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Physiology & Behavior, Vol. 61, No. 1, pp. 107-117, 1997 Copyright 1996 Elsevier Science Inc. Printed in the USA. All rights reserved 0031-9384/97 $17.00 + .00

ELSEVIER

PII S0031-9384(96) 00357-5

Olfactory Conditioning in the Honey Bee, Apis mellifera: Effects of Odor Intensity
S E E T H A B H A G A V A N 1 A N D B R I A N H. S M I T H

Department o f Entomology, 1735 Neil Ave, Ohio State University, Columbus, O H 4 3 2 1 0 - 1 2 2 0 U S A

R e c e i v e d 4 M a r c h 1996; accepted 23 J u l y 1996 BHAGAVAN, S. AND B. H. SMITH. Olfactoryconditioning in the honey bee, Apis mellifera: Effects of odor intensity. PHYSIOL BEHAV 61( 1 ) 107-117, 1997.--Any odor-guided behavior might require generalization and/or discrimination over a wide range of odorant intensities. Proboscis extension conditioning (PEC) and electroantennogram (EAG) assays were used to investigate stimulus-intensity dynamics during olfactory processing in the honey bee. Experiments that tested generalization involved conditioning to one odorant concentration and either testing with a different odorant or with different concentrations of the same odorant. At low training concentrations, responses to either a novel odorant or to higher concentrations of the same odorant resulted in strong generalization. At higher training concentrations, significantly less generalization was observed to a novel odorant or to lower concentrations of the same odor. EAG analyses indicate that asymmetric generalization could arise due to long-term adaptation of peripheral receptor neurons. Discriminationexperiments showed that relatively higher odorant concentrations associated with an appetitive reinforcer could usually be discriminated from a lower concentration that was associated with punishment, but not vice versa. Although sensory modulation in peripheral (sensory) processes might be sufficient to account for discrimination of a high from a low concentration, discrimination of low from high concentrations point to the involvement of central processes. Copyright 1996 Elsevier Science Inc. Olfaction Sensory adaptation Proboscis extension conditioning Stimulus generalization Honey bee Discrimination Electroantennogram

GENERALIZATION among similar but nonidentical stimuli can be an integral part of an animal' s survival strategy ( 15,21 ). From a behavioral standpoint, generalization refers to the tendency to respond to a stimulus that differs from the Conditioned Stimulus (CS) along a defined dimension (10) (e.g., intensity, frequency, etc.). Theoretically, strong generalization responses arise because a test stimulus activates neural representations that are similar in spatial and/or temporal pattern to those activated by the CS itself (16). An underlying gradient of similarity of neural representations would, in turn, generate a gradient of behavioral responses; the nighest level of response would occur to the CS, and increasing decrements in the response level would be evident toward progressively less similar test stimuli. Discrimination of differentially reinforced conditioned stimuli (e.g., a C S + and a C S - ) that differ along a defined stimulus dimension can be accounted for by a similar mechanism. Although discrimination produces a gradient of excitation around the representation of the CS+, an inhibitory gradient similar to the one just described is also established around the representation of C S - . The two gradients can interact if the C S + and C S - are similar enough in representation such that the gradients taper into one another (13). Therefore, a great amount of information can be gained about an animal's sensory system by studying patterns of stimulus generalization under a variety of training conditions (10,13).

Changes in patterns of generalization when training conditions are altered can indicate modulation of one or more elements in neural processing of the stimuli. For example, steep (very little generalization) vs. flat (strong generalization) gradients after excitatory conditioning (i.e., no explicit discrimination is reinforced by use of a C S - ) might occur due to neural representations set up across sensory afferents or in more central neuropils. Steeper gradients after discrimination conditioning might indicate the interaction of excitatory and inhibitory gradients within the central nervous system. Furthermore, the ability to discriminate a high intensity CS+ from low intensity C S - , but not vice versa, would indicate an inability to form explicit associations with different parts of the underlying neural representations that are active at different concentrations. That is, if the representation activated by the low-intensity CS is qualitatively different from that activated by a nigh-intensity CS, discrimination should be possible. On the other hand, if the low CS representation is qualitatively a subset of the nigh CS, the discrimination might be more difficult. We explicitly investigated these latter problems in regard to odorant-intensity discrimination in the honey bee. The effect of odor concentration on acquisition and generalization following excitatory or discrimination training was examined. Finally, an electroantennogram (20) assay was used to determine if gener-

] Reprint requests should be addressed to Seetha Bhagavan, Institute for Cognitive and Computational Sciences, The Research Building - EP07, Georgetown University Medical Center, 3970 Reservoir Rd., NW, Washington DC 20007-2197 USA. 107

108

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Excitatorv Conditioning, 100 8O 60 40 Z Z X l-Hexanol

Generalization Test 100 80

Excitatory Conditionj~ag l -Hexanol

Generalization Test

Training concentration

- - - LOW INT HIGH

- -

g
Z e z ~

60
40

Trainingconcentration LOW INT -HIGH

-

- -

20
0
I I I I I ! I I

2o
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I I I I I I I i I

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0.0002

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Geraniol 80 60 4O 20
I I I l | I

80 60 40 20

/
er
I

. . . . .

