Schematic drawing of the wind tunnel (length, 250 cm; width, 90 cm; height, 90 cm).

<p>Females were released from a platform 50(distance between sources, 20 cm) were placed at the upwind entrance to the wind tunnel. These consisted of filter papers loaded with synthetic flower odors. Headspace volatiles from non-flowering plants placed in a glass cylinder outside the tunnel were released close to the source of flower volatiles.</p

Attraction of <i>M. sexta</i> females to plant and flower odors.

<p>(a) No-choice experiment: Percentage of moths that flew upwind towards the presented odor source (duf) and reached the source with extended proboscis (sc). (b) Two-choice experiment with two single odor sources presented in the wind tunnel (20 cm apart). (b-i) Number of first source contacts. (b-ii) Total number of approaches per moth within 5 min. (c) Two-choice experiment, presenting a single flower blend stimulus and a combined flower and plant odor. (c-i) Number of first source contacts. (c-ii) Total number of approaches per moth within 5 min. Error bars depict the standard deviation.</p

Temporal features of spike trains in the moth antennal lobe revealed by a comparative time-frequency analysis

The discrimination of complex sensory stimuli in a noisy environment is an immense computational task. Sensory systems often encode stimulus features in a spatiotemporal fashion through the complex firing patterns of individual neurons. To identify these temporal features, we have developed an analysis that allows the comparison of statistically significant features of spike trains localized over multiple scales of time-frequency resolution. Our approach provides an original way to utilize the discrete wavelet transform to process instantaneous rate functions derived from spike trains, and select relevant wavelet coefficients through statistical analysis. Our method uncovered localized features within olfactory projection neuron (PN) responses in the moth antennal lobe coding for the presence of an odor mixture and the concentration of single component odorants, but not for compound identities. We found that odor mixtures evoked earlier responses in biphasic response type PNs compared to single components, which led to differences in the instantaneous firing rate functions with their signal power spread across multiple frequency bands (ranging from 0 to 45.71 Hz) during a time window immediately preceding behavioral response latencies observed in insects. Odor concentrations were coded in excited response type PNs both in low frequency band differences (2.86 to 5.71 Hz) during the stimulus and in the odor trace after stimulus offset in low (0 to 2.86 Hz) and high (22.86 to 45.71 Hz) frequency bands. These high frequency differences in both types of PNs could have particular relevance for recruiting cellular activity in higher brain centers such as mushroom body Kenyon cells. In contrast, neurons in the specialized pheromone-responsive area of the moth antennal lobe exhibited few stimulus-dependent differences in temporal response features. These results provide interesting insights on early insect olfactory processing and introduce a novel comparative approach for spike train analysis applicable to a variety of neuronal data sets

Significant differences found between corresponding wavelet coefficients in the time-frequency analysis of biphasic and excited PNs from sexually isomorphic and MGC AL neurons of <i>Manduca sexta</i>.

<p>^a All LNs, other MGC neurons and inhibited PNs exhibited no significant differences (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084037#s3" target="_blank">Results</a>).</p><p>^b Wavelet levels correspond to the following frequency bands: level 1: 22.86 to 45.71 Hz, level 2: 11.43 to 22.86 Hz, level 3: 5.71 to 11.43 Hz, level 4: 2.86 to 5.71 Hz, level 5 (scaling): 0 to 2.86 Hz.</p

Temporal Response Patterns in the MGC.

<p><b>A–B</b> Confocal micrographs of two male ALs of Z-strain <i>O. nubilalis</i>, each extracting an overlay of several optical orthogonal slices (38 in A, 20 in B). Neurobiotin-injected cells were stained with Alexa-conjugated Streptavidin and alpha-synapsin/Alexa for background staining. Pictures were obtained by confocal microscopy of two separate whole mount brain preparations using a 40×, 1.3 Oil DIC objective lens (Plan-Neufluar, Zeiss). Optical sections (1031/1024×1024 pixel) were taken at intervals of 0.9 µm (<b>A</b>) and 0.7 µm (<b>B</b>). <b>A</b> and <b>B</b> display PNs innervating the MGC (outlined with dotted line); soma in <b>B</b> indicated with asterisk; scale bar: 50 micrometers. <b>C–D</b> Raster plots showing spiking times in different MGC neurons with excited response type. (<b>C</b>) Raster plots of excited neurons in the <i>Ostrinia nubilalis</i> MGC stimulated with the mixture (upper panel; 10 ng total) and the individual pheromone components at the mixture concentration (lower panel). (<b>D</b>) Excited PNs in the <i>Manduca sexta</i> MGC stimulated with the mixture (upper panel; 100 ng loading) and the individual pheromone components at the mixture concentration (lower panel). Colored bars indicate significant differences as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084037#pone-0084037-g003" target="_blank">Figure 3</a>. Stimulus timing shown in grey.</p

