211

Biochlmica et Biophysica Acta, 437 (1976) 211--220

O Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 27921 APPARENT OPPOSING EFFECTS OF CYCLIC AMP AND DIBUTYRYLCYCLIC GMP ON THE NEURONAL FIRING OF THE BLOWFLY CHEMORECEPTORS

DARRYL L. DALEY a and JERRY S. VANDE BERG b a Department o f Entomology, Neural and Behavioral Biology Section, University o f Illinois, Urbana, Ill. 61801 and b Department o f Biological Sciences, Old Dominion Un&,ersity, Norfolk, Va. 23508 (U.S.A.)

(Received October 10th, 1975) (Revised manuscript received February 17th, 1976)

Summary Cyclic AMP and its dibutyryl derivative inhibit neuronal firing of the labellar sugar sensitive receptor o f the blowfly when applied in conjunction with the stimulant sucrose. Furthermore, simultaneous application of aminophylline (phosphodiesterase inhibitor) and sucrose or in combination with cyclic AMP caused a similar depression of the sugar receptors response. In contrast, dibut y r y l cyclic GMP elicited an increase in sugar receptor firing when applied with sucrose to the sugar receptor. Either 5'-AMP or 5'-GMP in combination with sucrose had no discernable effect on the sugar receptors response. Different ratio combinations of cyclic AMP and dibutyryl cyclic GMP showed the striking inhibitory effect of cyclic AMP upon the dibutyryl cyclic GMP elicited increases in receptor firing frequency. Therefore, it is suggested that these two nucleotides may be mediating different but complimentary aspects of sugar receptor function in a push-pull manner.

Introduction Although the role of cyclic GMP is still n o t understood, a function for this c o m p o u n d has been suggested according to a dualistic theory involving cyclic AMP in nerve tissue [1--5]. This concept suggests that cyclic GMP and AMP function through promoting opposing biological events within the same cell. Although data exist for and against this idea, it appears that the a m o u n t of supportive evidence is accumulating for its acceptance. Within the area o f chemosensory physiology, a role for cyclic nucleotides was initially suggested by high levels of adenyl cyclase activity in the papillae of bovine taste buds which were comparable to that found in the brain [6]. A

212 similar level of adenyl cyclase was also exhibited in the rabbit olfactory epithelium from which it was concluded that cyclic AMP may have an important role in the transduction process common to both sensory organs. In support of these conclusions, it was noted that cyclic AMP phosphodiesterase was inhibited by bitter taste stimuli such as caffeine and theophylline [7]. In the blood-feeding bug, Rhodnius, a chemoreceptive role for cyclic AMP was suggested in the mediation of the gorging response [8]. This response was significantly enhanced by theophylline and caffeine. A relatively recent report [9] suggests that chemoreception for the swallowing response o f the sea anemone, Anthopleura, is mediated by cyclic AMP control of Ca 2+ distribution in the oral disc and pharynx. This conclusion was based on the finding that activation of adenyl cyclase in the oral disc and pharynx by reduced glutathione was correlated with increased Ca 2+ binding in the particulate fraction containing adenyl cyclase. In our laboratory, recent cytochemical localization studies in the blowfly labellar chemoreceptor sensillum indicated that phosphodiesterase was specifically associated with axonal microtubules; whereas, the dendritic terminals, as well as some o f the supportive cells in the sensillum, exhibited adenyl cyclase activity [10] (Felt, B.T. and Vande Berg, J.S., unpublished). Recent radioimmunoassay studies involving this receptor system also showed high levels of endogenous cyclic AMP and GMP (Felt, B.T. et al., unpublished). In view o f these findings and those which suggest a dualistic function between these nucleotides, we decided to utilize the unique structural and functional features of the blowfly chemoreceptor system to electrophysiologically investigate the possibility of these cyclic nucleotides promoting opposite neuronal events within one of the four sensory nerve cells (i.e., what has been previously suggested as the sugar sensitive receptor cell). Materials and Methods

Animals and compounds Electrophysiological tests were conducted on adult, female blowflies (Phormia regina, Meigen) which were reared in the laboratory. Test flies varied from two to seven days and were maintained on a diet of powdered dry milk and sucrose (1 : 1) and water. All compounds, adenosine 3',5'-cyclic monophosphate (cyclic AMP); N 6, 02dibutyryl adenosine 3',5'-cyclic monophosphate (dibutyryl cyclic AMP); N6,O 2dibutyryl guanosine 3',5'-cyclic monophosphate (dibutyryl cyclic GMP 5'-adenosine monophosphate (5'-AMP); 5'-guanosine monophosphate (5'-GMP); and aminophylline, used in these experiments were obtained from Sigma Chemical Co., and stored in desiccator jars below 0 ° C.