Training trial

Conditioned Test stimulus

Novel

0.0002

0.0200

2.0000

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Test concentration (M)

FIG. 1. Stimulus acquisition and generalization across odorants as a function of concentration. Acquisition responses (left set of graphs) to different training concentrations of an odorant during excitatory conditioning in 6 independent treatment groups. Extinction responses (right set of graphs) to alternate presentations of conditioned and novel odorants at the training concentration were tested 1 h after conditioning. Geraniol served as the novel odorant when subjects were trained to 1-hexanol (top) and vice versa (bottom). Training concentrations are 0.0002 M (LOW), 0.02 M (INT), and pure odorant (HIGH) concentrations. Sample sizes for each graph at 0.0002, 0.02, and pure odorant concentrations are 40, 40, and 48 subjects, respectively.

FIG. 2. CS intensity effects on acquisition and generalization gradients across concentrations of the conditioned odorant. Same as in Fig. 1, except HIGH was 2.0 M concentration and, during extinction trials, subjects from each treatment group were tested with 0.0002, 0.02, and 2.0 M concentrations of the conditioned odorant. Test concentration presentations were pseudorandomized during the generalization test. Sample sizes at 0.0002, 0.02, and 2.0 M training concentrations are 30, 29, and 30 subjects, respectively, for the top set of graphs and 28, 29 and 30 subjects, respectively, for the bottom set of graphs. feeding) on the following morning were used for the experiment on that same day. Proboscis extension conditioning. Subjects that were motivated on the morning of the experiment were assayed for responses to odors using the proboscis extension conditioning paradigm (4,12). Two hours before an experiment, odor cartridges were prepared by placing either 3 #1 of a pure odorant or 5 #1 of an odorant diluted in hexane onto a strip of filter paper. This odor-laden filter paper was then inserted into a 1-ml tuberculin glass syringe. All trials were begun by placing a subject into the training arena, through which a slowly moving airstream was vented into an exhaust hood. Odor (conditioned stimulus, CS ) was delivered 30 s later by shunting air through the odor cartridge for 3 s by opening flow-valves via a computer-controlled relay. Using a Gilmont micrometer glass syringe (Gilmont Instruments, Barrington, IL), 2 #1 of 2 M sucrose solution (unconditioned stimulus, US ) was presented first to the antennae and, following subsequent proboscis extension, to the proboscis. Animals were fed for 2 s. In discrimination experiments, the appetitive US was 2 M sucrose solution, and 3.0 M salt solution drawn into a 1-ml glass tuberculin syringe served as the aversive US. The aversive US was presented to the antenna, but the subjects were never fed with the salt solution. Most experiments utilized forward-pairing conditioning trials, as just described, during which the onset of the CS preceded the presentation of US by 2 s. In addition to forward-pairing trials, several other types of trials were used in different subject groups for E A G 2 experiments. In back-

aiization gradients following excitatory conditioning might result, at least in part, from adaptation in the peripheral sensory system. METHODS

General Methods Subjects. Worker honey bees were collected from an outdoor colony during the summer and from a colony maintained in an indoor flight-room during the winter, These colonies were established with a single open-mated queen. For experiments conducted in summer, pollen foragers identified by the pollen on their hind legs were collected as they returned to the colony. In experiments performed in winter, bees were collected as they flew toward a light source above the flight cage and landed on the ceiling of the cage. Workers were collected individually in glass vials during early afternoon on the day prior to the experiment, and the vials were placed shortly thereafter in an ice-water bath. As soon as a bee stopped moving it was restrained in a small metal harness by placing a strip of duct tape between the head and thorax. Bees were fed 30 min later with a 2.0-M sucrose solution until satiation and left overnight. Only bees that vigorously extended their proboscides when tested for motivation (by presenting sucrose only to their antenna and not followed by

2 Abbreviations: PEC, proboscis extension conditioning; EAG, electroantennogram.

ODOR INTENSITY EFFECTS ON OLFACTORY CONDITIONING

109

0.50

0.22

1- H e x a n o l
0.45 ~-~ 0.40 ;> 0.35 0
O No odor exposure Forward-pairing

Geraniol 0.20 0.18 0.16 0.14

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FIG. 3. Electroantennogram responses to different concentrations of an odorant (listed top left in each graph) in 2 independent treatment groups. One group of 20 subjects received no prior conditioning experience and the second group of 20 subjects were conditioned to 2.0 M concentration of the odorant over 6 forward-pairing trials.

