Behavioral response facilitation upon odor repetition.

(a)Schematic of the innate palp-opening response (POR) experimental paradigm. Opening of the locust’s maxillary palps within 15 s of the odor onset was considered a POR.

(b)Schematic of the block odor stimulation protocol. Each block consisted of ten trials and a 4s odor pulse was presented in each trial. A 15-minute no-stimulation period separated the blocks.

(c)Response matrices are shown summarizing individual locust PORs (rows) for blocks of ten trials (columns). White boxes indicate PORs in a specific trial, while black boxes indicate the absence of PORs in that trial. Locusts were sorted such that the least responsive locusts are shown at the top and the most responsive ones are near the bottom. PORs varied between locusts, across trials.

(d)The probability of PORs across locusts (P(POR)) is shown as a function of trial number for two odorants: iaa and bza at high intensities (1% v/v). P(POR)s as a function of trial number is shown for four odorants (oct, hex, iaa, bza) at two different intensities (0.1% v/v – low and 1% v/v – high). The trials were split into early (trials 1-5) and late (trials 6-10) trials. While the P(POR) of both odorant intensities increased over trials, the P(POR) values for high-intensity odor exposures were notably higher than P(PORs) for low-intensity odor presentations.

(f) Bar plots compare the average P(POR) for early and late trials for the low- and the high-intensity presentations of the same odor. P(POR) values observed at high and low intensities of an odorant were similar for early trials (p>0.05, one-tailed t-test). For three out of four odors tested the P(PORs) for high-intensity odor exposures were significantly higher (p<0.05, one-tailed t-test). This suggests that locusts can better perceptually distinguish between high and low odor intensities during later trials.

Ensemble neural activity systematically changes across repeated encounters with an odorant.

(a)Schematic of the olfactory stimulation protocol. Each block consisted of twenty-five trials with a four seconds odor pulse delivered in each trial. The inter-trial interval was 56 s. Two datasets were collected. Each dataset consisted of five randomized blocks of four odorants (dataset 1: hex (H), hex (L), oct (H), oct (L), hex (H)-repeat; dataset 2: iaa (H), iaa (L), bza (H), bza (L), iaa (H)-repeat). A fifteen-minute no-odor stimulation period separated blocks of trials.

(b)Raster plots of eight representative projection neurons (PNs) in the locust antennal lobe are shown. Spiking activities are shown for twenty-five trials (rows) with earlier trials shown at the top and later trials at the bottom. The shaded region indicates the four seconds odor stimulation period, and the identity of the stimulus is indicated in each plot.

(c)Raster plots are shown for four representative PNs during two blocks of trials. The same odorant was presented in both blocks. Note that spiking activity changes are repeatable across the block of trials.

(d) Schematic showing how vesicular depletion and lateral inhibition facilitation models would change spiking activity in individual neurons. Y-axis represents the change in response over trials ((1st trial response – 25th trial response)/ (1st trial response + 25th trial response)). Positive numbers indicate that the first trial had a stronger response, hence response reduction. Negative numbers indicate that the last trial had a stronger response, hence response facilitation. Along the x-axis, the response in the 1st trial is shown. Vesicular depletion should impact the strongly activated neurons more, whereas lateral inhibition facilitation should progressively suppress weak responders.

(e)Following the schematic in panel d, response changes observed in PNs are shown. Only PNs with significant odor-evoked responses are included in this plot. Responsiveness of PN was determined based on whether their peak odor-evoked response in the first trial exceeded the mean + 6.5 SDs of spontaneous activity preceding odor stimulation in that first trial. In total hex (L/H), n = 26, 39 PNs; oct (L/H), n = 35, 28 PNs; iaa (L/H), n = 39, 35 PNs; bza (L/H), n = 33, 40 PNs were included in the plot. Different colors and symbols are used to denote the identity of the odorant and its intensity (low-intensity trials – triangles and high-intensity trials – circles). The dotted line shows the regression fit revealing a linear relationship, if any, between the activation strength in the first trial (x-axis) and the response reduction observed (y-axis). As can be noted the R2 values are low indicating that both vesicle depletion and lateral inhibition facilitation models fail to capture the adaptation trends in our datasets.

(f)Changes in PN spiking activity over trials during high-intensity odor exposures (y-axis) are plotted against response changes observed for the same PN during low-intensity exposures of the same odorant. The dotted line indicates the regression fit revealing a linear relationship between the response changes observed during high and low-intensity exposures of the same odorant (hex, R2=0.034; oct, R2=0.032; iaa R2=0.067; bza R2=0.0016). The poor linear regression fit here indicates that reductions in neural response amplitude for one odor intensity do not model reductions in response amplitude for another odor intensity.

Stimulus repetition and intensity decrements reduce spiking responses.

(a) Peristimulus time histograms (PSTHs) across all PNs (hex and oct, n = 80 PNs; iaa and bza, n = 81 PNs) for each odorant. PSTHs of trials 1 and 25 are shown.

(b) PSTHs across all PNs are compared between high and low-intensity odor exposures. Response during the first trial is shown.

(c) Summed spike counts (across PNs and four-second odor presentation) are calculated and shown as a function of the trial number. The dotted line indicates the spike count of the 25th trial of the high-intensity odor exposure.

Odor identity and intensity information is maintained across trials.