Recording technique An isolated head whs m o u n t e d in Cambasco mounting wax on a tilt-top tripod head under a modified AO binocular microscope. The electrophysiological recording method utilized was according to Hodgson et al. [11]. According to these procedures, an indifferent electrode (glass micro-pipette) containing 0.05 M NaC1 impaled the fly's head. The recording electrode, which served also as

213 the stimulating electrode, contained a test solution plus an e!ectrolyte and was advanced by a motorized micromanipulator over the tip of the largest labeUar se~ae (Group IV type setae [12]. This arrangement completed the electrical circuit for recording extracellular action potentials. The Ag/AgC1 wires, leading from both recording mid indifferent electrodes were connected to a Grass P-16 A.C./D.C. microelectrode amplifier via a high impedance probe. The o u t p u t of the P-16 amplifier was A.C. coupled to a Tektronix RM502A oscilloscope for display and photography by a Tektronix 125 Polaroid camera.

General experimental design Reference and recording electrodes contained 0.05 M NaC1 which was below threshold for both salt sensitive receptors and inhibition of the sugar response. The recording electrode, containing the electrolyte and 0.2 M sucrose, initially measured the inherent firing frequency of the test receptor. Removing the electrode and following a 5-min disadaptation period, a second recording c!ectrode containing 0.05 M NaC1, sucrose and a test c o m p o u n d was identically placed over the same test seta. Thus, the initial responses indicated the inherent firing of the sugar sensitive receptor and also served as a control to the test response. In each experiment, control data were displayed directly over the response o f the test compound.

Analysis Procedure With each experimental tracing, spikes were counted 1000 ms after stimulus onset (because of amplifier block when closing the circuit and "electrical noise" produced by the motorized drive} for a period o f 1500 ms. From these data, percent increases or decreases to various test compounds were calculated with respect to inherent firing frequency of the sugar receptor over the time interval. In each series of experiments involving a particular compound or combination of compounds, the mean and standard error o f the mean of percent increase or decrease was determined. Results While there are at least four different chemical modalities to which the chemoreceptor sensillae of the blowfly responds, this study was limited to the effects of several compounds on the sugar sensitive receptor. Such responses to various stimuli can be distinguished by amplitude o f the action potential. Further selection was obtained by using a subthreshold concentration o f NaC1 which allowed only occasional large amplitude spikes (salt spikes) to interpose the tracings. Thus, unless each receptor is excited by an adequate stimulus, they apparently remain silent. Most of our recordings contain only responses from the sucrose sensitive receptor and small amplitude spikes associated with the water receptor. The effects of various concentrations o f cyclic nucleotides on the sucrose sensitive receptor neuron are shown in Table I. All figures are expressed as averaged values from at least 8 different chemoreceptor sensillae. In instances

214

TABLE

I

EFFECTS OF CYCLIC NUCLEOTIDES ON THE SUCROSE SENSITIVE RECEPTOR NEURON Compound

Increase in response (percent)

Cyclic AMP (5 • 10 -s M) (1 • 10 -4 M)

(5 • 10 -4 M) (5 • 10 -3 M) Dibutyryl

cyclic (5 • (5 • (5 •

Aminophylline

AMP 10 -5 M) 10 -4 M) 10 -3 M)

(2 raM)

Aminophylline (2 raM) and cyclic AMP (5 • 10 -4 M)

D e c r e a sin e response (percent)

No effect

S.E,M.

