We generated transgenic mice that express ChR2-EYFP in all mature olfactory sensory neurons using the olfactory marker protein (OMP) promoter, such that all glomeruli were light-sensitive (). We characterized the olfactory receptor ChR2 mice (ORC) mice in vitro
using acute brain slices and laser scanning photo-stimulation8
. Whole-cell patch clamp recordings from mitral cells () showed reliable light-evoked currents only in response to photo-stimulation of the glomerular layer. These responses were blocked by ionotropic glutamate receptor antagonists CNQX and APV (, N=5 cells). Furthermore, when stimulating at sub-glomerular inter-foci spacing (15 μm), excitatory currents () were obtained by stimulation of only a single glomerulus for each recorded cell (). Fluorescent dye (Alexa 546) loaded into the recorded mitral cell confirmed that its apical dendrite projected to the same glomerulus whose stimulation evoked currents, as indicated by the circle in (right panels). Increasing the stimulation intensity led to larger currents (Supplementary Fig. 1a
), as well as spread of the hotspot to areas adjacent to the input glomerulus (Supplementary Fig. 1b
), presumably due to activation of ChR2 in axons of passage. These experiments confirmed that activation of ChR2 in olfactory sensory axons within single glomeruli could depolarize their terminals effectively, causing glutamate release to activate postsynaptic neurons.
OMP-ChR2 (ORC) transgenic expression pattern; Laser scanning photo-stimulation (LSPS) identifies parent glomeruli for mitral cells in ORC mice olfactory bulb slices
Identification of parent glomeruli of M/T cells in vivo by photostimulation
We adapted a digital micro-mirror device (DMD) from a commercial DLP projector (see Methods) to construct an instrument that allowed us to illuminate the olfactory bulb surface with arbitrary light patterns (at ~470 nm, see Methods) and photo-activate glomeruli in vivo (). We could therefore optically control neuronal activity at sub-glomerular resolution, with each pixel on the DMD corresponding to a ~5 μm spot on the bulb surface.
DMD patterned illumination in ORC mice maps the parent glomeruli of M/T cells in vivo
We simultaneously recorded M/T cell activity using tetrodes that were inserted 250–300 μm deep into the olfactory bulb of anesthetized ORC mice (see Methods). The surface of the bulb was tessellated into a square grid and each square pixel was illuminated sequentially by single focal spots of light in a pseudo-random spatial order (). The stimulating light spots were of the same size, or smaller (20–75 μm) than the average mouse glomerulus (~75 μm)6,9
. Light stimulation induced rapid and reliable changes in firing, peaking at 25–50 ms and lasting for 100 ms on average ().
For each single unit (, left panel) satisfying our selection criteria (see Methods), we constructed a two-dimensional light activation map (2DLAM) at subglomerular resolution (75 μm down to 20 μm) (, center and right panels, see Methods). The light intensity was successively lowered until no light triggered activity was seen in any unit being recorded (down to intensities lower than 2 mW/mm2). This ensured that we were operating in the regime of minimal glomerular activation () with negligible non-specific activation of axons of passage, as observed in acute slice experiments.
Since any given M/T cell in the adult mouse olfactory bulb receives excitatory inputs from only one glomerulus1
, we interpreted the hotspots to be parent glomeruli for the corresponding M/T cells. This was supported by the observation that reducing light levels led to a single hotspot on the 2DLAMs for all 40 units that were light responsive. In addition, the average size of the hotspot at the lowest intensity of stimulation (73.6 ± 3.9 μm, N=40) was comparable to the size of the average mouse glomerulus (74.2 ± 1.2 μm), as measured in OMP-synaptopHluorin (OMP-spH) mice10
(572 glomeruli, 9 hemibulbs, ), in agreement with anatomical studies9
. Further, taking advantage of YFP expression in glomeruli, we overlaid functional hotspots (N=9) obtained from light mapping onto multiphoton z-stack projections of images of YFP glomeruli taken in the same animal (see Methods). The hotspots co-localized well with anatomical glomeruli ( and Supplementary Fig. 2a
) and the FWHM of the hotspots fitted within the anatomical boundaries of matching glomeruli (). Furthermore, the jitter between the centroids of hotspots and anatomical glomeruli was significantly smaller than the corresponding glomerular widths (, see Methods). The parent glomerulus for each isolated M/T unit was thus identified optically, just as it was done in vitro
Functional hotspots correspond to anatomically identified glomeruli
Glomeruli are generally laid out in a single row on the bulb surface, but occasionally they can be stacked on top of each other. Since it could affect the correct assignment of parent glomeruli to M/T units, we quantified the frequency of this over-stacking. Multiphoton z-stacks of ORC-M and OMP-spH glomeruli obtained via the same surgical configuration used for the physiology experiments showed that only ~6% of glomeruli overlapped on the dorsal surface (5.94 ± 0.45%, 558 glomeruli, 4 ORC-M hemibulbs and 6.02 ± 1.16%, 875 glomeruli, 7 OMP-spH hemibulbs, and Supplementary Fig. 2b
Identification of sister and non-sister M/T cells by light
Using tetrodes or dual-tetrodes11
, we recorded odor responses simultaneously from multiple M/T units with defined parent glomerular identities. On average, we were able to isolate ~4 single units per recording site. We compared all possible pairs of light responsive units obtained at each recording site (N=35 pairs) by taking the difference of 2DLAMs corresponding to each unit (). In some pairs, the hotspots were clearly spatially separated (), while in others they were overlapping (). Thus, the units shown in appeared to receive inputs from different neighboring glomeruli, whereas the pair plotted in shared the same glomerular territory.
