Associative conditioning tunes transient dynamics of early olfactory processing

Abstract

Odors evoke complex spatiotemporal responses in the insect antennal lobe (AL) and mammalian olfactory bulb. However, the behavioral relevance of spatiotemporal coding remains unclear. In the present work we combined behavioral analyses with calcium imaging of odor induced activity in the honeybee AL to evaluate the relevance of this temporal dimension in the olfactory code. We used a new way for evaluation of odor similarity of binary mixtures in behavioral studies, which involved testing whether a match of odor-sampling time is necessary between training and testing conditions for odor recognition during associative learning. Using graded changes in the similarity of the mixture ratios, we found high correlations between the behavioral generalization across those mixtures and a gradient of activation in AL output. Furthermore, short odor stimuli of 500 ms or less affected how well odors were matched with a memory template, and this time corresponded to a shift from a sampling-time-dependent to a sampling-time-independent memory. Accordingly, 375 ms corresponded to the time required for spatiotemporal AL activity patterns to reach maximal separation according to imaging studies. Finally, we compared spatiotemporal representations of binary mixtures in trained and untrained animals. AL activity was modified by conditioning to improve separation of odor representations. These data suggest that one role of reinforcement is to "tune" the AL such that relevant odors become more discriminable.

Figure 1.

Transitions in ratios of binary mixtures define smooth transitions in odor perception. A, Top row of boxes represents a schematic of the experimental protocol. Bees were trained to the CS+ = 10:0 (pure 1-hexanol) and CS− = 0:10 (pure 2-octanone) or vice versa over the indicated number of trials. During training (white boxes) bees were reinforced with 0.4 μl of 2 m sucrose on every CS+ trial; CS− trials were unreinforced. The two graphs to the left show percentage of proboscis extension at each trial during training. The top and bottom graphs show two different groups in which the odors were counterbalanced as the CS+ and CS−. During testing (gray boxes to the right), different ratios of odors (1-hexanol: 2-octanone) were presented unreinforced in a randomized order. The graph shows the duration of the proboscis extension response (see Materials and Methods) during testing (mean ± SE) as a function of different test ratios. Dotted versus solid lines represent responses from the two groups shown to the left. Two-way repeated measures ANOVA was performed for the following factors: ratio, NS; conditioning treatment, NS; interaction ratio × treatment, p < 0.001, N = 19 bees for 10:0+, 19 bees for 0:10+. B, top row, Schematic of experimental protocol for bees trained either CS+ = 9:1, CS− = 1:9 or vice versa. White boxes intermingled with test trials indicate retraining trials (see Materials and Methods). Left graphs represent percentage proboscis extension during training; retraining trials performed in between recall testing (right) are separated by the gray line. Right graphs represent duration of proboscis extension response during testing (mean ± SE) as a function of different test ratios. Two-way repeated measures ANOVA was performed as above: ratio NS; conditioning treatment, NS; ratio × treatment p = 0.006, N = 28 bees for 9:1+, 32 bees for 1:9+. In all cases, sampling time (odor delivery time) was 4000 ms for both conditioning and testing.

Figure 2.

Excitation and inhibition interact to produce to produce a peak shift in the response. Top, Schematic of experimental protocol for bees trained to the CS+ using a 5:5 mixture and CS− either 1:9 or 9:1. Test odors were presented in random order and intermingled with retraining trials. Bottom. Duration of the proboscis extension response for 5:5+ versus 9:1− (dotted line) and 5:5+ versus 1:9− (full line) as a function of different 1-hexanol: 2-octanone test ratios. Sampling time (odor delivery time) was 4000 ms for both conditioning and testing. Two-way repeated measures ANOVA was performed for the following factors: test ratio, NS; (GLM a priori contrasts between 3:7 and 9:1; p = 0.01); conditioning treatment, NS; interaction ratio × treatment, p = 0.02; (GLM a priori contrasts between 3:7 and 9:1; p = 0.0004). (GLM = general linear model). N = 36 bees for 5:5+ versus 1:9−; and N = 39 bees for 5:5+ versus 9:1−. Acquisition curves are shown in supplemental Figure S1, available at www.jneurosci.org as supplemental material.

Figure 3.

Neural representation of binary mixtures 1-hexanol: 2-octanone in PNs of the AL showed a gradient of activation along different ratios. A, Anatomical view (raw fluorescence) of the antennal lobe (left) and correlated image (right) (see Materials and Methods) showing the 17 glomeruli identified in this study. B, Color-coded odor response patterns across the dorsal glomeruli in the AL from a representative individual honeybee. PNs were labeled by backfilling with fura 2-dextran. All the ratios from 1-hexanol (10:0) to 2-octanone (0:10) are shown. The maps are represented according to a common intensity scale (right). Each image represents an average of the glomerular activity between 375 and 625 ms after odor onset. C, Glomerular response delta (340/380) percentage of selected individual glomeruli (mean ± SE, N = 9) to different ratios of test odors. Mean of glomerular activity between 325 and 625 ms after stimulus onset is shown. In all cases asterisks indicate significant differences by repeated measures ANOVA (*p < 0.05, ***p < 0.001).

