets of physiochemical factors govern stages of transport and odorant-receptor interaction. Consequently, the verbal response - the indicator used in human psychophysical studies of structure-activity relations - reflects the end product of events whose separate contributions are unknown. What is needed in interpreting such data, is a means of segregating and manipulating separate phases of the process or of components of the chemosensory system and assessing their relative influences on the final measured response. To do so we must turn to animal studies. Thus, in the appropriate preparation, it is possible to eliminate certain transport factors; to employ an aqueous rather than a vapor phase to transport odorants to the olfactory surface; to study separately the response characteristics of subpopulations of receptors differing in their structure-activity relations (including the separate contributions of the olfactory receptors and the highly chemosensitive endings of the trigeminal nerve in the nasal mucosa), and to take advantage of the various anatomical and functional features peculiar to specific animal groups such as the extreme absolute sensitivity to certain odorants shown by the dog. The power of the comparative approach to structure-activity relations can be illustrated with selected examples drawn from electrophysiological studies in fish, amphibians and mammals; and behavioral studies in mammals. What follows is a selective and not a comprehensive review of relevant comparative research. +

This chapter not subject to U.S. copyright. Published 1981 American Chemical Society

In Odor Quality and Chemical Structure; Moskowitz, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

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ODOR QUALITY AND CHEMICAL STRUCTURE

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Electrophysiological studies i n f i s h The most extensive s t u d i e s on s t r u c t u r e - a c t i v i t y r e l a t i o n s i n o l f a c t i o n - apart from those on humans - have been on f i s h . This i n t e r e s t r e l a t e s p a r t l y to the commercial importance of t h i s group. But there are advantages i n d e l i v e r i n g odorants i n the aqueous phase: s o r p t i o n onto the f l u i d s e c r e t i o n s (mucus) covering the o l f a c t o r y surface i s l i k e l y to be l e s s than occurs with gaseous odorants and the odorant p a r t i t i o n c o e f f i c i e n t f o r water/ mucus w i l l be c l o s e r to one than would be the case f o r air/mucus. For example, carvone i s s t r o n g l y sorbed anteromedially when flowed i n the vapor phase over the frog's o l f a c t o r y epithelium and has a r e l a t i v e l y long r e t e n t i o n time on t h i s t i s s u e (_1,2) . The same compound i n the aqueous phase (Ringer's s o l u t i o n ) was flowed over the frog's o l f a c t o r y o l f a c t o r y epithelium p r i o r to washing with t r i t i a t e d N-ethylmaleimide (NEM - a group s p e c i f i c p r o t e i n reagent). S i t e s protected by carvone from a t t a c k by NEM were subsequently found d i s t r i b u t e d evenly across the epithelium. This suggests that s o r p t i v e e f f e c t s do not c o n t r o l odorant d i s t r i b u t i o n i n the aqueous phase ( 3 ) . I t i s true that most of the compounds that normally e x c i t e the o l f a c t o r y organ i n f i s h d i f f e r from those to which a i r breathing vertebrates are exposed, but there i s no evidence that the b a s i c transduction mechanisms i n a i r and water d i f f e r s i g n i f i c a n t ly. I t i s known, f o r example, that the same odorants d e l i v e r e d i n the aqueous phase, a r e as e f f e c t i v e as when d e l i v e r e d i n the vapor phase as judged by the slow voltage s h i f t recorded when a macroelectrode t i p was p o s i t i o n e d i n the nasal c a v i t y of a box t u r t l e during odorant s t i m u l a t i o n (k_) . A f u r t h e r advantage i n using f i s h i s anatomical. In a i r breathing vertebrates the o l f a c t o r y chamber extends from the r e s p i r a t o r y airway; i n most f i s h , however, i t i s a separate organ divorced from r e s p i r a t o r y f u n c t i o n s . This f e a t u r e , and the presence of an aqueous medium, allows us to p l a c e a c o n d u c t i v i t y e l e c t r o d e at the i n l e t and one at the o u t l e t of the nasal chamber. I f e l e c t r o l y t e s are used as odorants, t h e i r a r r i v a l and departure from the chamber can then be measured by c o n d u c t i v i t y changes. Since c o n d u c t i v i t y i s p r o p o r t i o n a l to concentration we can speci f y odorant concentration, w i t h i n known l i m i t s , c l o s e to the r e ceptors - something which cannot be done with the i n t a c t n a s a l chamber i n a i r - b r e a t h i n g v e r t e b r a t e s . I t i s a l s o p o s s i b l e to d e l i v e r the odorant i n a way that c l o s e l y i m i t a t e s that i n which i t normally a r r i v e s ( 5 ) . Among the most e f f e c t i v e o l f a c t o r y s t i m u l i f o r f i s h are amino a c i d s . For example, thresholds of 10" have been reported f o r L-glutamine i n the Conger e e l (6) and of 3.2 x 10" i n the A t l a n t i c salmon (7) and c a t f i s h (8). This s e n s i t i v i t y i s presumably r e l a t e d to the widespread d i s t r i b u t i o n of amino acids i n

