Dynamic Impairment of Olfactory Behavior and Signaling Mediated by an Olfactory Corticofugal System

Activation of AON neurons in awake animals is not perceived as an odorant equivalent cue

To explore possible behavioral effects of AON activation on odorant perception, we initially asked whether photostimulation of ChR2-expressing neurons in the AON could serve as an odorant equivalent cue. The AON as a sensory olfactory cortical area receives direct MTC input from the OB. Since optogenetically induced “illusory” sensory perception has been reported for different sensory areas (O'Connor et al., 2013; Guo et al., 2015), we tested whether AON activation might be able to evoke odor sensations in mice. We used a go/no-go odor discrimination assay in which water-restricted head-fixed mice freely running on a Styrofoam ball (see Materials and Methods) were exposed to different odors (S⁺), all of which were rewarded (Fig. 2B). In each behavioral session, 4-8 odorants were randomly picked of a repertoire of 36 odorants, so that over time, each mouse encountered all odorants. Within the session, the selected odorants were applied in random order (50-210 trials [S⁺ and S⁻ presentations] per session; 30-90 min duration). S⁺/S⁻ presentation was also randomized (50/50 distribution). During this period, mice learned to lick to any given odor stimulus and to refrain from licking to clean air (S⁻, blank trial). When mice performed reliably above criterion (>80% accuracy; odor hits + correct rejections/number of trials; see Materials and Methods), we tested whether unilateral photostimulation of ChR2-expressing AON neurons would elicit licking responses when applied without sensory input (3 ChR2, 3 uninjected controls and 1 EYFP control, 32 sessions). Therefore, we modified the behavioral paradigm such that, in ∼10% of S⁺ trials, we presented a blank (clean air) instead of an odorant (gray bars in the S⁺ condition; Fig. 2B) and licking to these trials would have been rewarded. As expected, mice did not lick to S⁺ blank trials (Fig. 2B,C). When we coupled the AON photostimulation to a subpopulation of S⁺ blank trials (gray bars with blue surrounding), mice equally failed to respond, suggesting that activating AON was not able to elicit an odor percept. In order to ask whether AON activation might trigger odor perception, this low number of optogenetic stimulation trials was necessary as mice had to stay trained to associate water reward with real odor presentation. Importantly, mice were not trained to report to optogenetic stimulation since it has been shown that with a sufficiently high trial number (≥650 stimulations) animals can be trained to report essentially any kind of rewarded stimulus in any brain area, for example, from optogenetic stimulation of one OB glomerulus (Smear et al., 2013) to electrical activation of single cells (Histed et al., 2013). Lick responses in blank trials did not differ significantly from responses in blank + photostimulation trials within and between individual ChR2 or control animals (Fig. 2C; % blank responses 2.11 ± 0.79 control, 4.17 ± 4.17 ChR2; % blank + photostimulation responses 0.63 ± 0.63 control, 0 ChR2; p = 0.29, Kruskal–Wallis test). We performed the experiments using four different optogenetic pulse protocols. The absence of a licking response in the photostimulation trials is unlikely to be caused by a lack of motivation as general performance accuracy (odor hits + correct rejections/number of trials) was high in all mice, and smaller differences in accuracy levels were not significant within and between individual ChR2 and control mice (94.56 ± 1.89% control, 92.9 ± 0.34% ChR2 [mean ± SEM]; p = 0.09, Kruskal–Wallis test). Mice also failed to respond to AON stimulation when this paradigm was repeated for 6 consecutive days, showing that mice did not report optogenetic stimulation with this amount of trials.

• Download figure
• Open in new tab
• Download powerpoint

Figure 2.