23.53 27.76 33.66 12.89

+0.67 Y0.83 ±1.39 ±0.60

22.39 27.15 16.05

-+0.21 -~ 1 . 9 8 ±1.44

34.43

+-1.00

40.84

± 1.92

Dibutyril cyclic GMP (1 • 10 -s M) (1 • 10 -4 M)

(5 • 10 -4 M) (1" 10 -3 M)

15.64 21.74 24.41 36.37

+~1 . 4 4 tl.66 +~0.93 .1.56

5 ' - A M P ( 5 • 10 - 4 M )

No effect

5 ' * G M P (1 • 1 0 - 3 M )

No effect

Ratios of dibutyryl cyclic GMP: cyclic AMP ( 2 0 : 1) (i0 : i) ( 2 : 1) (1 : 5)

31.32 16.14 14.04 24.34

z2.21 -+0.66 ~ 1.21 -*1.52

where a c h e m o r e c e p t o r neuron did n o t respond initially to stimulation, it was rejected and a n o t h e r preparation was tested. The chemosensory response was inhibited in every instance by a c o n c u r r e n t application o f cyclic AMP with 0.2 M sucrose. Fig. l a represents the inherent firing f r eq u en cy of one r e c e pt or neuron to 0.2 M sucrose, T he effect o f an exogenous application o f 5 . 10 -4 M cyclic AMP and sucrose on the response of this same r e c e p t o r shows a 33.66 -+ 1.39% (mean -+ S.E.M.) decrease in impulse firing. Increased chemosensory inhibition was correlated with increasing concentrations o f cyclic AMP except at the 5 • 10 .3 M level (Table I). In this instance, inhibition was less than that n o t e d by any ot her concentration. Simultaneous application o f the dibutyryl derivative o f cyclic AMP at various concentrations depressed the response of the sugar sensitive n e u r o n in a way quite similar to cyclic AMP (Table I). Increasing concentrations o f this c o m p o u n d could be associated with increasing inhibition with an apparent reversal also at the 5 • 10 .3 M level. Aminophylline (2 mM) (inhibitor of the degradating enzyme, phosphodiesterase) like cyclic AMP and its d i b u t y r y l derivative depressed the response o f the

215

b Fig. 1. (a) I n h e r e n t r e s p o n s e t o 0 . 2 M s u c r o s e . CO) R e s p o n s e t o c o n c u r r e n t a p p l i c a t i o n o f AMP and 0.2 M sucrose. The observed depression was 33.66 ± 1.39% ( m e a n ± S.E.M.) tions. The vertical bar equals 0.2 mV and the horizontal bar equals 1000 milliseconds T h e r e g i o n b e t w e e n t h e w h i t e a r r o w h e a d s r e p r e s e n t s t h e 1 5 0 0 m s p e r i o d u s e d in s p i k e

5 • 1 0 "~ M c y c l i c in e i g h t prep~Lraf o r Figs. 2 a n d 3. c o u n t s f o r all fig-

~re~

b Fig. 2. (a) C o n t r o l r e s p o n s e t o 0 . 2 M s u c r o s e . Co) R e s p o n s e to s i m u l t a n e o u s a p p l i c a t i o n o f 2 m M a m i n o p h y l l i n e a n d 0 . 2 M s u c r o s e . T h e o b s e r v e d d e p r e s s i o n w a s 3 4 . 4 3 -+ 1 . 0 0 ( m e a n ± S . E . M . ) f o r e i g h t p r e p a r a tions.

8

b Fig. 3. (a) C o n t r o l r e s p o n s e t o 0 . 2 M s u c r o s e . ( b ) R e s p o n s e t o c o n c u r r e n t a p p l i c a t i o n o f 1 • 10 -3 M d i b u tyryl cyclic GMP and 0.2 M sucrose. The observed effect was a 36.37 ± 1.56 ( m e a n ± S.E.M.) gain over the c o n t r o l r e s p o n s e in e i g h t p r e p a r a t i o n s .

216

sucrose receptor (Fig. 2a and b). When this level of aminophylline was combined with 5 • 10 -4 M cyclic AMP and sucrose, the response was further depressed to 4O.84%. In contrast to the general results o f cyclic AMP and its dibutyryl derivative, concurrent applications of various concentrations o f dibutyryl cyclic GMP with 0.2 M sucrose caused an increase in firing frequency in the labellar sugar sensitive receptor (Fig. 3a and b). The increase appeared to be positive with each increased level o f dibutyryl cyclic GMP. Dibutyryl cyclic GMP did not show a particular optimal level for this enhanced firing over the range o f concentrations tested. In another series of experiments which were designed to determine the effects that various combination ratios of dibutyryl cyclic GMP and cyclic AMP have on the sugar receptor response, a constant level o f dibutyryl cyclic GMP with various concentrations o f cyclic AMP were added in conjunction with the stimulant, sucrose. The constant level o f dibutyryl cyclic GMP (1 • 10 -3 M) maintained in these experiments was based on previous concentration studies (Fig. 3) which indicated this a m o u n t caused the greatest increase in receptor firing.