In Odor Quality and Chemical Structure; Moskowitz, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

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f i s h s k i n e x t r a c t s , which e l i c i t f r i g h t and alarm r e a c t i o n s i n other f i s h of the same species (e.g. 9 ) ; i n mammalian s k i n , which a c t as a r e p e l l a n t to salmon (10,-13); and i n substances that a t t r a c t or e l i c i t feeding behavior i n f i s h (14,15). I t i s not s u r p r i s i n g , t h e r e f o r e , that most s t r u c t u r e - a c t i v i t y s t u d i e s on f i s h o l f a c t i o n have centered on amino a c i d s . Despite the range of species that have been i n v e s t i g a t e d , the v a r i e t y of techniques used and the presence of some species d i f f e r e n c e s i n response (see, f o r example, 6), there i s considerable agreement between workers on the f a c t o r s that govern neural r e s p o n s e , i r r e s p e c t i v e of whether a c t i v i t y i s recorded at a p e r i pheral or higher l e v e l (16-19,6). For example, ^-amino acids e l i c i t the maximum responses, and the most e f f e c t i v e member of a c h i r a l p a i r i s the L-isomer. (Of these, L-glutamine or La l a n i n e are the most powerful s t i m u l i f o r the majority of species so f a r t e s t e d , but not f o r a l l ) . An amino a c i d c o n s i s t s of an asymmetrical carbon center surrounded by four f u n c t i o n a l groups: (1) ^-amino (2) primarycarboxyl (3) ^-hydrogen and (4) a s i d e chain, R:

Response amplitudes can be reduced by s u b s t i t u t i n g other funct i o n a l groups (-H, -CHo, -OH) f o r the -amino group; by methyla t i o n or a c e t y l a t i o n or the ^-amino moiety; by s u b s t i t u t i o n of the ^-hydrogen; o r , i n some cases, at l e a s t , by r e p l a c i n g the primary-carboxyl group. The most e f f e c t i v e amino acids are g e n e r a l l y those with 5-6 carbon atoms and with l i n e a r and uncharged s i d e chains. Amida t i o n g r e a t l y increases the e f f e c t i v e n e s s of a s p a r t i c and g l u t amic a c i d , and s u l f u r - c o n t a i n i n g amino acids are a l s o p a r t i c u l a r l y strong e x c i t a n t s . However, Caprio (19) has concluded t h a t , i n general, the S atom may be equivalent to another C atom i n the chain. The above i n t e r p r e t a t i o n s of the data do not consider the a l t e r n a t i v e i m p l i c a t i o n s of a m u l t i p l e receptor s i t e model f o r the odorant-receptor i n t e r a c t i o n . In such a model the response e l i c i t e d by a l i g a n d r e s u l t s from the simultaneous b i n d i n g of s e v e r a l groups rather than one. Thus i f one group i s modified i t may a l t e r the odorant molecule i n such a way that i t no longer binds to other s i t e s c o n t r i b u t i n g to the response. Hara (17) has proposed a model of the amino a c i d receptor s i t e c o n s i s t i n g of two charged s u b s i t e s , one c a t i o n i c and one a n i o n i c , capable of i n t e r a c t i n g with i o n i z e d ^-amino and p r i mary-carboxyl groups of amino a c i d molecules. He assumes that the L-isomers have more ready access to the receptor and accounts oc