Optogenetic AON activation is not perceived as an odorant equivalent cue. A, Top, Schematic of experimental approach. Bottom, Overview of optical fiber tip locations within the AON of ChR2 (blue) and control (gray) mice. D, Dorsal; M, medial. B, Representative part of one behavioral session (left, ordinate displays trial number in chronological order). Mice were trained in a go/no-go odor paradigm and discriminated rewarded odorants (S⁺, yellow squares, odor duration 4 s) from clean air (S^–, “blank,” gray squares) by licking in response to the S⁺ and refraining from licking to the S^–. Three to eight odorants (randomly picked out of a repertoire of 36 total odorants) were applied in random order. For simplification, yellow squares represent all odorants in this session. Middle, Right, Behavioral session subtrials ordered by S⁺ (middle) and S^– (right) trials. Solid black dot represents first licks in each trial. Light dots represent subsequent licks. Each trial is categorized according to the classification depicted on the left. Briefly, a response on a S⁺ trial (hit) and no response during an S^– trial (correct rejection) were categorized as correct responses (black dots at the end of the odor presentation); no response on a S⁺ trial (miss) or a response during an S^– trial (false alarm) was categorized as incorrect responses (red dots at the end of the odor presentation). Approximately 10% of S⁺ trials consisted of a blank presentation, and licking to these trials would have been rewarded. Blue box represents blank + AON photostimulation trials (in S⁺ condition). Mice did not lick to S⁺ blank or S⁺ blank + photostimulation trials. C, Odor response accuracy and % responses in blank and blank + photostimulation trials for control and ChR2 animals. No significant difference was observed within and between individual ChR2 or control mice (n = 7 mice, 32 sessions). D, Part of a representative “Novel odor trial” session. Pink represents the novel odorant. Yellow represents “familiar” odorants. The animal responded to the novel odorant in the first trial. No difference in licking response to novel or familiar odorants was observed. ns, not significant.

Likewise, the lack of lick responses in blank + photostimulation trials was not because of a failure to generalize for odorants since both ChR2-expressing (4 animals, 10 sessions) and control mice (3 uninjected animals, 6 sessions) directly licked to any not previously encountered odor (Fig. 2D).

To exclude the possibility that the lack of lick responses in blank + photostimulation trials was because of the unilateral AON stimulation, which might be perceived differently compared with bilaterally sampled S⁺ odorants, we also performed bilateral AON stimulations in a separate cohort of animals (2 ChR2 mice and 1 EYFP control mouse, 18 sessions; see Fig. 4). As with unilateral stimulation, no significant differences between responses in blank trials and responses in blank + photostimulation trials could be observed (% blank responses 1.78 ± 1.67 control, 4.34 ± 1.21 ChR2; % blank + photostimulation responses 3.33 ± 3.16 control, 2.1 ± 2.08 ChR2; p = 0.81, Kruskal–Wallis test). General performance accuracy was high and not significantly different within and between ChR2-expressing and control mice (95.89 ± 1.14% control, 93.79 ± 0.67% ChR2 [mean ± SEM]; p = 0.65, Kruskal–Wallis test).

In conclusion, our data suggest that AON activation does not seem to trigger odor perception.