Figure 4.

Spatiotemporal response patterns show a smooth transition along ratios from one single component to the other. A, Top and middle panel, Odor responses (% delta 340/380) in eight representative glomeruli to ratios 9:1 (top) and 1:9 (bottom) over 125 ms time steps from just before odor delivery through 2750 ms for a control bee. Different colors correspond to different glomeruli (see legend). Stimulus duration (1000 ms) is marked by the shaded area. Bottom panel, Euclidean distance 9:1 to 1:9 over time for the same control bee. B, Left, Odor specific trajectories for two control bees; seven of nine bees follow the same general pattern. To generate this figure the original 17-dimensional space has been projected onto the first two principal components for each bee. Under these conditions, 86.2–97.5% of the variance is explained for each bee. All ratios were presented to each bee and Ca²⁺ transients were recorded at fixed time intervals (125 ms). Accordingly, the distance between different colored data points represents the divergence of the odor representations over time. Trajectories depart rapidly from baseline and slow down when they approach odor-specific regions. PC1 and PC2 indicate principal component factors 1 and 2 respectively. Right, PCA of odor-evoked activity patterns for a control “average” bee obtained by averaging 9 control bees by the activity of 17 glomeruli along 27 125 ms-time intervals. The response of each glomerulus was used as a dimension for the analysis. C, Euclidean distances between ratio 10:0 (pure 1-hexanol) and all the other test ratios based on a 17-dimensional space over 125 ms time steps from just before odor delivery through 3000 ms after odor onset. Stimulus duration (1000 ms) is marked by the shaded area.

Figure 5.

Differential conditioning increases the distance between spatiotemporal patterns. Worker honeybees were differentially trained to 9:1+ and 1:9−. Nine hours after conditioning, brains were treated as above with fura-2 and 8–12 h later were imaged as described in Materials and Methods. These bees were evaluated in parallel to the untrained “control” bees reported in Figure 4. PCA analysis was done jointly for untrained and trained animals, allowing a direct comparison of the two groups. A, Left, Odor specific trajectories for two trained bees. Seven of eight bees follow the same pattern. The PCA of odor-evoked activity patterns account for 88.2–95.4% of the variance for each bee. PC1 and PC2 indicate principal component factors 1 and 2 respectively. Right, PCA of odor-evoked activity patterns for a trained average bee (N = 8). The relative positions of odor representations between trained and control groups (compare with Fig. 5B, right) suggest the modification of odor representation through conditioning. The first two components account for 97% of the whole variance of the trajectories. B, Relative euclidean distances between ratios 9:1 and 1:9 (i.e., CS+ vs CS−) based on a 17-dimensional space over 125 ms time steps from just before odor delivery through 3000 ms after odor onset. Stimulus pulse (1000 ms) is marked by the shaded area. Close and open symbols indicate respectively trained and untrained bees. Asterisks indicate significant differences between trained and control bees (Mann–Whitney U test, p < 0.05).

Figure 6.

Training times <500 ms lead to poor discrimination when test time does not match training time. A, Schematic of experimental protocol for bees trained to CS + 9:1 and CS− 1:9 (ratio 1-hexanol: 2-octanone). t_TR indicates odor sampling time during training (either 200, 500, 1000, or 2000 ms). During testing trials, the odors were presented in a random sequence. SV indicates stimulation by solvent. Thus, each set of honeybees was trained using one sampling time and then tested with each of the four times. Acquisition curves for each set are shown in supplemental Figure S2, available at www.jneurosci.org as supplemental material. Sample sizes were: N = 37 for 200 ms, N = 30 for 500 ms, N = 39 for 1000 ms, and N = 40 for 2000 ms. B, Duration of the proboscis extension response when odor sampling time during training matched test time, i.e., honeybees were trained and tested with the same sampling time. Hatched bars indicate response levels during the test with 10 μl of solvent (mineral oil). C, Duration of the conditioned response for all combinations of training and testing times (i.e., bees were trained with a specific stimulus length and tested with the same or with a different one). Gray bars denote data already shown in Figure 3B (presented here for comparison). In all cases, asterisks indicate significant differences by Wilcoxon matched pairs test between 9:1 and 1:9 (*p < 0.05, **p < 0.005).