ward-pairing groups, the US preceded the CS by 1 s. In the unpaired group, subjects received explicitly unpaired presentations of CS and US in 2 successive repeats of the following sequence: US, CS, CS, US, CS, US, US, CS. In this group, trials consisted of presentation of the CS or the US on each trial, but never both together. In CS-only groups, subjects received only 3 s odor stimulation. Subjects in the US-only groups received only 2 s sucrose presentations, first to antenna and, finally, followed by feeding. To maintain the same number of total trials in all groups (i.e., placements into the training arena), subjects in forward-pairing, backward pairing, C Sonly and US-only groups received placement (P) trials. During the placement trials, subjects were placed in the training station, but received no stimulation with odorants or the US. These trials were interspersed with stimulation (S) trials in 2 successive repeats of the following sequence: S, P, P, S, P, S, S, P. After conditioning was complete, a series of extinction trials, during which subjects received only odor presentations, was used to test responses to odorants. The sequence of presentation of test odorants across subjects was alternated over 2 test trials or pseudorandomized over 3 test trials. The intertrial interval (ITI) for any given experiment was the same for the acquisition and extinction phases using the PEC paradigm. Subjects were always

tested 1 h after they were trained, to minimize nonassociative effects such as sensitization. EAG recordings. Electroantennograms were obtained from antennae of isolated heads using the protocol described in detail elsewhere (3). Briefly, the cut tip of one antenna was inserted into a glass pipette that was filled with saline and was in contact with the recording electrode. The other antenna was removed to prevent disturbance due to movement. A ground electrode was inserted into the saline-filled petri dish. Electrodes were connected to a DC amplifier and the unfiltered output was amplified ( 10 ) and fed into an oscilloscope for viewing responses. Odor cartridges were prepared by placing 5 #1 of an odorant diluted in hexane to obtain a given concentration on a strip of filter paper. The latter was then inserted into a 2 inch Teflon tube ( 5 / 3 2 " - i.d.) that was capped with a barbed polypropylene fitting. These cartridges were replaced with new ones after 5 stimulations with each cartridge to control for stimulus depletion over time. For EAG recordings, the antenna was continuously subjected to air at a rate of 97 ml/min to adapt out the mechanosensory receptors. During stimulation, the rate of air flow through a cartridge was 129 ml/min. Background recordings to air and solvent were obtained by shunting air through a cartridge that was either empty or carried a strip of filter paper with solvent on it, respectively.

110

BHAGAVAN AND SMITH

0.48
1-Hexanol

0.30
O No odor exposure Forward-pairing Odor (CS) only Unpaired exposure

i
Geraniol
O V [] No odor exposure Forward-pairing Backward-pairing Odor (CS) only Sucrose (US) only Unpa~-ed exposure

0.44 0.40 > 0.36 r~ 0.32 Z ca., 0.28 r/) ~ r~ 0.24

0.28 0.26 0.24 0.22 0.20 0.18 0.16 0.14 -

~7 Backward-pairing [ ] Sucrose (US) only

0.20 0.16 0.12 0.08

0.12
0.10 0.08

0.06 0.04 0.04 ~ 0.00 0.001 0.01 0.1 0.02 0.00

|ll
0.001

,1

nn,,,i 0.01 0.1

Concentration (M)

Concentration (M)

FIG. 4. EAG responses to different concentrations of an odorant in 12 independent treatment groups. For each odorant (listed top left in each graph), 5 treatment groups of 12 subjects each were conditioned to 2 M concentration of odorant using either forward-pairing trials, backwardpairing trials, CS only, US only, or explicitly unpaired presentationsof CS and US. One treatment group of 12 subjects that received no conditioning prior to testing served as control.

Each antenna received 6 recorded stimulations, each lasting for 1 s. The initial 2 stimulations with air and solvent were for determination of background EAG responses. Test odorants were presented in a pseudorandomized sequence. The last stimulation with solvent was to determine any change in background response from that recorded earlier in the test sequence. A 2-min interval separated each of these 6 stimulations. Odorants. Odorants used in this study were geraniol and 1hexanol (99% purity; Sigma, St. Louis, MO). Geraniol is a monoterpenoid component of the honey bee Nasonov pheromone (17), as well as a common component of floral odors (11). These odorants were serially diluted in hexane from 2.0 M stock solutions to obtain low, intermediate, and high (0.0002, 0.02, and 2.0 M, respectively) concentrations for this study. The highest concentration of each odor reaches saturation in EAG analyses, whereas the lower concentrations yield EAG responses that are above background, but below saturation (3). In one set of experiments, the pure odorant was used. Statistical analyses. Acquisition and EAG data were analyzed using nonparametric statistical tests (22). Comparisons between summed numbers of conditioned responses (proboscis extension to CS prior to US onset) for each subject across 6 acquisition trials were made between different treatment groups. For exam-