AON activation impairs odor detection in awake behaving animals

Next, we asked whether photostimulation of ChR2-expressing neurons in the AON might affect behavioral odor responses. Experiments were partially conducted in the same animal cohort used for the previous behavioral experiments. A similar go/no-go odor discrimination assay was used in which mice were trained to lick to different odors (S⁺) and refrain from licking to clean air (S⁻). Each behavioral session consisted of an initial baseline session (duration range from 10 to 30 min; 20-100 trials) in which the mouse's response to different odors (S⁺) and clean air (S⁻) was determined. Both experimental groups (ChR2-injected and control animals) performed reliably above criterion (80% accuracy). Following this baseline period, one odor was randomly chosen and AON photostimulation was coapplied with that odor (Fig. 3A; 4s duration, starting simultaneously; 20 Hz with 25 ms pulses, for 4s), adopted from Choi et al. (2011). Odor + AON stimulation trials were interleaved with S⁻ and odor only trials (Fig. 3A; 8 ChR2, 4 uninjected controls, and 1 EYFP control, 126 sessions). During this period, the photostimulation intensity was gradually increased (range 1-10 mW). Plotting lick latency as a function of laser power (Fig. 3B) revealed a photostimulation power-dependent increase in lick latency (0.51 ± 0.03 to 1.5 ± 0.31 s; photostimulated odor, p = 0.03, Wilcoxon signed rank test) that was restricted to the odor coupled to photostimulation in the ChR2 group (0.46 ± 0.02 to 0.56 ± 0.11 s; nonphotostimulated odors, p = 0.36, Wilcoxon signed rank test). A further slight increase in photostimulation intensity caused a failure of licking responses (0.51 ± 0.03 to 4 s [licktimeout]; photostimulated odor, p = 0.03, Wilcoxon signed rank test) of only that particular odor coupled to light stimulation (0.46 ± 0.02 to 0.58 ± 0.1 s; nonphotostimulated odors, p = 0.46, Wilcoxon signed rank test; Fig. 3B). This effect was not observed in control animals (0.45 ± 0.01 to 0.43 ± 0.02 s; photostimulated odor, p = 0.44; 0.45 ± 0.02 to 0.45 ± 0.01 s; nonphotostimulated odors, p = 0.9, Wilcoxon signed rank test). “Suprathreshold” photostimulation significantly inhibited responses to odor presentation (measured in percentage of the total number of trials in this condition) in all ChR2 mice compared with control animals (Fig. 3C, blue bars; 97.59 ± 1.58% control, 1.93 ± 0.66% ChR2 [mean ± SEM]; p = 5 × 10⁻¹⁰, Kruskal–Wallis test, nonpaired post hoc test). The reduction of a licking response in these “suprathreshold” photostimulation trials was unlikely to be the result of a declining motivation as general performance accuracy as well as the percentage of responses during odor presentation in subthreshold stimulation trials did not differ significantly within and between ChR2 and control mice (Fig. 3C, dark and light gray bars; accuracy: 96.61 ± 0.37% control, 91.1 ± 0.76% ChR2; p = 0.08, subthreshold stimulation: 97.01 ± 1.1% control, 92.74 ± 1.67% ChR2; p = 0.13 Kruskal–Wallis test). Additionally, odor presentation without photostimulation led to a normal response in ChR2 mice. Control mice did not show any effects on photostimulation regardless of the applied laser power (range 1-10 mW) as did ChR2 mice when trained to a nonolfactory detection task (data not shown), showing that mice could perform well during light stimulation. Inhibition of licking response to odor presentation during “suprathreshold” photostimulation trials was also observed in bilaterally stimulated mice (Fig. 4; 100% control, 2.5 ± 2.05% ChR2 [mean ± SEM]; p = 1.25 × 10⁻⁹, Kruskal–Wallis test, nonpaired post hoc test).

• Download figure
• Open in new tab
• Download powerpoint

Figure 3.

Optogenetic AON activation suppresses odorant detection. A, Representative go/no-go behavioral session. Mice were trained in a go/no-go odor paradigm and discriminated rewarded odorants (S⁺, yellow squares, odor duration 4 s) from clean air (S^–, “blank,” gray squares) by licking in response to the S⁺ and refraining from licking to the S^–. Three to eight odorants (randomly picked out of a repertoire of 36 total odorants) were applied in random order. For simplification, yellow squares represent all odorants in this session. One odor was chosen, and AON photostimulation was coapplied with that particular odor (4 s duration, starting simultaneously). Selected odors changed between sessions. Optical stimulation trials were randomly interspersed with trials with no stimulation (S⁺ and S^–). Photostimulation intensity was gradually increased from trial to trial (range 1-10 mW). Unless noted otherwise, all photostimulation trials before odor suppression are categorized as subthreshold stimulations (blue represents suprathreshold trials). Lick responses are only depicted in the magnification on the right. AON photostimulation strongly suppresses odorant detection. B, Lick latency (s) plotted as a function of laser power (mW). Increased laser power led first to an increase in lick latency before causing complete suppression of odorant-evoked licking. C, Odor response accuracy and % responses during subthreshold/suprathreshold odor + photostimulation trials for control and ChR2 animals. Suprathreshold stimulation selectively suppresses odorant detection in all tested ChR2 mice. There was no difference in task accuracy and % responses in subthreshold trials within and between control and ChR2 mice (n = 13 mice, 126 sessions). ***p < 0.0005, ns, not significant.