35--

30-

2=--

20--

15--

I0--

5--

I

I 5x10 "5

I

I

l x l O -4

Concentration

I

5 x l O -4 of

cAMP

I 5xlO 3

(Molar)

Fig. 4. (a) P e r c e n t d e c r e a s e o f t h e sugar r e c e p t o r s r e s p o n s e t o f o u r c o n c e n t r a t i o n s o f c y c l i c AMP. T h e p o i n t s on t h e g r a p h r e p r e s e n t t h e m e a n v a l u e s f r o m e i g h t e x p e r i m e n t a l p r e p a r a t i o n s . B r a c k e t s r e p r e s e n t t h e r a n g e of v a l u e s (± S.E.M.).

217

40

35~

30

25

o

20

15

10

I

I

I

I l x l O "4

1 x l 0 -5 Con~e:ltration

of

Dibutyryl

I 5x10"4

I lxlO "3

cGMP (M)

Fig. 5. P e r c e n t i n c r e a s e o f t h e s u g a r r e c e p t o r s r e s p o n s e t o f o u r c o n c e n t r a t i o n s o f d i b u t y r y l c y c l i c GMP. T h e p o i n t s o n t h e g r a p h r e p r e s e n t the m e a n v a l u e s f r o m e i g h t e x p e r i m e n t s . B r a c k e t s i n d i c a t e d t h e r a n g e of v a l u e s (-+ S.E.M.).

Th e e f f e c t o f 1" 10 -3 M d i b u t y r y l cyclic GMP and 5" 10 -s cyclic AMP (20 : 1) p r o d u c e d an average increase in r e c e p t o r firing o f 31.32% (Table 1). However, this increase was significantly less than the response gain elicited when onl y d i b u t y r y l cyclic GMP with sucrose was applied to the sugar sensitive neuron. Ratios o f 10 : 1 and 2 : 1 p r o d u c e d a f ur t her inhibitory influence on the enhanced firing effects o f di but yr yl cyclic GMP. The final test mixture, a 1 : 5 ratio, p r o d u c e d a 24.34% increase over the cont rol response. The inhibitious effects o f cyclic AMP at this concent r a t i on was n o t as great as the previous level. This response was compatible with the limited inhibition elicited by this concentration in earlier studies o f when onl y cyclic AMP was administered to the sugar sensitive neuron. Control tests, with (5 • 10 -4 M) 5'-AMP and (1 • 10 -~ M) 5'-GMP (breakdown products o f cyclic AMP and d i b u t y r y l cyclic GMP) on norm al firing o f the sugar r e c e p t o r when stimulated with 0.2 M sucrose, exhibited no significant decrease or increase in firing frequency.

218

Discussion Previous research efforts suggest that stimulus reception and regulation of chemosensory nerve function are meadiated by cyclic nucleotides, specifically cyclic AMP [7--10,13]. The results from our investigation expand the role for cyclic AMP in chemoreception and include cyclic GMP as having a distinct and opposite effect on the chemoreceptor response. The response to three of four concentrations of cyclic AMP tested were nearly linear with respect to increasing nucleotide concentrations. The fourth concentration (5 • 10 -3 M) which deviated significantly from the linearity of the previous three indicates that there is a reversal in the response. This finding is particularly significant since it reflects a trend in the firing pattern from an exogenous application of only cyclic AMP and sucrose and suggests that 5 • 10 -4 M appears to be a near o p t i m u m concentration for the observed inhibitive effect. In contrast 5'-AMP, the degradation product of cyclic AMP, had no effect on the receptor firing. Thus it would appear that cyclic AMP alone is mediating the specific inhibitory effect. Additionally, with regard to molecular effects, it was observed that the response to concurrent applications of dibutyryl cyclic AMP and 0.2 M sucrose are strikingly similar to those n o t e d with cyclic AMP. In this respect it appears that both cyclic nucleotides may be acting in an analogous manner. It is further observed that simultaneous applications o f 2 mM aminophylline (inhibitor of phosphodiesterase) with 0.2 M sucrose also decreased the firing frequency of the sucrose sensitive receptor. Based on previous studies, this effect is probably due to increased levels of endogenous cyclic AMP which resuited from inhibition of phosphodiesterase activity. In support o f these data, a complimentary effect was noted when aminophylline and 5 . 1 0 -4 M cyclic AMP in combination with the stimulant, sucrose, were exogenously applied to the sucrose receptor. Thus, the depression by aminophylline and cyclic AMP was greater than when either compound, alone, was applied with sucrose. Similarly it has shown that aminophylline hyperpolarized the retinular cells of Limulus when applied at concentrations of 2 mM or less [20] while Vande Berg [22] n o t e d that theophylline, a compound quite similar to aminophylline, reduced the spike amplitude, as well as conduction velocity in the frog sciatic nerve. In addition, he reported a synergistic effect with cyclic AMP and theophylline together. Thus, these data and those from the present study support the ideas that the observed effects are most likely endogenous in nature, whether mediated by an exogenous application of cyclic AMP, or by application o f a c o m p o u n d which indirectly elevates endogenous levels (via aminophylline). The response to concurrent application of various concentrations of dibutyryl cyclic GMP and 0.2 M sucrose was an increase in the firing frequency and totally counter to those found with cyclic AMP or its dibutyryl derivative. Although the effect o f increasing concentration on the responses was not completely linear (see Fig. 5), there occurred no reversal of effects like that observed with cyclic AMP and its dibutyryl derivative. The highest concentration tested (1 • 10 -3 ) caused a greater increase in firing frequency with respect to the other three concentrations tested. Exogenous application o f 5'-GMP, the