pie, each subject could have 0 to 6 responses, depending on the number of trials during which it responded. For EAG data, response to an odor stimulation was subtracted from the response to the solvent for each subject within a group, and these differences were averaged for each treatment group. The response to an odor stimulation was recorded as zero when the response to the solvent slightly exceeded the response to odorant stimulation. All analyses of EAG data compared these adjusted responses in each treatment group. Mann-Whitney (U) or Kruskal-Wallis (H) test analyses were used for comparing differences in responses between 2 or 3 groups, respectively. For data obtained from extinction trials, we analyzed the percent proboscis extension responses. Subjects within each group for an experiment were classified according to whether or not they responded to the test stimulation. To test if the degree of association between the response (C) to a test odorant (B) differed for different training conditions (A), a log-linear model (AB. AC- BC; three-factor effects) (22) was fitted. According to this model, a significant test statistic (Pearson's chi-square), which denotes the 3-factor interaction term, denotes that the degree of association (interaction) between any pair of factors would depend upon the different levels of a third factor. In addition, for data in Fig. 2 (top panel), Pearson's chi-square anal-

O D O R INTENSITY EFFECTS ON O L F A C T O R Y C O N D I T I O N I N G

111

100 90 80 70 60 50 40 30

Geraniol

----

0.02 M 2.00M

o
Z X

20 10 0
1 2 3 4 5 6 7 8 9

10

100 90 80 70 60 50 40 30 20 10 0

1-Hexanol
- - -

0.02 M 2.00M

i|

10

Trial FIG. 5. Acquisition curves for discrimination conditioning to a low concentration of an odorant associated with an appetitive reinforcer (straight lines) and a high concentration of the same odorant associated with an aversive reinforcer (broken lines). Differentially reinforced concentrations are indicated in each graph. Top, geraniol (n = 23); bottom, 1hexanol (n = 25).

Discrimination experiments. In the following set of experiments, subjects received 20 forward-pairing trials (ITI = 3 min). For 10 of those conditioning trials, a given concentration of an odorant was followed by sucrose feeding ( C S + ) . In the remaining 10 trials, a different concentration of the same odorant was followed by presentation of salt solution to the antenna ( C S - ) . The sequence of presentation of CS + and C S - was identical for each subject: 2 and 1 half successive sequences of C S + , C S - , C S - , C S + . C S - , C S + , C S + , C S - . Nine subjects were tested daily and odors were counter-balanced over a 2-day period. To prevent decrease in stimulus concentration due to use of the same odor cartridge over 20 trials, each odor cartridge was replaced after use for 10 trials. In all but the initial set of discrimination experiments, subjects were tested with solvent and 2 novel concentrations (0.002 and 0.00002 M ) of the same odorant beginning 1 h after completion of conditioning. PEC - EAG experiments. An experiment was performed to test whether or not behavioral effects on generalization gradients might be due to modulation of sensory receptors. Four independent groups of subjects were tested with geraniol or 1-hexanol, using the EAG protocol. Two groups of subjects were conditioned to 2.0 M concentration of geraniol or 1-hexanol over 6 forward-pairing trials prior to being tested. The ITI was 6 rain, as in the second set of generalization experiments. The second group of subjects was tested for EAG responses without prior conditioning to 1-hexanol or geraniol. Ten subjects (5 per group) were used daily. Geraniol and 1-hexanol were tested on alternate days. The experiment was repeated in summer with 4 additional conditioning groups (backward-pairing, CS only, US only, and an unpaired group) for each odorant. The CS, either geraniol or 1-hexanol at 2 M concentration, was presented at 6 rain ITIs as in the second generalization experiment described above. Twelve subjects (2 per group) were tested daily. As in the previous experiment, 1-hexanol and geraniol were tested on alternate days. Subjects that received any type of training were tested for EAG responses 1 h later.
RESULTS

yses (22) were performed to test the association between extinction responses under different training condition to the intermediate test concentration. All analyses were performed with S Y S T A T (version 5.0) for the Macintosh.