• Download figure
• Open in new tab
• Download powerpoint

Figure 4.

Bilateral AON photostimulation in odor and blank trials. Odor response accuracy and % responses in odor, odor + photostimulation, blank, as well as blank + photostimulation trials for EYFP control and ChR2 mice (suprathreshold photostimulation; same laser intensity in odorant and blank trials). No significant differences between responses in blank trials and responses in blank + photostimulation trials could be observed. Photostimulation in odor trials, however, suppressed odorant detection in all tested ChR2 mice. Task accuracy and % responses in odor/blank trials within and between EYFP control and ChR2 mice were not statistically different (n = 3 mice, 18 sessions). ***p < 0.0005, ns, not significant.

We also tested whether AON photostimulation is able to suppress responses to different odorants. Therefore, in separated sessions, different odorants were coupled to the photostimulation. Licking responses could not only be inhibited by AON photostimulation for all tested monomolecular odorants but also for an odor mixture (Table 1; 8 ChR2 animals, 97 sessions, 12 monomolecular odorants, one mixture of 4 odorants; each odorant was tested in at least 4 sessions). In sessions where we switched optogenetic stimulation rapidly between up to 6 odorants, we were able to demonstrate that licking to all stimulation coupled odorants could be inhibited, even within a single behavior session (Fig. 5A). This demonstrates that (1) AON-mediated inhibition of odor responses is not odor-specific; (2) the AON inhibits odor responses on a fast timescale; and (3) there is no training effect involved. As shown previously, animals were able to generalize to a novel odor stimulus (Fig. 2D), which renders it unlikely that the absence of licking in odor + photostimulation trials is caused by an AON-mediated change in/or novel odor percept. Photostimulation in control animals had no significant effect on odor responses (Table 1; 4 controls, 101 sessions, 12 monomolecular odorants, one mixture of 4 odorants). We also examined whether the effect of AON activation could be overcome by stronger sensory input. For this, we set the laser strength to the minimal light intensity necessary for inhibiting odor detection at a concentration of 0.5% (saturated vapor pressure). Odor concentration was then gradually increased from 0.5% to 4.5% (Fig. 5B). AON photostimulation significantly inhibited odor detection over this whole concentration range (lick delay [seconds, mean ± SD] of ChR2 mice [4 animals, 40 sessions] vs photostimulated controls [3 animals, 53 sessions]; 4 s = no lick, 0.1%, 4 ± 0 s vs 0.52 ± 0.05 s, p = 5.8 × 10⁻⁴; 0.5%, 3.97 ± 0.06 s vs 0.58 ± 0.57 s, p = 2.5 × 10⁻⁴; 1%, 4 ± 0 s vs 0.83 ± 0.91 s, p =1 × 10⁻⁴; 2%, 4 ± 0 s vs 0.4 ± 0.08 s, p = 4.6 × 10⁻⁵; 3%, 4 ± 0 s vs 0.4 ± 0.05 s, p = 4.5 × 10⁻⁵; 4%, 3.81 ± 0.36 s vs 0.56 ± 0.32 s, p = 0.009; Mann–Whitney U test for ≥4 sessions per odorant concentration).

• Download figure
• Open in new tab
• Download powerpoint

Figure 5.