Light mapping sorts M/T cells into sister and non-sister pairs
We needed an objective criterion to classify the recorded M/T units into `sister' cells or `non-sister' cells (). For each pair, we calculated the Euclidean distance between the centers of the hotspots, normalized by the mean width (FWHM) of the two hotspots considered (). Our results indicate that the size of glomeruli is equal to or larger than 1 FWHM of the functional hotspots (, ). Therefore, pairs of M/T units whose parent glomeruli were placed less than one mean FWHM apart were classified as sisters, and the rest as non-sisters. Two distinct populations emerged (), unambiguously separable by the FHWM criterion. Out of the 35 M/T pairs considered, 20 were found to be sisters. In a second strategy, we computed the correlation between the 2DLAMs for each M/T pair (Supplementary Fig. 3
, see Supplementary Information
); this approach yielded the same results. The surprisingly high proportion of sister pairs is due to two factors: heterogeneity in light excitability of glomeruli and pre-selection bias to overcome the intrinsically low chance (20–30%) of recording sister pairs (see Supplementary Information
). We conclude that the physical separation between 2DLAM hotspots can be used to determine which M/T units share input from the same parent glomerulus.
Odor response diversity in sister M/T cells
We next investigated the odor response properties of these M/T unit pairs to a set of 42 odor stimuli. M/T firing is often locked to respiration12
(), that is, M/T cells tend to spike preferentially at a particular phase of the respiratory cycle. In response to an odor, M/T cell firing rates can increase or decrease from resting values, and the timing of spikes in relation to respiration can be altered13,14,15
. We divided each cycle of respiration into 5 bins and populated these bins with spikes (). The resulting vector, referred to as the phase tuning curve, was obtained for each M/T unit separately for the air period and odor period, across many respiratory cycles (), for all 42 odors. For example, the unit shown in spiked reliably at the beginning and towards the end of the respiratory cycle during fresh air, but shifted its phase preference to a different point in the cycle, and underwent a reduction in firing rate once allyltiglate was presented (, lower panel).
Examples of similarities and differences in odor responses of sister M/T cells
Responses to multiple odors were compared in simultaneously recorded sister M/T cells (). In the example shown, p-anis aldehyde increased the firing rates of both units, with odor triggered spikes occurring at all phases of the respiratory cycle. For heptanal, however, the firing rate increased for unit 1 but was suppressed for unit 2. In response to 2-heptanone, although both units increased their firing rates, the phases of the respiratory cycle at which they predominantly spiked were different. Thus, we observed similarities and differences in the odor responses of sister M/T cells.
We next systematically analyzed the activity of both sister and non-sister M/T unit pairs, focusing in particular on changes in firing rate and phase tuning.
Odors induce correlated changes in the firing rate of sister M/T cells
For each M/T unit, we calculated the average change in firing rate upon odor presentation for all 42 stimuli, and constructed a firing rate based odor response spectrum (F-ORS, , see Methods). Sister M/T units tended to be similar in their firing rate changes () as quantified by the Pearson correlation coefficient between the F-ORSs (0.68 ± 0.05, N=20, ). In contrast, the F-ORSs of non-sister pairs were diverse () and had lower correlations (0.23 ± 0.11, N=15, p = 2.4 × 10−4, two-sample unpaired t test, ). To obtain a measure of reliability for individual units across different trials, we split the odor repeats and calculated `self' F-ORS correlations, whose average value was 0.67 ± 0.04 (N=40). Sister pairs' F-ORS and `self' correlations were not significantly different (p = 0.81, also true when using matched number of trials, data not shown). However, as observed in the examples shown (, heptanal and 2-heptanone; , arrows), some odors did trigger different changes in the firing rates of sister cells.
Sister M/T cells have correlated changes in odor induced firing rates
Sister M/T cells are desynchronized by odors
For each M/T cell, we constructed phase tuning curves during Air and Odor () for all stimuli. As a measure of similarity, we computed the correlation coefficient between the phase tuning curves in Air and Odor periods for each stimulus. We termed this the phase response, analogous to a firing rate response. The phase responses for all 42 odors then yielded a phase odor response spectrum (P-ORS) () for each cell. For a given stimulus, a high value of the phase response (close to 1) would indicate that odor presentation did not cause a substantial change in the phase tuning curve compared to the preceding air period.