219 degradation product of cyclic GMP, in contrast, had no discernible effect on the sugar receptors firing. Again it appears that a cyclic nucleotide (dibutyryl cyclic GMP) is mediating the specific effect. While a second messenger concept cannot be verified from these data, it is shown, however, that with sucrose stimulation, there is a specific depression of receptor behavior with cyclic AMP and an apparent amplification with dibutyryl cyclic GMP. Since various insect receptors are stimulated with molecular amounts of stimuli an amplification system affecting membrane permeability and subsequent selective ion transport may be an important feature of the chemotransductive process. These data also show that one particular neuron (sucrose receptor} responds in an excitatory manner to dibutyryl cyclic GMP and in an inhibitory manner to cyclic AMP. A similar situation was demonstrated when dibutyryl cyclic AMP caused a depolarization o f post-ganglionic neurons in the rabbit, while cyclic AMP caused a hyperpolarization of the same neurons [3]. The effects o f dibutyryl cyclic GMP in combination with various concentrations o f cyclic AMP with a constant level o f sucrose were most interesting. As the concentration o f cyclic AMP was increased in combination with a constant level of dibutyryl cyclic GMP, the presence of cyclic AMP reduced the increase in firing frequency as compared to the response from dibutyryl cyclic GMP. This antagonistic effect supports the concept o f a biological dualism between these two compounds. A strikingly similar situation was presented in a review on the role o f cyclic nucleotides in visual excitation [5]. This review suggested different y e t complimentary roles for cyclic AMP and GMP in the regulation of the visual excitation system. At approximately the same time this investigation was completed, Stone et al. [4] reported that cyclic AMP and cyclic GMP in the pyramidal tract neurons in the rat cerebral cortex functioned as reciprocal intracellular second messengers for norepinephrine and actycholine, respectively. Cyclic AMP as norepinephrine was shown to inhibit neuronal firing in PT cells, while acetylcholine and cyclic GMP excited neuronal activity. It was stated that this study [4] was the first to report functional evidence favoring the Yin-Yang hypothesis in a vertebrate central nervous system. The role o f cyclic nucleotides (cyclic AMP and GMP) in the sucrose sensitive receptor of the blowfly is n o t yet clear. These compounds are definitely exerting opposite effects, though their exact mode of action remains to be determined. The olfactory receptor model previously proposed by Riddiford [23] and its analogy to the hormone (or neurotransmitter) cyclic AMP system has much appeal. The alternative mode o f action, that of modulation o f certain events surrounding receptor potential electrogenesis remain quite plausible. While this d i c h o t o m y remains unsettled and the results presented are too preliminary to define a model for chemoreceptor function, the data do suggest a push-pull type of system for cyclic nucleotide action, in the blowfly sensillum, is definitely possible. References 1 G o l d b e r g , N . D . , O ' D e a , R . F . a n d H a d d o x , M.IC ( 1 9 7 3 ) in A d v a n c e s in C y c l i c N u c l e o t i d e R e s e a r c h ( G r e e n g a r d , P., a n d R o b i s o n , G . A . , eds), Vol. 3, pp. 1 5 5 - - 2 2 3 , R a v e n Press, N e w Y o r k

