Olfactory cortical neurons read out a relative time code in the olfactory bulb

Journal name:
Nature Neuroscience
Volume:
16,
Pages:
949–957
Year published:
DOI:
doi:10.1038/nn.3407
Received
Accepted
Published online

Abstract

Odor stimulation evokes complex spatiotemporal activity in the olfactory bulb, suggesting that both the identity of activated neurons and the timing of their activity convey information about odors. However, whether and how downstream neurons decipher these temporal patterns remains unknown. We addressed this question by measuring the spiking activity of downstream neurons while optogenetically stimulating two foci in the olfactory bulb with varying relative timing in mice. We found that the overall spike rates of piriform cortex neurons (PCNs) were sensitive to the relative timing of activation. Posterior PCNs showed higher sensitivity to relative input times than neurons in the anterior piriform cortex. In contrast, olfactory bulb neurons rarely showed such sensitivity. Thus, the brain can transform a relative time code in the periphery into a firing rate–based representation in central brain areas, providing evidence for the relevance of a relative time–based code in the olfactory bulb.

At a glance

Figures

  1. Characterization of responses to single spot optogenetic activation of olfactory nerve input.
    Figure 1: Characterization of responses to single spot optogenetic activation of olfactory nerve input.

    (a) Experimental setup. Light (cyan) was focused on the surface of the olfactory bulb. Mice expressing ChR2 in ORNs (Omp-ChR2) were used. Spiking activity was recorded extracellularly from mitral and tufted (M/T) cells in the olfactory bulb (1) and neurons in the aPC (2) and pPC (3). OB, olfactory bulb; OE, olfactory epithelium; PD, photodiode. (b) Fluorescence image of the olfactory bulb in an Omp-ChR2 mouse. Yellow indicates the location of ChR2 tagged with a yellow fluorescent protein (ChR2-YFP). ChR2 was located exclusively in the glomerular layer (GL). EPL, external plexiform layer; GRL, granule cell layer; MCL, mitral cell layer. Scale bar represents 100 μm. Sections from n > 20 mice were examined. (c) Left, two-dimensional light activation map for an example neuron in olfactory bulb. Each pixel represents the average firing rate change relative to baseline caused by activation of each olfactory bulb spot. The values were obtained using a 100-ms window after light onset, averaged over 20 randomly interleaved repetitions. The yellow and cyan represent an increase and decrease from baseline, respectively. The range of the color scale corresponds to ±5 s.d. of the baseline activity. The total area scanned for an experiment was determined by the size of the craniotomy. Middle and right, peri-event time histograms (PETHs, mean ± s.e.m.) and raster plots of excitatory and inhibitory spots. Each tick mark represents one spike. The timing of light stimulation is indicated by the cyan bar. The locations of the spots are indicated on the light activation map on the left. This neuron was excited by two spots and inhibited by one spot (*P < 0.05, t19 > 2.1, t test, corrected for multiple comparisons using Bonferroni correction, n = 20 repetitions). (d) Results from an example PCN. An olfactory bulb was optically stimulated while the activity of a PCN was recorded. This neuron was excited by six spots (*P < 0.05, t14 > 2.3, t test, Bonferroni correction, n = 15 repetitions). None of the spots caused a significant inhibitory response in this neuron (P > 0.05, t test). PC, piriform cortex. (e) Percentage of excitatory spots in single-spot scanning experiments (*P < 0.05, t test, n = 15–20 repetitions, n = 29 OBNs, n = 25 aPC neurons, n = 22 pPC neurons). The central mark indicates the median and the edges of the box are 25th and 75th percentiles. Each experiment contained 42−60 total spots. Excitatory spots were more prevalent in aPC and pPC than in olfactory bulb (olfactory bulb versus aPC, Z = 3.75, P = 0.00017; olfactory bulb versus pPC, Z = 2.1, P = 0.031; Mann-Whitney U test). Dotted lines inside the bars represent the FDRs. The vertical line indicates the maximum and minimum values of non-outliers. Points were considered to be outliers if they were larger than b + 1.5(ba) or smaller than a − 1.5(ba), where a and b are the 25th and 75th percentiles, respectively. *P < 0.05, ***P < 0.001. (f) Percentage of inhibitory spots in single-spot scanning experiments. Data are presented as in e. Inhibitory spots were more prevalent in olfactory bulb than piriform cortex (olfactory bulb versus aPC, Z = −4.6, P = 3.6 × 10−6; olfactory bulb versus pPC, Z = −4.2, P = 1.8 × 10−5; Mann-Whitney U test).