Acquisition and Generalization Across Concentrations

The first set of generalization experiments measured the effect of training odorant concentration on generalization from the CS to a different odorant (Fig. 1 ). Subjects in 6 independent treatment groups acquired the conditioned response to all concentrations of 1-hexanol and geraniol within a few trials (left set of graphs). Levels of conditioned responding were not different between subjects trained to pure odorant, intermediate, and low concentrations of 1-hexanol (top panel; H = 0.907, NS) or geraniol (bottom panel; H = 1.042, NS). That is, all lines reach the same approximate asymptotic level of responding by about the second or third trial. Extinction responses (Fig. 1, right set of graphs) across treatment groups measured 1 h after acquisition present a somewhat different picture. When 1-hexanol was the conditioning odorant, there was no significant interaction between different training concentrations and the responses to conditioned and novel (geraniol) odorants (top panel, (X 2 = 8.08, N S ) . That is, the 3 lines are statistically parallel even though there is a trend toward a higher response to 1-hexanol when the high training concentration was used. When geraniol was used for conditioning (bottom panel), there was significant interaction between the different training conditions and the responses to conditioned and novel (l-hex-

Experimental Designs Generalization experiments. The first set of experiments tested the effect of training concentration on generalization to a different odorant. Subjects collected in summer were trained in 6 forward-pairing (ITI = 30 s) trials. Pure odorant, 0.02, and 0.0002 M concentrations of 1-hexanol or geraniol were used as the CS in different groups of subjects. During extinction trials, subjects within each of the 6 groups were tested twice, once each with 1-hexanol and geraniol at the CS concentration. The second set of experiments tested generalization across a range of concentrations of the conditioned odorant. Subjects from different groups were trained in 6 forward-pairing trials to geraniol or 1-hexanol. In this experiment, we used a 6-min ITI to match the ITI used in E A G analyses. The CS was a 0.0002, 0.02, or 2.0 M concentration of odorant. One hour after conditioning, subjects from each group were tested with 0.0002, 0.02, and 2.0 M concentrations of the same odorant that was used during the training phase.

112

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AND SMITH

Geraniol L+/H- Discrimination

-

_--

8.

.8

90&M

Generalization test
100 90 80 100 80 B ___ solvent 0.0002 M 70 60 50 40 30 20 ___ solvent 0.02 M 10 0
Training experience

I
:;

l C

solvent

o.obOO2

0.002

Test concentration

(M)

Trial
FIG. 6. Acquisition curves for discrimination conditioning to geraniol at appetitively reinforced LOW concentrations (straight lines) and aversively reinforced HIGH stimulus concentrations (broken lines). (A) n = 15; (B) n = 15; (C) n = 30. Extinction responses (graph on right) to pseudorandomized presentations of solvent and 2 novel concentrations (0.00002 and 0.002M) were recorded from each treatment group 1 h after discrimination conditioning.

anol) odorauts (x2 = 11.06, p < 0.05). In this case, the lines are not parallel. After training to an intermediate or high concentration of geraniol, the response to geraniol is higher than the response to the novel (1-hexanol) odorant. However, when the low training concentration was used, the gradient is flat. The second set of generalization experiments measured generalization across concentrations of the conditioned odorant (Fig. 2). Summed conditioned responses in the acquisition phase did not differ between 2 different groups of subjects trained to low and intermediate concentrations of 1-hexanol (top left set of graphs; LOW vs. INT: U = 439.5, NS). However, conditioned responding at either of those concentrations was significantly lower than the acquisition responses to high concentration of lhexanol (LOW vs. HIGH: U = 434.5, p < 0.05; INT vs. HIGH: U = 415.0, p < 0.05). Responses to geraniol (bottom left set of graphs) at the low concentration were significantly lower than responses to intermediate (LOW vs. INT: U = 244.5, p < 0.05)
and to high concentrations 0.05 ) . Acquisition curves (LOW vs. HIGH: U = 183.0, p < to intermediate and high concentra-

tions of geraniol = 363.0, NS).

did not differ significantly

(INT vs. HIGH: U

To test whether or not differences in acquisition reflect differences in the buildup of associative strength, all subjects were tested with the same concentrations ( 18). There was significant interaction effect between extinction responses (Fig. 2, right set of graphs) to test concentrations given different training conditions, when geraniol was used as the conditioning odorant (bottom panel: (x2 = 9.60, p < 0.05). The generalization gradient across concentrations was flat in the group trained to a low concentration of geraniol. That is, generalization from a low to a higher concentration occurred. However, when trained to an intermediate or high concentration, the generalization gradients were not flat. Subjects did not easily generalize from higher concentrations to a low concentration. For 1-hexanol, although there was no significant interaction between extinction responses to test concentrations across training condition (top panel: (x2 = 6.61, NS), there were significant differences in responses to intermediate test concentration across training conditions (x * = 7.66, p < 0.05). That is, when subjects were trained to a high concentration, the response level to an intermediate test concentration was significantly lower compared to the response levels to the same test concentration after subjects