Optogenetic AON activation modulates odorant detection independent of odor identity and concentration. A, Lick responses to different odorants could also be inhibited within a single behavior session by quickly switching optical stimulation between odorants. Representative part of one behavioral session showing suppression of odor detection for two different odorants. In contrast to previous plots, individual odorants are color-coded. B, AON photostimulation significantly inhibits odor detection over a large concentration range. Representative part of a training session in which odor concentration was gradually increased from 1% to 4.5%. Suppression of odor detection occurred at all tested concentrations. C, Timing of AON photostimulation affects behavioral responses. Decreasing the photostimulation time relative to the odor duration led to an immediate response (lick latency [s]) at the end of the photostimulation in ChR2 animals. Blue box represents photostimulation duration. Black bar represents odor stimulation length. Longer stimulation times also increased the number of missed trials (red dotted line). D, AON photostimulation in S^– odor trials. Blank response accuracy and % responses in odor and odor + photostimulation trials for control and ChR2 mice. Animals trained to report the absence of odorants did not lick in odor + AON stimulation trials. No significant difference was observed within and between individual ChR2 or control mice (n = 4 mice, 20 sessions). *p < 0.05. **p < 0.005, ns, not significant.

Finally, we investigated the relative timing of AON photostimulation effects on behavioral responses by varying stimulation times from the default settings (4 s both, given simultaneously). Using a paradigm of decreased photostimulation time relative to the odor duration, we found that ChR2 mice (3 animals, 12 sessions) immediately licked at the end of the photostimulation: a 2, 3, and 4 s optical stimulation, which was followed by several seconds of “unmasked” odor, resulted in a lick delay of 2.38 ± 0.08, 3.52 ± 0.08, and 4.87 ± 0.22 s, respectively (Fig. 5C). Lick delays of ChR2 mice at any photostimulation length were significantly different compared with control mice (3 animals, 17 sessions) (lick delay [seconds, mean ± SD] of ChR2 mice vs photostimulated controls; 2 s photostimulation 2.38 ± 0.08 vs 0.42 ± 0.07 s, p = 0.004; 3 s photostimulation 3.52 ± 0.08 vs 0.46 ± 0.06 s, p = 0.02; 4 s photostimulation 4.87 ± 0.22 vs 0.43 ± 0.04 s, p = 0.03; Mann–Whitney U test). Interestingly, ChR2 mice failed significantly more often to lick in response to 4 s photostimulation trials compared with shorter stimulation lengths (Fig. 5C; % missed trials; 2 s photostimulation 9%, 3 s photostimulation 7%, 4 s photostimulation 61%; p = 2.5 × 10⁻⁹, Kruskal–Wallis test, nonpaired post hoc test). This suggests that longer photostimulation produces longer-lasting effects. In contrast to that, varying the odor length at a fixed photostimulation had no significant effect on lick latency (laser 2 s, lick delay: 2.7 ± 0.18 s [4 s odor], 3.07 ± 0.24 s [6 s odor], 2.5 ± 0.11 s [8 s odor], p = 0.12, Kruskal–Wallis test; data not shown).

Taken together, we found that, in mice trained to associate water rewards with odorants, optogenetic AON stimulation alone was not able to elicit a behavioral response. AON stimulation during odor presentation, however, strongly suppressed licking responses.

AON stimulation changes odor perception

So far, our data rather favor the interpretation of an AON-mediated suppression of odor detection compared with a modulation of odor perception (e.g., intensity, timing). To distinguish between these two possibilities, we trained a separate cohort of animals (2 ChR2 mice, 1 uninjected control, and 1 EYFP control mouse, 20 sessions) to lick to blank stimuli (S⁺) while refraining to lick to any given odorants (S⁻). After reaching criterion performance, we stimulated the AON during S⁻ odor presentation (and licking to these trials would have been rewarded): if AON stimulation completely suppresses odorant perception, odor + AON stimulation should be perceived as clean air and mice should lick. If stimulation alters the perception of the odor, the mice should not lick. We found that mice did not start to lick to odor + photostimulation trials (Fig. 5D). Lick responses in odor trials did not differ significantly from responses in odor + photostimulation trials within and between individual ChR2 or control animals (Fig. 5D; % odor responses 9.21 ± 0.6 control, 12.24 ± 1.62 ChR2; % odor + photostimulation responses 3.93 ± 1.07 control, 0 ChR2; p = 0.09, Kruskal–Wallis test). General performance accuracy was high and did not differ significantly within and between individual ChR2 and control mice (91.35 ± 0.76% control, 86.91 ± 1.85% ChR2 [mean ± SEM]; p = 0.19, Kruskal–Wallis test). Mice also failed to respond to odor + AON stimulation when this paradigm was repeated for 6 consecutive days. These results render it unlikely that mice perceive odor + AON stimulation as clean air. Instead, AON stimulation might modulate odor perception so profoundly that mice, trained to respond to odors, do not recognize it as a go stimulus anymore.