In Odor Quality and Chemical Structure; Moskowitz, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

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f o r t h i s by p o s t u l a t i n g that the two subsites are arranged around the t h i r d c e n t r a l s u b s i t e i n such a way that i t accommodates the ^-hydrogen atom of an amino a c i d molecules. Since the fourth rc-amino moiety g r e a t l y i n f l u e n c e s s t i m u l a t i n g e f f e c t i v e n e s s he proposes that there i s a f u r t h e r region which recognizes t h i s moiety and accounts f o r d i s c r i m a t i n g amino-acid q u a l i t y . Caprio (19), however, has argued that the b i n d i n g of the primary c a r boxyl group may not be p r i m a r i l y i o n i c . In the rainbow t r o u t , o l f a c t o r y bulb neurones seem to d i s criminate between various chemical s t i m u l i having only s l i g h t l y d i s s i m i l a r molecular s t r u c t u r e s and conformations. In f a c t , s e v e r a l c e l l s , i n one study, gave opposite responses to members of enantiomeric p a i r s of amino a c i d s : the L-isomer g e n e r a l l y e x c i t e d while the D-isomer i n h i b i t e d the c e l l (18). There are three problems i n p a r t i c u l a r that complicate i n t e r p r e t a t i o n of much of the data on s t r u c t u r e - a c t i v i t y r e l a t i o n s i n o l f a c t i o n . F i r s t , the d i f f e r e n t techniques used o f t e n y i e l d data that are not s t r i c t l y comparable. Recordings from a s i n g l e or a few receptors, f o r example, are more r e l i a b l e i n d i c a t o r s of the odorant-receptor i n t e r a c t i o n than are recordings of the massed a c t i o n of many neural elements i n the o l f a c t o r y bulb. Thus discrepancies among r e s u l t s are to be expected. Second, many workers record without regard to the existence of topographic d i f f e r e n c e s i n the s e n s i t i v i t y of the system to d i f f e r e n t odorants. For example, DtJving et a l (20) showed that b i l e acids e l i c i t e d responses ( i n the o l f a c t o r y bulb of chars and g r a y l i n g s ) which d i f f e r e d s p a t i a l l y from those of two amino a c i d s . A t h i r d d i f f i c u l t y i s that many workers i n v e s t i g a t e the r e sponse to only one concentration of each odorant. But i t i s w e l l known that some odorants can increase neural a c t i v i t y at low concentrations and suppress i t at higher concentrations (21,20,5). This r a i s e s the p o s s i b i l i t y that the r e l a t i v e s t i m u l a t i n g e f f e c tiveness of a group of odorants e s t a b l i s h e d at one concentration, i s not the same as that e x i s t i n g at another l e v e l . The point i s w e l l i l l u s t r a t e d i n a study by Meredith (22,_23,j>) . The aim was to e s t a b l i s h and analyse response s i m i l a r i t i e s of s i n g l e bulbar neurones i n the g o l d f i s h when stimulated s u c c e s s i v e l y by seven amino acids - each a c i d being presented i n not one, but two d i f f e r e n t concentrations. The compounds used, t h e i r s t r u c t u r e s and c e r t a i n p h y s i c a l p r o p e r t i e s are shown i n Table I. Response s i m i l a r i t y was measured by c o r r e l a t i n g temporal patterns of c e l l f i r i n g r a t e (rather than maximum f i r i n g r a t e which was l e s s c h a r a c t e r i s t i c of odor type and concentration) using the Spearman Rank Order C o r r e l a t i o n (p). (A mean s i m i l a r i t y measure f o r a given stimulus p a i r was determined by f i n d i n g the average f i r i n g r a t e across a l l u n i t s t e s t e d . Guttman-Lingoes nonmetric mulitdimensional s c a l i n g procedure was a p p l i e d to the matrix representing a l l p a i r s of mean s i m i l a r i t y measures r e s u l t ing i n the arrangements i n F i g u r e ( 1 ) . In these p l o t s the rank order of distances between points i s the inverse of the rank

In Odor Quality and Chemical Structure; Moskowitz, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

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Table I.