O D O R INTENSITY EFFECTS ON O L F A C T O R Y C O N D I T I O N I N G

113

1-Hexanol L+/H- Discrimination

100 80 60 40 20 O 0 100 80 60 40
r,g3

Generalization test
100

i
B

Training e x p e r i e n c e

90
80

o A (9 B C
o

r.#3

solvent 0.0002 M

70

[--,

60
50 40 30 20
C 0 0

20 0 100 80 60 40 20 0
I I
I I I I I I I I

solvent 0.02 M

10
0
I I I

solvent

0.00002

0.002

Test concentration (M)

l0

Trial
FIG. 7. Same as Fig. 6, except 1-hexanol was the odorant used in this study, and sample sizes are 15 subjects for each graph.

were trained to low or intermediate concentrations of 1-hexanol. The significance of this effect provides at least some indication of asymmetric generalization such as that observed for geraniol. Lack of significant interaction could be explained by the lack of differences in response levels to low or high test concentrations under different training conditions.

Electroantennograms Based on Conditioning Experience

To test whether the effect of concentration during the acquisition phase on response to test odorant concentrations might be due to sensory adaptation, we compared E A G responses to test concentrations between groups of subjects that received no training and groups that were trained to a high concentration of 1hexanol or geraniol. Subjects conditioned to the high concentration of an odorant over 6 forward-pairing trials showed significantly lower E A G responses to 0.02 M concentration of the same odorant than subjects that did not receive any prior conditioning experience (Fig. 3; 1-Hexanol: U = 273.50, p < 0.05; geraniol: U = 101.00, p < 0.05). There were no significant differences between treatments in E A G responses to 0.0002 or 2.0 M test concentrations of either odorant (1-hexanol: U = 209.00, NS; 170.5, NS, respectively; geraniol: U = 182.0, NS; 209.0, NS, respectively). Therefore, E A G response levels to an

intermediate concentration decreased when subjects were trained to a high concentration of odorant. This decrease in E A G responses to an intermediate test concentration might be due to associative processes involved specifically in associating the high training concentration with reinforcement during forward-pairing conditioning trials. Alternatively, preexposure to a stimulus during these conditioning trials might also result in a nonspecific change in sensitivity of sensory receptors. To test the latter hypothesis, we repeated the experiment using, in addition to the forward-pairing and untrained groups, backward-pairing, CS-only, US-only, and unpaired groups. For 1-hexanol (Fig. 4, left panel), EAG responses to each of the 3 test concentrations were not significantly different across the different treatment groups (Fig. 4, LOW: H = 3.39, NS; INT: H = 1.106; HIGH: H = 3.778, N S ) . However, electroantennogram responses to 0.02 M concentration of geraniol were significantly different (Fig. 4, right panel; H = 14.100, p < 0.05). Subjects that received any kind of exposure to stimuli used for conditioning had significantly lower EAG responses to 0.02 M concentration than subjects from the untrained group (comparisons between E A G responses in untrained and each of the following treatment groups: forward-pairing, U = 88.0, p < 0.005; backward-pairing, U = 84.0,p < 0.05; CS only, U = 86.5,p < 0.05;

114

100
80 60 40 20

B H A G A V A N A N D SMITH

Geraniol H+/L- Discrimination

0.0002 M

Generalization test
"-''''''" 100 90 80 70 60 50 40 30 20 C 0.02 M solvent 10 0
I I I

o z

0
100 80 60 B 0.0002 M solvent

Training experience O A O B

r~ rj
r/)

40 20 0 100

80
60 40 20

solvent

0.00002

0.002

Test concentration (M)

I I I I I I I I I I

Trial
FIG. 8. Same as Fig. 6, except the HIGH concentration of geraniol was appetitively reinforced and the LOW concentration was aversively reinforced. (A) n = 18; (B) n = 15; (C) n = 18.

US only, U = 88.0, p < 0.005; unpaired, U = 73.0, p < 0.10). There were no significant differences in E A G responses to geraniol at 0.0002 or 2.0 M test concentrations between treatments (H = 5.725, NS for 0.0002 M; H = 3.777, NS for 2.0 M ) . Thus, in 3 of 4 replications, E A G responses to a test concentration lower than the training concentration showed a decrement in treatment groups relative to the control (untrained) group. Because this decrement occurred in all groups that received some form of sensory experience, these results are due to a nonassociative process.