220

2 Krause, E.G., Halle, W. a n d Wollenberger, A. ( 1 9 7 2 ) i n Advances in Cyclic N u c l e o t i d e R e s e a r c h . Physiology a n d P h a r m a c o l o g y o f Cyclic AMP ( G r e e n g a r d , R., Paoletti, R. a n d Robison, G.A., eds.), Vol. 1, pp. 3 0 1 - - 3 0 5 , R a v e n Press, New York 3 McAfee, D.A. a n d G r e e n g a r d , P. ( 1 9 7 2 ) Science t78, 3 1 0 - - 3 1 2 4 Stone, T.W., T a y l o r , D.A. a n d B l o o m , F.E. ( 1 9 7 5 ) Science 1 8 7 , 8 4 5 - - 8 4 7 5 B i t e n s k y , M.W., Miki, N., Marcus, F.R. a n d Keirns, J.J. (1973) Life Sci. 13, 1 4 5 1 - - 1 4 7 2 6 K u r i h a r a , K. ( 1 9 7 2 ) F E B S Lett. 27(2), 2 7 9 - - 2 8 1 7 K u r i h a r a , K. ( 1 9 7 2 ) Biochem. B i o p h y s . Res. C o m m u n . 4 8 ( I ) , 3 0 - - 3 4 8 S m i t h , J.J.B. a n d Friend, W.G. ( 1 9 7 2 ) J. Inst. Physiol. 18, 2 3 3 7 - - 2 3 4 2 9 G e n t l e m a n , S. a n d Mansour, T. ( 1 9 7 4 ) Biochim. Biophys. A c t a 3 4 3 , 4 6 9 - - 4 7 9 I 0 V a n d e Berg, J.S. ( 1 9 7 5 ) J. Inst. Physiol. 2 1 , 4 5 5 - - 4 6 2 11 H o d g s o n , E.S., Lettvin, J.Y. a n d R o e d e r , K.D. ( 1 9 5 5 ) Science 122, 417 12 Wilczek, M, ( 1 9 6 7 ) J. Morphol. 122, 1 7 5 - - 2 0 1 13 Price, S. ( 1 9 7 3 ) Nature 241, 5 4 - - 5 5 14 Breckenridge, B.McL., Burn, J.H. a n d M a t s c h i n s k y , F.M. ( 1 9 6 7 ) P r o c . Natl. Acad. Sci. U.S. 57, 1 8 9 3 - 1897 15 Krishna, G., Weiss, B. a n d Brodie, B. ( 1 9 6 8 ) J. P h a r m a c o l . Exp. Ther. 48, 3 0 - - 3 4 16 Siggins, G.R., H o l l e r , B.J. a n d B l o o m , F.E. ( 1 9 6 9 ) Science 165, 1 0 1 8 17 Siggins, G.R., Oliver, A.P., Hoffer, B.J. a n d Bloom, F.E. ( 1 9 7 1 ) Science 171, 1 9 2 - - 1 9 4 18 Miller, W.H., G o r m a n , R.E. a n d Bitensky, M.W. ( 1 9 7 1 ) S c i e n c e 174, 2 9 5 - - 2 9 7 19 Miki, N., Keirns, J.J., Marcus, F.R. a n d B i t e n s k y , M,W, ( 1 9 7 4 ) E×O. Eye Res. 1 8 , 2 8 1 - - 2 9 7 20 Wulff, V.J. ( 1 9 7 3 ) Vis. Res. 13, 2 3 3 5 - - 2 3 4 4 21 H a r d m a n , J.G., R o b i s o n , G.A. a n d S u t h e r l a n d , E.W. ( 1 9 7 1 ) A n n u . Rev. Physiol 33, 3 1 1 - - 3 3 6 22 V a n d e Berg, J.S. ( 1 9 7 4 ) E x p e r i e n t i a 30, 1 0 2 5 - - 1 0 2 6 23 R i d d i f o r d , L.M. ( 1 9 7 1 ) in G u s t a t i o n a n d O l f a c t i o n (Ohloff, G. a n d T h o m a s , A.F., cds), pp. 3 8 2 - - 3 8 4 , A c a d e m i c Press, New Y o r k