Physico-chemical Constants and Structures of Amino Acids Substance MW Symbol Glycine

75.1 G

Structure

Substance M W Symbol

Structure

H-CHC0 H

CH -CH S0 H I NH

2

2

I

NH

Taurine 125.1 T 2

2

3

2

Alanine

89.1 A

CH -CHC0 H 3

2

NH /3-alanine 89.1

B

105.1 S

165.2

P

Pentanoic acid 11021 —

-Butanol

STRUCTURE

H

'

H

0 CHjCh CH CH -C-OH 2

2

2

- .• I Insol. si. sol. sol. very sol. miscible

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Dimethyl sulfide 62 | 37 |

u

Trimethylamine 59 3 | | ^Heptanal

1114 1531 M 0

CHj

1

CH,SCHj

CHJNCHJ

CH CH CH CH CH CH -C-H 3

2

2

2

2

2

0 Phenylethanone 120 202|

^ 2-Pro pa none

COCH

3

CH3-C-CH3

Compound 100 150^^/3-lonone Molecular weight-^ / / / Boiling point ' / / Solubility in water '/ Solubility in alcohol '



192 |140|_J a-Terpineol

11541220l '• CH,

i I

B

^

O

0

6



58 |56^^JCyclopenianone| 84 1311

H

JCOH 3 "XH C

3

insol. si. sol. sol. very sol. miscible

Geraniol

154 2301

il521285J ki Isoeugenol

^ Vanillin

1164[134| L

^^CH OH 2

HjC

?

CHj

CHO

H

CH=CH-CH

3

3-Phenyl _ |148 Napthalena |196| - | L. - propanoic acid ' ..^•M

Cantharadin 0

OCH,

?

128 218|

8

1

5

4

^

00

I

CH, CH 3

BOCC-C- "

N 0

6

H

6

3

N

Figure 2. Structures and selected properties of odorants used to stimulate olfactory epithelium of tiger salamander. Odorants are classified according to their relative effectiveness in stimulating different epithelial regions (33).

In Odor Quality and Chemical Structure; Moskowitz, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

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12.

Posterior stimulants

_

100 150 n-Octane Compound Molecular Weight^ / / / Boiling Point / / / Solubility in water '/ Solubility in alcohol ' • insol.

U

i

B

si. sol.

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1

s o l . very sol miscible

Hexachloro-

114 126

|l02|l02| !• D-Limonene

136 178

Uniform stimulants H Cyclohexanone 98

1 5 5

BM

O ^n-Pentyl acetate 130 149

CH

J|

3

CHsCOOCsHu

CI3CCCI3

HC 3

a -Pinene

136 1561 gJD-Camphor

CH

2

152 204 !• n-Decyl acetate 1230 244

CH

3

CHj CH C0 (CH ) CH 3

Q

CH

2

2

9

3

^r^-CH,

3

Figure 2, Continued

In Odor Quality and Chemical Structure; Moskowitz, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