  2. Acquisition of TTCs for olfactory bulb and PCNs.
    Figure 2: Acquisition of TTCs for olfactory bulb and PCNs.

    (a) Experimental design for testing temporal sensitivity. Each spot was illuminated for 83.3 ms (five projector frames at 60 Hz). Two spots in the olfactory bulb were illuminated with varying orders and lags. Lags used for the main experiment were 17, 33, 50 and 67 ms. In some experiments, larger lags were also included. For each lag, we tested the response to activation of spot A followed by B (Aright arrowB, positive Δt) and the reversed order (Bright arrowA, negative Δt). We also tested the response to each spot alone (A or B) and to simultaneous activation of the two spots (A and B, Δt = 0). (be) Example raster plots of one olfactory bulb (OB, b), one aPC (c) and two pPC neurons (d,e). Cyan and magenta bars indicate the timing of light stimulation of spots A and B, respectively. Each dot represents a spike and each row represents one trial. Black lines separate between the different lags. The Δt values are indicated on the right. The experiments shown in b and c did not include Δt = ±83 ms. (fi) TTCs of the neurons shown in be. The total spike counts in the 200-ms analysis window were used to calculate the firing rate. The baseline firing rates in the 200-ms time window before optical stimulation were subtracted. Magenta, spot A alone; cyan, spot B alone; blue, positive Δt (Aright arrowB); red, negative Δt (Bright arrowA); black, simultaneous stimulation of A and B (Δt = 0). Data are presented as mean ± s.e.m. (n = 40 repetitions). The dashed horizontal lines represent the sum of the responses to spot A alone and B alone (that is, r(A) + r(B)).

  3. Piriform cortex neurons show higher sensitivity to the lag of olfactory bulb stimulation.
    Figure 3: Piriform cortex neurons show higher sensitivity to the lag of olfactory bulb stimulation.

    (a) Analysis of TTCs. For each spot pair, two slopes were obtained by regressing the TTCs at the negative (red) and positive (blue) Δt separately with straight lines (r = b + at|). The unit of slopes is spikes per s per ms. Error bars represent s.e.m.; n = 40 repetitions. (b) Distributions of slopes of TTCs for olfactory bulb, aPC and pPC neurons. Thin lines indicate the distribution of the slopes with the data shuffled with respect to Δt. The distribution of the slopes of the fitted lines was mostly near zero for OBNs (–0.0005 ± 0.5, t test against zero, t151 = 0.14, P = 0.89, n = 152 fitted lines), whereas aPC and pPC neurons' slopes were significantly shifted below zero (aPC, −0.069 ± 0.09, t test against zero, t227 = 11.07, P = 4.6 × 10−23, n = 228 fitted lines; pPC, −0.042 ± 0.06, t257 = 9.8, P = 1.0 × 10−19, n = 258 fitted lines). The distribution of the slopes for OBNs was similar to those obtained in the surrogate data, suggesting that the variability in slopes originates mostly from the finite number of trials in the data. In contrast, the distribution of the slopes for PCNs was shifted compared with trial-shuffled surrogate data, and the mean slopes were significantly smaller than those of the surrogate data (P < 0.001, Kolmogorov-Smirnov test for both aPC and pPC, n = 228 and 258 fitted lines for pPC and pPC, respectively), whereas that of olfactory bulb was not (P = 0.63, Kolmogorov-Smirnov test, n = 152 fitted lines). (c) Box plots of the slopes of TTCs in the three brain regions. The mean slopes of aPC and pPC neurons were significantly negative (P < 0.001 for both aPC and pPC, t test against zero). The average slopes of OBNs were significantly different from those of aPC and pPC neurons (Z > 6.2, P < 3.5 × 10−10, for both olfactory bulb versus aPC and olfactory bulb versus pPC, Mann-Whitney U test), whereas aPC slopes were also significantly different from pPC slopes (Z = −2.82, P = 0.0047, aPC versus pPC, Mann-Whitney U test). Data presented as in Figure 1e. **P < 0.01, ***P < 0.001. (d) An example TTC to illustrate the analysis. r(A), response to A; r(B), response to B; r(A and B), response to simultaneous activation of A and B (Δt = 0). The gray dashed line indicates the arithmetic sum of the response to spot A and B (r(A) + r(B)). (e) Comparison between the sum of the responses to spots A and B (r(A) + r(B)) and the actual response for simultaneous presentation of spots A and B (r(A and B), Δt = 0). Each circle represents a spot pair. Open and filled dark circles indicate supralinear facilitation or suppression (P < 0.05, t test, not corrected (open) or corrected (filled) for multiple comparisons, n = 40 repetitions). In aPC and pPC neurons, the response to r(A and B) tended to be larger than that to r(A) + r(B). (f) Percentage of spot pairs in which the responses to two-spot stimulations (r(A, B)) were greater than r(A) + r(B) (that is, supralinear, t test, P < 0.05, n = 40 repetitions, solid lines) or smaller (sublinear, t test, P < 0.05, n = 40 repetitions, dashed lines) for a given lag (Δt). The average trial-shuffled control values in all three brain areas resulted in ~3% (range: 1–8%).