Significant differences found in the PSTH analysis of biphasic and excited PNs from sexually isomorphic and MGC AL neurons of <i>Manduca sexta</i>.

<p>^a All LNs, other MGC neurons and inhibited PNs exhibited no significant differences (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084037#s3" target="_blank">Results</a>).</p

Example of DWT analysis for two traces.

<p>Left, response to a mixture, and right, response to a single component at mixture concentration. Stimulus period shown as gray bar. Raw traces (<b>A</b>) were transformed into sums of delta functions (<b>B</b>) from which instantaneous rate functions (in Hz) were determined. Dotted lines in (<b>A</b>) indicate set spike threshold. The rate functions were then separated into 128 bins (<b>C</b>) and used as input to the DWT, which decomposed the binned data into 4 frequency band levels 1–4 and one approximation (or scaling) level 5 (<b>D</b>). Color code indicates the power spectral density (PSD) corresponding to the resultant wavelet coefficients (<b>E</b>). The instantaneous spiking rate for each trace was thus decomposed in specific frequency bands and time windows. Finally, the corresponding PSD values (<b>E</b>) were statistically compared using a Mann-Whitney U test (complete data sets shown as rasters in <b>F</b>). The time windows associated with each wavelet coefficient were considered significant (red bars on the abscissa) when the uncorrected p values were smaller than the <i>crit_p</i> value obtained using a FDR level <i>q</i> set to 0.10 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084037#pone-0084037-t001" target="_blank">Table 1</a>).</p

Temporal Response Patterns in sexually isomorphic AL neurons.

<p>(<b>A</b>) Raster plots showing the response of PNs to a mixture of 2–7 components and the single odorants at mixture concentration for biphasic (top) and excited (bottom) response types. (<b>B</b>) Raster plots showing the response of PNs to single odorants at low (1×10⁻⁴) vs. high (2–7×10⁻⁴) concentration as displayed as in <b>A</b>. Rasters include every recording from all sampled PNs for a given stimulus and response type. Each raster provides an example case on top showing the membrane potential recorded from same PN for the two different types of stimuli compared. Top row of colored bars on the abscissas indicate time windows where significant or marginally significant DWT differences in the temporal response patterns were found (red, FDR≤0.10; yellow, FDR&lt;0.25, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084037#pone-0084037-t001" target="_blank">Table 1</a>). In the second row of colored bars, time windows exhibiting significant differences via PSTH analysis are shown (orange, FDR≤0.10; green, FDR&lt;0.25; note that FDR = 0.28 for comparison in <b>B</b> lower panel, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084037#pone-0084037-t002" target="_blank">Table 2</a>). Stimulus timing is shown as a gray vertical bar.</p

Basic temporal response types.

<p>(A) A biphasic neuron from <i>M. sexta</i> stimulated with a mixture (upper panel) and a single component at the mixture concentration (lower panel). (B) An excited neuron stimulated at low (upper panel) and high (lower panel) concentrations of a compound. (C) An Inhibited neuron stimulated with low (upper panel) and high (lower panel) concentrations of a compound. Stimulus elapse shown in grey. The panels on the right show confocal micrographs of the three respective female <i>M. sexta</i> AL neurons, each extracting a single optical orthogonal slice (soma with asterisks, left images). Neurobiotin-injected cells were stained with Alexa-conjugated Streptavidin. Pictures were obtained by confocal microscopy of three separate whole mount brain preparations using a 10×, 0.45-NA objective lens (C-Apochromat, Zeiss). Optical sections (1024×1024 pixel) were taken at intervals of 0.8 µm. A and B display LNs, while C shows a multiglomerular PN; scale bar: 50 micrometers.</p