u

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p e n t y l a c e t a t e g e n e r a l l y s t i m u l a t e s maximally along a d i s t i n c t r i d g e extending a n t e r i o r l y f o r about 2mm from the d-limonene s e n s i t i v e zone located p o s t e r i o r l y . F o c i f o r peak s e n s i t i v i t y to eugenol and iso-eugenol are a l s o c l e a r l y segregated (34). In t h i s study odorants were d e l i v e r e d by way of a microp i p e t t e p o s i t i o n e d l e s s than a mm from the o l f a c t o r y s u r f a c e (a m o d i f i c a t i o n of a technique described by Kauer and Moulton (35)) . This e l i m i n a t e s any p o s s i b i l i t y that the d i f f e r e n c e s reported r e s u l t e d from a d i f f e r e n t i a l s o r p t i o n of odorant molecules by the mucus. In the behaving animal, however, odorants flow over the mucus a n t e r i o r l y to p o s t e r i o r l y . Because d i f f e r e n t odorants have d i f f e r e n t mean r e l a t i v e r e t e n t i o n times on the o l f a c t o r y mucosa O ,36) they may c r e a t e d i f f e r e n t gradients of e x c i t a t i o n across i t (except f o r those with r e l a t i v e l y short mean r e t e n t i o n t i m e s ) . To what extent such f a c t o r s c o n t r i b u t e to the o v e r a l l s p a t i a l p a t t e r n of e x c i t a t i o n i s not yet c l e a r (37). Cone entra t ion-response r e l a t ions Despite the apparently widespread conformity of many sensory f u n c t i o n s to the Weber-Fechner or Steven's Power Laws, the r e l a t i o n s between stimulus i n t e n s i t y and response magnitude can sometimes be more complex. For example, d i s c o n t i n u i t i e s i n t h i s r e l a t i o n are a s s o c i a t e d with dual somesthetic receptor f u n c t i o n s (38) and with dual f u n c t i o n s of a s i n g l e receptor type i n the r e t i n a (39). Should such d e v i a t i o n s occur i n o l f a c t o r y functions, they may not have been i d e n t i f i e d i n many s t u d i e s because of the very assumption that a simple r e l a t i o n must e x i s t between conc e n t r a t i o n and response. T h i s assumption determines the concent r a t i o n s at which response measures are made - a number which may be inadequate to r e v e a l any d e v i a t i o n s from a simple r e l a t i o n . A l t e r n a t i v e l y , i f they do appear, they may be dismissed as s t a t i s t i c a l l y i n s i g n i f i c a n t a b e r r a t i o n s . Yet, even i f a curve r e f l e c t e d only l i g a n d b i n d i n g i t i s u n l i k e l y to be simple. In a v a r i e t y of n e u r a l and other t i s s u e s , b i n d i n g curves are i n f l u enced by v a r i o u s froms of c o o p e r a t i v i t y (binding, e f f e c t and i n t e r m o l e c u a l r c o o p e r a t i v i t y ) . For example, i n b i n d i n g coopera t i v i t y the presence of l i g a n d molecules already bound can a l t e r the a f f i n i t y of the r e c e p t o r f o r a d d i t i o n a l l i g a n d b i n d i n g (40). But i n f a c t , f u r t h e r complexity may be imposed by events preceding and succeeding odorant b i n d i n g . The most s i g n i f i c a n t of these are transport f a c t o r s and n o n l i n e a r transform f u n c t i o n s w i t h i n the c e n t r a l nervous system ( i n the case of measures taken at bulbar or higher l e v e l s ) . I t has a l s o been suggested that enzymes, which are probably present i n the mucus, may degrade odorant molecules d i f f u s i n g towards b i n d i n g s i t e s ( N i c o l l i n i 41). Thus as G e t c h e l l and G e t c h e l l (42) have noted, p e n t y l acetate may degrade to pentanol and a c e t i c a c i d . These events could f u r t h e r d i s t o r t the concentration-response curve. In view of these f a c t o r s i t i s not s u r p r i z i n g that s i n g l e

In Odor Quality and Chemical Structure; Moskowitz, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