Discrimination Conditioning Discrimination of a low CS+ from a high CS-. Discrimination of an appetitively reinforced low concentration (L + ) from an aversively reinforced high concentration ( H - ) of the same odorant was rare, and depended on the magnitude of differences between the 2 concentrations of the conditioned stimuli (Figs. 5, 6A, B, C and 7A, B, C ) . For geraniol, subjects discriminated solvent as L + from geraniol as H - at 0.02 M (Fig. 6C, U = 581.5, p < 0.05), but not other combinations of L + and H (Fig. 5, top panel; U = 240.0, NS; Fig. 6A, U = 74.5, NS; 6B,

U = 115.5, NS). Subjects discriminated between 0.02 and 2.0 M concentrations of 1-hexanol as L + and H - , respectively (Fig. 5, bottom panel; U = 411.0, p < 0.05), but not other combinations of L + and H - (Fig. 7A, U = 116.5, NS; B, U = 115.0, NS; C, U = 100.5, N S ) . In summary, in 1 of 4 cases in which the L + concentration was not a solvent, subjects discriminated between L + and H - concentrations. Furthermore, discrimination was evident in only 1 of 4 cases in which L - was solvent. Discrimination of a high CS+ from a low C S - . Discrimination of appetitively reinforced high concentrations ( H + ) and aversively reinforced low concentrations ( L - ) of the same odorant was more c o m m o n at the same concentrations for either 1-hexanol or geraniol (Figs. 8A, B, C and 9A, B, C ) . Acquisition responses to 0.02 M as H + were significantly higher than that to 0.0002 M as L - (Fig. 8A, U = 277.0, p < 0.05; Fig. 9A, U = 520.0; p < 0.05) and also higher than responses to solvent as L - (Fig. 8C, U = 287.0, p < 0.05; Fig. 9C, U = 239.0, p < 0.05). Subjects did not discriminate between 0.0002 M concentration as H + and solvent as L (Fig. 8B, U = 112.0, NS; Fig. 9B, U = 147.0, N S ) . In contrast to L + / H - discrimination, in 4 of 6 cases H + / L - discrimi-

ODOR INTENSITY EFFECTS ON OLFACTORY CONDITIONING

115

1-Hexanol H+/L- Discrimination

100 80 60 A 0.02 M 0.0002 M

G e n e r a l i z a t i o n test
20
z

100

0
100 80
B - -

"
solvent
0.0002 M

9080706050403020-

Training experience OA
OB C

60
40 20

0
100
80

o~

0.02 M solvent

100I

60 40 20
0
I I I I I I I I I I

solvent

0.00002

0.002

Test concentration (M)

10

Trial
FIG. 9. Same as Fig. 8, except the odorant used in this study was 1-hexanol.(A) n = 28; (B) n = 16; (C) n = 18.

nations were observed. The only 2 cases in which discrimination did not occur used the lowest (0.002 M) concentration of odorant vs. solvent.

Patterns of Generalization After Discrimination Conditioning

The pattern of generalization to solvent and 2 novel concentrations of a conditioning odorant did not result in significant interaction from different discrimination training experience (Figs. 6 - 9 , graphs on the right). Generalization gradients were flat and did not differ to solvent, 0.00002, and 0.002 M concentrations when subjects were trained to discriminate L+ from H concentrations of geraniol (Fig. 6, (X 2 = 1.18, NS ) or 1-hexanol (Fig. 7 ) (X 2 = 4.7, NS). When subjects were trained to discriminate H+ from L - concentrations, generalization gradients tended to increase with concentration, but did so in a statistically identical fashion regardless of the concentration chosen for H+ and L - (Fig. 8, X 2 = 1.22, NS; Fig. 9, X2 = 3.25, NS).
DISCUSSION

A general conclusion from this study is that CS intensity and training conditions during acquisition influence generalization gradients established due to conditioning. That is, lev-

els of conditioning supported by a CS intensity during excitatory or discrimination conditioning yield different patterns of generalization. In the first set of generalization experiments (Fig. 1), CS concentrations did not influence the rates or asymptotes of acquisition during conditioning. Nevertheless, generalization to a novel odorant changed as a function of CS concentration for geraniol, but not for 1-hexanol. In other words, the pattern of generalization shifted from fiat to steep gradients as the training odorant concentration shifted from a low to a high concentration. In contrast, levels of conditioning supported by different CS concentrations were significantly different in the second experiment (Fig. 2). In that experiment, rates of acquisition, and possibly also asymptotic levels of conditioning, differed as a function of CS concentration. However, comparison of response levels during acquisition trials is inadequate as a sole means for judging response strength because subjects are stimulated differently (18). To more completely analyze differences in response strength, subjects must also be tested under a common condition. When that kind of test was performed during the generalization test with the high concentration of the same odorant used for conditioning, subjects still displayed different response levels in 1 case (geraniol). Thus, it is reasonable to conclude that CS