  4. Order-specific responses of PCNs.
    Figure 4: Order–specific responses of PCNs.

    (a) TTC of an aPC neuron. Data are presented as in Figure 2. The responses to Bright arrowA stimulation (red) remained flat for all lags, whereas the responses to Aright arrowB (blue) decreased as the lag increased. This asymmetry was captured by both the global and lag-specific tests (PA = 0.00013, F1,446 = 14.8, ANCOVA; Δt = ±67, P = 0.0023, t88 = 3.1; Δt = ±83 ms, P = 0.00040, t88 = 3.7; t test, n = 45 repetitions for both). PA indicates the P value in ANCOVA. The P values for lag-specific comparisons are shown only when they are smaller than the criterion (t test, corrected for the number of |Δt| values, Bonferroni correction). Data are presented as mean ± s.e.m. (b) TTC of a pPC neuron. The responses to Bright arrowA were similar to r(A) + r(B) (the gray dashed line), whereas the responses for the opposite order (Aright arrowB) decreased as the lag increased (F1,316 = 5.4, PA = 0.019, ANCOVA, n = 40 repetitions). (c) TTC of a pPC neuron. The responses to Bright arrowA were generally weaker than those to Aright arrowB. This asymmetry was captured by the global tests (PA = 0.041, F1,316 = 4.2, ANCOVA). The lag-specific differences at Δt = ±67 and ±50 ms (Δt = ±67, P = 0.023, t78 = 2.3; Δt = ±50, P = 0.025, t78 = 2.3; t test, n = 40 repetitions) did not cross the criterion (P < 0.0125, Bonferroni corrected; Supplementary Fig. 5). (d) TTC of a pPC neuron. This neuron responded maximally when spot B was stimulated 67 ms after spot A (P = 0.0055, t78 = 2.8, t test between Δt = 67 ms and Δt = 0 ms, n = 40 repetitions) and did not respond to activation of either of the spots or to simultaneous stimulation of both spots. This asymmetry was captured by the lag-specific test (Δt = ±50, P = 0.00042, t78 = 3.6; Δt = ±67 ms, P = 0.0062, t78 = 2.8; t test, n = 40 repetitions for both). The global test was not significant (PA = 0.062, F1,316 = 3.5, ANCOVA). (e) Left, TTC of a pPC neuron. This neuron responded strongly only when A started 50–83 ms after spot B (Δt = ±50, P = 0.038, t78 = 2.1; Δt = ±66, P = 0.00034, t78 = 3.7; Δt = ±83, P = 0.0062, t78 = 2.8; t test, n = 40 repetitions for all). The responses peaked at 67 ms (P = 0.00099, t78 = 3.4, t test between Δt = 67 ms and Δt = 0 ms, n = 40 repetitions). Right, TTC of the shown at left, but tested with a different set of lags in an independent experiment. The peak at Δt = 67 ms was reproduced (P = 0.0027, t78 = 3.1, t test between Δt = −67 ms and Δt = 0 ms, n = 40 repetitions), response decreased with longer Δt. (f) Percentage of order-sensitive cases calculated in terms of spot pairs (white bars) and neurons (gray bars) in each brain area (Bonferonni corrected t test for all ±Δt and PA < 0.05, ANCOVA, n = 76, 114, 129 spot pairs and n = 45, 46, 63 neurons for olfactory bulb, aPC and pPC, respectively). Error bars represent s.e.m. based on the binomial model. Dashed lines inside the bars represent the FDRs resulting in only ~8% of order-sensitive cases in all three brain areas. (g) Percentage of order-sensitive responses as a function of the distance between the spots. Error bars represent s.e.m. based on the binomial model. The fraction of asymmetric TTCs in piriform cortex did not depend on the distance between the two spots as far as 1 mm on the olfactory bulb surface, indicating that order-sensitive temporal interactions occur between glomeruli that are widely distributed in the olfactory bulb. This also indicates that order sensitivity was not a result of an artifact caused by activation of adjacent spots through scattered light. (h) Percentage of cases in which the response to lagged stimulation (either Aright arrowB or Bright arrowA) was significantly higher than the response to A and B. The results were obtained in terms of the number of spot pairs (white bars) and neurons (gray bars). Error bars represent s.e.m. based on the binomial model. Dashed lines inside the bars represent the FDRs. ***P < 0.001 (binomial test against trial-shuffled controls, n = 76, 114, 129 spot pairs).