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c e l l s i n the g o l d f i s h o l f a c t o r y bulb y i e l d curves w i t h a v a r i e t y of shapes. Some, f o r example, show monotonic response f u n c t i o n s while others show i n i t i a l l y i n c r e a s i n g and then decreasing f i r i n g r a t e s as c o n c e n t r a t i o n i s increased ( F i g . 3 ) . Some non-monotonic response f u n c t i o n s were a l s o recorded i n f r o g and salamander o l f a c t o r y r e c e p t o r s ( 4 3 , 4 4 ) . The average of c o n c e n t r a t i o n responsecurves showing a v a r i e t y of forms, such as those i n F i g . 3 , could be a r e l a t i v e l y complex f u n c t i o n . But whatever the reason, f o r some odorants at l e a s t , curves derived from l a r g e populations of receptors do show marked notches. They appear i n data generated both e l e c t r o p h y s i o l o g i c a l l y and p s y c h o p h y s i c a l ^ ( F i g . 4 ) . In the case of the psychophysical curve f o r °=-ionone seen i n data from dogs, the notch i s h i g h l y s i g n i f i c a n t s t a t i s t i c a l l y and d i v i d e s the curve i n t o a slowly descending upper limb, best f i t t e d by a p a r a b o l i c f u n c t i o n , and a r a p i d l y descending lower limb, best f i t t e d by a cubic f u n c t i o n ( F i g . 5 ) . In an homologous s e r i e s of a l i p h a t i c acetates the p o s i t i o n of t h i s notch on the curve ascends w i t h i n c r e a s i n g chain l e n g t h , and i t has been suggested that the notch may r e f l e c t the independent c o n t r i b u t i o n s of two types of receptors - the response of one, c o n t r o l l i n g the lower limb of the curve, and that of the other, c o n t r o l l i n g the form of the upper limb ( 2 4 ) . An a l t e r n a t i v e e x p l a n a t i o n , however, i s that the a f f i n i t y of a s i n g l e type of s i t e f o r the odorant changes as a c r i t i c a l c o n c e n t r a t i o n i s reached. The form of the concentration-response curve o f f e r s a potent i a l approach to grouping odorants, and Mathews (48) has made a promising s t a r t i n t h i s d i r e c t i o n . He recorded the averaged act i v i t y from bundles of r e c e p t o r nerve f i b e r s i n the r a t . The seven odorants he tested f e l l i n t o three groups according to the slope and form of the curves that they e l i c i t e d . Members of the f i r s t group were n-pentyl a c e t a t e and two compounds with a pepperm i n i t y odor: menthone and 2-sec b u t y l hexanone. T h e i r curves showed c l e a r notches and a c c e l e r a t e d n e g a t i v e l y towards t h e i r asymptotes. The second group contained l i n a l o o l and dimethyl benzyl carbonyl a c e t a t e - compounds w i t h a f l o r a l odor and p o s i t i v e l y a c c e l e r a t i n g curves. In the t h i r d group were camphor and i s o - b o r n e o l , both w i t h a camphoraceous odor and a curve c o n s i s t ing of a lower n e g a t i v e l y a c c e l e r a t i n g limb and a l i n e a r upper limb. Further evidence that the slope of the concentration-response curve may be r e l a t e d i n a p r e d i c t a b l e way to the physiochemical p r o p e r t i e s of the odorant molecule comes from a study of the r e l a t i v e d e t e c t a b i l i t y of members of an homologous s e r i e s of a l i p h a t i c acetates ( 4 9 ) . P r o b i t r e g r e s s i o n l i n e s were f i r s t derived from the concentration-response data f o r each member of the s e r i e s . The slopes of these l i n e s , when p l o t t e d a g a i n s t l o g carbon chain l e n g t h , y i e l d e d an approximately l i n e a r r e l a t i o n . One consequence of the r e l a t i o n i s that each of s e v e r a l p r o b i t r e g r e s s i o n l i n e s i n t e r c e p t s one or more other l i n e s . Thus the r e l a t i v e e f f e c t i v e n e s s of these compounds depends on the p e r f o r -

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mance l e v e l that i s chosen as a b a s i s f o r comparison. For example, i f a 50% c o r r e c t response score i s taken as the c r i t e r i o n (chance being 50% c o r r e c t ) , the r e l a t i o n between performance and response i s l i n e a r . I f , however, an 85% c o r r e c t c r i t e r i o n i s chosen a p a r t i a l l y c u r v i l i n e a r r e l a t i o n emerges (49). The dependence o f response s i m i l a r i t i e s (determined e l e c t r o p h y s i o l o g i c a l l y ) on c o n c e n t r a t i o n was discussed above i n r e l a t i o n to a group of amino a c i d s . Thus the physicochemical p r o p e r t i e s o f an odorant that c o n t r o l i t s r e l a t i v e s t i m u l a t i n g e f f e c t i v e n e s s a t one c o n c e n t r a t i o n are not n e c e s s a r i l y those c o n t r o l l i n g e f f e c t i v e ness a t another c o n c e n t r a t i o n . Acknowled %ment s We thank Dr. Michael Meredith f o r permission to i n c l u d e f i g u r e s and data from a p r e s e n t a t i o n and f o r c r i t i c a l l y reading the manuscript. We a l s o thank Dr. Susan Schiffman f o r permission to i n c l u d e the r e s u l t s of a multidimensional a n a l y s i s of the data and f o r h e l p f u l d i s c u s s i o n and comments on the manuscript. Part of the work described here was supported by NIH grant No. 5 R01 NS 10617-04 and AFOSR grant No. 77-3162.

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October 13, 1980.

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