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B H A G A V A N A N D SMITH

concentration can affect the rate and/or asymptotic level of conditioning. An increase in the rate at which the CS enters into an association with the US at a high CS intensity, which is a function of concentration, can be accounted for by models of conditioning (19). However, these models of conditioning do not always account for a difference in asymptotic response levels at different CS intensities. Differences in odor concentration during conditioning influence the expression of generalization to different concentrations of the same odor during generalization testing (Fig. 2). Our data show that, at low training concentrations, generalization to a range of test concentrations resulted in flat gradients. That is, when trained to a low concentration, subjects responded equally well to higher concentrations. On the other hand, at high training concentrations, levels of response to the high concentration were significantly higher than response levels to the lower concentrations, which resulted in steep gradients. Thus, subjects generalize from low to high concentrations but not vice versa. This low-to-high, but not high-to-low, generalization to the same series of test concentrations fits well with development of an adaptation level as proposed by Helson (9). According to this model, an adaptation level is established around the training concentration following conditioning. In essence, the adaptation level means that responses to test concentrations lower than the training concentration are adapted out, and test concentrations higher than the training concentration elicit response levels that are equivalent to that obtained for the training concentration. Such differences in adaptation levels would, therefore, generate fiat gradients to test concentrations higher than the training concentration, and steep gradients to lower test concentrations. This adaptation level could arise in one or more peripheral or central loci. Our EAG data indicate involvement of the antennal system. E A G responses to test concentrations were compared between 1 group of subjects that received no training and another group of subjects that were trained to 1 of the test concentrations. Significantly lower responses were observed in the latter group to a test concentration that was lower than the CS concentration. Further E A G analyses revealed that, depending on the odorant and its concentration range, any kind of sensory experience, including exposure to the CS only, US only, or even unpaired presentations of the CS and the US, results in significantly lower responses to test concentrations lower than the CS concentration. These EAG results argue for a peripheral mechanism of establishing different adaptation levels potentially via a shift in the sensitivity of sensory receptors in the antennal system. Variation in sensitivity of olfactory receptor neurons due to sensory experience has been observed in other studies as well (8,23). Changes in receptor neurons could be due to direct factors, such as intracellular changes in second-messenger systems, protein phosphorylation, or release of nitric oxide, or due to indirect factors such as hormonal changes (1,2,5,6,14). Peripheral mechanisms of sensory modulation might not account for all changes in patterns of generalization resulting from

conditioning experiments. This was demonstrated by investigating the pattern of generalization to a range of concentrations following discrimination conditioning to 2 different concentrations of the same stimulus; 1 concentration associated with an appetitive reinforcer and the other with an aversive reinforcer. The general conclusion from these experiments is that discrimination between 2 concentrations depends on the odorant and the magnitude of difference between the 2 conditioned concentrations of this odorant. There was no significant interaction between responses to test concentrations under either different H + / L - or different L + / H - discrimination training conditions. However, for geraniol, but not for 1-hexanol, the general pattern of generalization shifted from steep gradients following H + / L - discrimination to more or less flat gradients following L + / H - discrimination. The adaptation-level model could be extended to explain generalization gradients for excitatory conditioning, as well as gradients established to H + / L - discrimination conditioning. That is, if an adaptation-level is established around the H + , responses to either L - during acquisition trials or lower test concentrations during extinction trials should be adapted out. Thus, H + / L discrimination should be easier than L + / H - discrimination. This pattern is reflected in our data on discrimination conditioning, where H + / L - discrimination was observed in 4 of 6 instances and L + / H - discrimination was only observed in 2 of 8 cases. On the other hand, the adaptation of peripheral receptors cannot be easily invoked to explain L + / H - discrimination, however infrequent it may be. This result from our data constitutes the strongest evidence that a central mechanism might contribute to the establishment of generalization gradients. For example, relative differences in sizes of active areas within the brain might be involved in the discrimination of a L + from a H - . Such differences in sizes or numbers of neural elements due to differences in CS concentration have been reported in the salamander olfactory bulb (7). Discrimination conditioning studies in vertebrates have focused on interactions between excitatory and inhibitory gradients of associative strength to C S + and C S - to explain generalization gradients to test concentrations. Such interactions may be giving rise to the patterns of generalization seen in our L + / H - experiments. Thus, our data suggest that CS intensity influences the extent of generalization to novel intensities of odor by establishing an adaptation level around the CS intensity. This adaptation level is subject to change following stimulus experience via modulation of sensory receptor sensitivity in the antennal system. Future experiments will be aimed at determining the time-course of longterm adaptation, as well as the rote of other factors in modulating sensory receptor sensitivity that leads to this phenomenon. ACKNOWLEDGEMENTS Supported by a grant from NIMH (to B. H. S.). The authors wish to thank Dr. C. D. Derby and B. Gerber for their valuable comments on an earlier draft of this manuscript.

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