  5. Delayed inhibition shapes the responsiveness of PCNs.
    Figure 5: Delayed inhibition shapes the responsiveness of PCNs.

    (a) Top left, TTC of an example pPC neuron (shown in Fig. 2d,h). Top right panels, PETHs of the responses. The response decreased steeply with increasing Δt for Aright arrowB, but not for Bright arrowA. The dashed lines in the right two panels represent the expected firing rate changes in response to the corresponding spot (A or B). The second stimulation was effective in evoking responses with Δt = −83 ms (third panel), but not with Δt = 83 ms (fourth panel, black arrow). Bottom left, TTC of a pPC neuron. Bottom right panels, PETHs of the responses. With Δt = ±83 ms, the response to Aright arrowB differed significantly from that of Bright arrowA (P = 4.5 × 10−5, t78 = 4.3, t test). Note that second spot stimulation did not elicit the expected responses in both orders (middle and right panels, black and gray arrows). (b) TTC of a pPC neuron that was tested with longer lags (Δt = 100, 133, 167 and 200 ms). The TTC was asymmetric at Δt = ±100 ms (black arrow, P = 2.3 × 10−5, t78 = 4.5, t test, n = 40 repetitions), but similar for Δt = 200 ms (P = 0.72, t test, t78 = 0.36). With Δt = −100 ms, stimulation of the second spot A did not elicit a strong response (third panel, Bright arrowA, red line and black arrow). However, with a larger lag (Δt = ±200 ms), the response to spot A resumed (red line in the fourth panel). (c) Percentages of lag-specific asymmetry in TTCs for each lag for all PCNs. Data are presented as mean ± s.e.m. based on the binomial model.

  6. Rate code conveys relative timing information progressively more at the central areas.
    Figure 6: Rate code conveys relative timing information progressively more at the central areas.

    (a) Classification success rates based on three different decoding methods. A linear classifier was trained to classify the neuronal responses of a population of neurons as belonging to either positive or negative Δt. A classifier was first trained using all but 10% of the trials, including all Δt, and the remaining 10% of the trials were used to test the performance of the classifier (a leave-10%-out procedure). The result was obtained using neural activity representing 100 spot pairs randomly sampled from the data obtained from 45, 47 and 63 neurons in olfactory bulb (OB), aPC and pPC, respectively (see Online Methods). The mean classification success rate was obtained from 500 repetitions using different random sets of test trials. Rate code is based on the number of spikes evoked in the analysis window of 200 ms. Rise time code is based on the time at which the number of spikes in a window of 20 ms became ±2 s.d. higher (or lower) than the baseline. Latency to first spike time is defined as the time of the first spike from stimulation onset. ***P < 0.001 (binomial test). Data are presented as mean ± s.e.m. Dotted lines inside the bars represent the FDRs. (b) Classification success rates as a function of the number of spot pairs. Data are presented as mean ± s.e.m. (n = 500 repeats). The lower and upper dotted horizontal lines represent the minimum and maximum FDRs.

  7. Order selectivity is largely preserved across different respiration phases.
    Figure 7: Order selectivity is largely preserved across different respiration phases.