Odors disrupt phase correlations of sister and non-sister M/T cells
How similar are phase responses for sister and non-sister pairs? We found that for sister pairs the average P-ORS correlation was only 0.18 ± 0.07 (N = 20, ), higher, but not significantly different from the average correlation for non-sister pairs (0.05 ± 0.06, N = 15, p = 0.16). These low correlation values were not due to lack of reproducibility of phase response spectra across trials, since `self' P-ORS correlation was 0.61 ± 0.05 (N = 40), significantly higher than both sister and non-sister P-ORS correlations (p < 10−5 and p < 10−7 respectively). Thus, unlike in the case of firing rate changes described above (), odors induce differential phase responses in sister as well as in non-sister M/T pairs.
How do odors induce distinct phase responses in sister cells – do they start with similar phase tuning curves that diverge upon odor stimulation, or do they start with different phase tuning curves even at rest (Air)? To determine the phase relationship between units, we calculated the correlation coefficient between the phase tuning curves of the two units of an M/T pair, for all stimuli used. We calculated this inter-unit phase similarity (PS) separately for Air and Odor periods (). In both the example shown and over the entire population of sister M/T cells, the average phase similarity for all stimuli was high during Air, and was significantly reduced when odors were presented (, Avg. PSAir = 0.45 ± 0.02, Avg. PSOdor = 0.27 ± 0.02, N=20 pairs times 42 odors, p < 10−7, two-sample K-S test). This drop in phase similarity upon odor presentation was not due to a lack of reproducibility of phase tuning curves across trials since the `self' phase similarity was high in both Air and Odor conditions (Avg. `Self'-PSAir = 0.45 ± 0.01, Avg. `Self'-PSOdor= 0.45 ± 0.01, p = 0.05, two-sample K-S test, ). Non-sister pairs had a broad, poly-modal distribution of phase similarities at rest (Air), with modes at positive and negative similarity values (), clearly different from the distribution of sister pairs (p = 0.004, two sample K-S test). This implies that different pairs of non-sister units fired consistently with different phase lags. Odor presentation flattened the distribution of phase similarity between the non-sister M/T units ().
We obtained similar results if we focused on just the mean firing phase instead of the entire phase tuning curve for each stimulus (Supplementary Fig. 4
and Supplementary Information
). We also examined the spiking relationship (Supplementary Fig. 5
and Supplementary Information
) between pairs of M/T units using the more commonly-used spike time correlation analysis. This analysis indicated that odor presentation led to significant broadening of the peak and a drop in peak height of the M/T units' auto-correlograms, as well as of sister pairs' cross-correlograms (Supplementary Fig. 4a,c,d
). Closer inspection revealed periodic modulation of spike timing in the beta and gamma frequency range for some M/T pairs, as described before16
(Supplementary Fig. 6
), but on average we did not detect significant power in these bands via coherence measurements at a population level (see Supplementary Information
Odorants tend to activate multiple glomeruli. To investigate the effects of activating only the parent glomerulus on the phase properties of sister M/T pairs, we used the minimal light stimulation strategy ( and ) to modulate activity of single glomeruli (see Methods), by presenting light pulses continuously for 200 ms periods. As expected, light activation of individual glomeruli significantly increased the firing rate of sister M/T units compared to baseline (Avg. 2.63 ± 0.47 Hz in Air versus 9.10 ± 0.72 Hz in Light period, p < 0.001 by two-sample paired t-test). The phase similarity between sister pairs was indistinguishable between Air and Light conditions (Avg. PSAir = 0.43 ± 0.14, Avg. PSLight = 0.42 ± 0.12, N=20 pairs, p=0.88 two-sample K-S test, ), even in instances when light stimulation changed the phase preference of the sister M/T units ().
Data described in this section indicate that sister M/T units are entrained by respiration to fire synchronously at rest, but become desynchronized (in terms of their firing relation to respiration) by odors. However, light activation of just the parent glomerulus altered the phase properties of sister units in similar manner. Non-sister M/T units fire with consistent phase lags with respect to each other when at rest. Upon odor stimulation, these predictable phase relationships are also disrupted.
Odor induced firing rate and phase changes are independent
Are the differential firing rate and phase responses in sister M/T units ( and ) we observed caused by the same odors? To answer this question, we identified odors that affected one unit in a distinct manner compared to the other (see Methods).
More odors had differential effects on phase than on firing rate in sister pairs (18.7 ± 3.0 % versus 9.3 ± 2.3 % of odors, p = 0.02, Wilcoxon signed-rank test, ), as anticipated from the data shown in and . Importantly, the percentage overlap between odors that caused differential responses in firing rate and phase was 1.6 ± 0.6 %, not different from chance (1.4 ± 0.5 %; , p = 0.68, by Wilcoxon signed rank test, and see Supplementary Information
). For non-sister M/T pairs similar percentages of odors caused both firing rate and phase similarity changes (19.1 ± 3.9% versus 18.1 ± 5.4%), also overlapping only to chance levels (p = 0.95, Wilcoxon signed-rank test). It is noteworthy that similar percentage of odors caused a decrease in phase similarity for sister (18.1 ± 3.0 %) and non-sister pairs (19.1 ± 3.9 %, p = 0.51, two-sample K-S test).
Odors trigger firing rate and phase changes in an independent manner
These results demonstrate that odors can induce differential changes in the phase of sister M/T cells without inducing differential changes in firing rates. Therefore, changes in phase and firing rate are independent.