(a)A schematic showing how the ensemble neural activity might change between two odorants and across multiple trials or repetition. The combination of neural activated should differ between different odorants, and therefore the odor identity should be represented by population response vectors that differ in their direction. Repetitions should reduce response strength without altering the combination of neurons activated. If this were the case, the later repetitions of the same odorant would evoke a response that can be represented by vectors that maintain directions while progressively becoming lesser in length or magnitude.

(b)A schematic coding scheme for achieving adaptation invariant intensity coding. The combination of neurons activated changes markedly with odor identity and subtly with odor intensity. These responses become less intense without altering the combination of neurons activated. If this were the case, then the ensemble vector direction would change with both odor identity and intensity, and the vector direction robustly maintained even though the vector length continues to change with repetition.

(c)Trial-by-trial odor-evoked ensemble PN response trajectories after dimensionality-reduction are shown for hex and oct. Only high-concentration exposures of both odorants are included in this plot. For each odor, the temporal response trajectories for the 1st, 5th, 10th, 15th, 20th, and 25th trials are shown (color gradient from light to dark as trial number increases). Note that the odor-response trajectories become increasingly smaller during later trials, but the direction of the trajectories is robustly maintained.

(d)Trial-by-trial response trajectories for low concentrations exposures of the hex and oct are shown. Similar convention as panel c. (f)Similar plot as in panel c, but now comparing the odor-evoked response trajectories elicited by hex at high and low intensities.

(g)Similar plot as in panel d but comparing the response trajectories elicited by oct at high and low intensities.

High and low stimulus intensities activate distinct ensembles.

(a)Correlations between neural responses observed in different trials are shown. Each pixel/matrix-element represents a similarity between mean neural responses in one trial versus those in another trial. Diagonal blocks reveal the correlation between trials when the same odorant at a specific intensity was repeatedly presented.

(b)A dendrogram was generated using a correlation distance metric comparing trial-by-trial ensemble spiking activities evoked by two different stimuli at two different intensities (see Methods). Two major response clusters that correspond to stimulus identity and intensity were identified. The number at the leaf node represents the trial number.

Neural adaptation inversely correlates with behavioral facilitation.

(a) The probability of odor-evoked POR (P(POR)) for a given trial is plotted against the total spike counts elicited during the trial or repetition number. Therefore each point represents a single trial. Symbols with light and dark colors are used for differential trials of high and low-concentration odor exposures. The line represents the regression fit between the behavioral and neural responses (hex (L), R2 = 0.65; hex (H), R2 = 0.79). The negative slope of the regression line indicates that while the neural responses diminish over trials, the behavioral responses increase over trials.

(b-d) Similar plots as in panel a but for oct (L/H), iaa (L/H), and bza (L/H).

Adaptation results in odor-specific neural response reductions and behavioral output facilitation.

(a)Two blocks of trials were used. First, a block of 30 trials where one odorant (hex) was presented in all trials except the 26th trial (the catch trial). During the catch trial, a deviant stimulus (iaa or app) was presented. After a 15-minute no-odor reset period, a second block of ten trials of the deviant stimulus was presented (trials 31 – 40). This was done to determine the unadapted (first trial) and adapted (later trials) responses of the same set of PNs to the stimulus used in the catch trial.

(b)Summed spike counts (during 4 s odor presentation period) across all PNs were calculated and shown as a function of trials (hex repetitions). Repeated presentations of odor A resulted in a substantial reduction in odor-evoked spike counts. However, the presentation of the deviant odor (iaa) during the catch trial (trial # 26) resulted in a marked increase in spike counts. The response strength was similar to the non-adapted responses (Trial # 31) for iaa (H). (right) Similar results are shown when hex was repeatedly presented and app was used as the deviant stimulus. Note that the response to app during the catch trial did not recover as was noted for iaa (left).

(c)Correlations between odor-evoked responses with hex response in the first trial are shown as a function of trial number.

(d)A similar catch-trial paradigm was followed for behavioral experiments. Each locust was presented with a repeating odorant for eight trials. A deviant stimulus was presented in the ninth trial. This was followed by six more repetitions of the recurring stimulus.

(e)Response matrix summarizing POR responses of individual locust PORs are shown. Same convention as in Fig. 1. (f)The P(POR) across locusts was calculated and plotted as a function of the trial number. Notably, when the deviant stimulus was presented there is a marked decrease in P(POR). The dotted line indicates the unadapted P(POR) value for the deviant stimulus.

POR responses of locusts across trials and odor intensities.

(a) POR responses of each locust (rows) across trials (columns) are shown for both high and low-intensity hexanol exposures. Black boxes indicate no POR responses, and white boxes indicate POR responses in that trial. The probability of palp-opening responses across locusts is shown as a function of trial number. The average POR in the first five and last five trials is compared between high and low-intensity hexanol exposures (p>0.05, one-tailed t-test).

(b-d) Similar plots characterizing locust POR responses for the other three odorants.

Combinatorial response patterns across two intensities of the same odorant.

Comparison of odor-evoked responses between high and low intensities exposures of all four odorants used in the study. Spiking activities of individual PNs were summed over the entire odor presentation window, sorted based on their response to the high-intensity presentations, and plotted to reveal the combinatorial response. Mean ± S.D. across the twenty-five trials is shown.