    (a) Raster plot of a mitral and tufted cell in response to single spot optical stimulation. Each tick mark represents one spike. The dark gray areas indicate inhalation periods and the light gray areas indicate exhalation periods. Trials are sorted by the timing of inhalation onset. The timing of light stimulation (duration = 83 ms) is indicated by the cyan area. This neuron fired preferentially during inhalation periods. (b) Firing rates in a 200-ms window (indicated at the top of a) as a function of inhalation onset timing relative to light onset (cyan). The data with no light stimulation (black) was obtained by randomly assigning light onset relative to the respiration cycle. (c) Two examples comparing TTCs at specific respiration phases. The data were parceled into four groups depending on the onset of light stimulation (as indicated in d). The shapes of TTCs were similar across the four groups. (d) Correlation of TTCs. A TTC was obtained for each of the four groups, as in c. The correlation between this TTC and the TTC computed with all other trial groups was obtained. The bar graphs show the median correlation across all spot pairs in each brain area. TTCs were obtained only if at least ten trials were available for all of the four groups (aPC, n = 112 TTCs; pPC, 95 TTCs). Data are presented as mean ± s.d. (e) Percentage of neuron spot pairs that were modulated by the lag and/or the respiration phase (two-way ANOVA, F3,199 > 2.6 and/or F4,199 > 2.4, P < 0.05, for 4 and 5 Δt, respectively, uncorrected for multiple comparisons). Many neurons were modulated by the respiration phase (white bars). Many neurons in aPC and pPC were modulated by the lag between two spot activations, but neurons in olfactory bulb were not (black bars; binomial test, P < 0.001 for both aPC and pPC compared to olfactory bulb). Error bars represent s.e.m. based on the binomial model. The number of TTCs used in the analysis was 134, 224 and 190 in the olfactory bulb, aPC and pPC, respectively. (f) The variance of neural responses explained by different factors per neuron (two-way ANOVAs, the average variance explained by the lag, respiration phase or both). Respiration phase explains on average ~10% of the variance in olfactory bulb and aPC. The lag between spot activations explain more of the variance in PCNs than in OBNs (aPC, Z = −3.2, P = 0.0011; pPC, Z = −4.3, P = 0.000016; compared with olfactory bulb, Mann-Whitney U test). Data are presented as mean ± s.e.m. (n = 134, 224 and 190 TTCs in the olfactory bulb, aPC and pPC, respectively).

  8. Direct activation of mitral and tufted cells produced consistent results.
    Figure 8: Direct activation of mitral and tufted cells produced consistent results.

    (a) Experimental design. Data are presented as in Figure 1a. (b) Characterization of Tbet-cre; ChR2loxP/loxP mouse. Left, a section from a Tbet-cre; LacZloxP/loxP mouse. Blue signals (LacZ staining) depict the location of cell bodies. Mitral and tufted cells (arrowheads) are stained. Right, fluorescent image of an olfactory bulb section. Red indicates the location of ChR2 tagged with a red fluorescent protein (tdTomato). Sections from n = 2 and n > 20 mice were examined for LacZ staining and tdTomato fluorescence, respectively. EPL, external plexiform layer; GL, glomerular layer; GCL, granule cell layer; MCL, mitral cell layer; OB, olfactory bulb. Scale bar represents 100 μm. (c) Percentage of order-sensitive responses in olfactory bulb (n = 17 neurons and 28 responding spot pairs) and PCNs (n = 34 neurons and 67 responding spot pairs). The results were obtained in terms of spot pairs (white bars) and neurons (gray bars). The data from aPC and pPC were pooled. Dashed lines inside the bars represent the FDRs. Error bars represent s.e.m.

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Author information

Affiliations

  1. Center for Brain Science, Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA.

    • Rafi Haddad,
    • Anne Lanjuin,
    • Venkatesh N Murthy &
    • Naoshige Uchida
  2. Allen Institute for Brain Science, Seattle, Washington, USA.

    • Linda Madisen &
    • Hongkui Zeng

Contributions

R.H. and N.U. conceived the experiment. R.H. performed the experiment. A.L. generated and characterized the Tbet-cre; ChR2loxP/loxP mice, and L.M. and H.Z. generated and characterized the ChR2loxP/loxP mice. V.N.M. provided the Omp-ChR2 mice. R.H. and N.U. wrote the paper and A.L., V.N.M. and H.Z. provided feedback on the manuscript.

Competing financial interests

The authors declare no competing financial interests.

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    Supplementary Figures 1–10

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