Optogenetic activation of AON axons in the OB has an inhibitory effect on spontaneous and inhalation-evoked MTC spiking

The observed changes in behavior could be caused by a large number of modulatory changes along the different levels of odor processing since the AON is abundantly connected with olfactory as well as nonolfactory brain centers. We next switched to electrophysiological recordings to elucidate possible mechanisms of AON stimulation-dependent change in odor perception. Because of the dense top-down projections from the AON to the OB, which constitutes the first relay station of olfactory information in the brain, we tested for direct AON-mediated modulation of OB output activity.

For this, we stimulated AON fibers while recording electrical activity in the OB of anesthetized mice. Direct stimulation of AON fibers in the OB should prevent interfering effects from other brain areas, whereas the anesthetized preparation offers tight sensory stimulation control. We directed 473 nm light (at similar power levels used in behavioral experiments; 1-10 mW total power) onto the dorsal OB surface while recording multichannel electrical activity from dorsally located presumptive MTCs in anesthetized, double-tracheotomized mice (Fig. 6K) (see Materials and Methods).

• Download figure
• Open in new tab
• Download powerpoint

Figure 6.

Optogenetic activation of AON axons in the OB inhibits spontaneous and inhalation-evoked MTC spiking. A, Top, Exemplary recording of digitized spike waveforms from presumptive MTCs, both with a clear odor response. For unit classification (e.g., the degree of sniff modulation; see Materials and Methods), odorants were applied before the optogenetic stimulation experiments (no sniff, sniff, odor). Action potential waveforms with a signal-to-noise ratio of at least 4 SD above baseline were saved to a disk (sample rate 24 kHz) and further isolated using offline spike sorting. Bottom left, Isolation of waveforms into two different units by principal components 1 and 2. Bottom right, Spike waveforms of the isolated units. B, Exemplary interspike intervals (ISI). Top, ISI distribution of a single-unit. Inset, Sniff-triggered spike histogram of MTC spikes aligned to the start of inhalation depicting clear sniff modulation. Bottom, ISI distribution of a multi-unit. Many events were detected at intervals <2 ms and are therefore in violation of the refractory period for a single neuron. Inset, Sniff-triggered spike histogram of the same unit showing no sniff modulation. C, Raster plot and spike histogram of spontaneous spiking of two presumptive MTCs in the absence of inhalation (no sniff condition). The spike rate was calculated per 100 ms bin. Spike rate decrease during optical stimulation of the dorsal OB (“stim,” blue-shaded box). Inset, Spike waveform of the recorded units. D, Plot of spontaneous MTC firing activity in the 9 s prior (“no stim”) and during light stimulation (“stim”) for all tested units (n = 51, 8 mice). Firing activity of most units was reduced during photostimulation. All units were subjected to a unit-by-unit test for significant effects of AON stimulation. Open circles represent units that showed significant light-evoked changes. Blue filled circles represent example units in C. Nine showed a statistically significant reduction in firing rate, and only two showed an increase. Inset, Spiking activity was also reduced when exclusively stimulating AON-derived fibers in the contralateral OB in unilateral injected animals (n = 13 units from 2 mice). E. Time course of light-evoked (blue bar) changes in spontaneous MTC firing (mean ± SEM across all units). Trace represents change in mean spike rate in 1 s bins relative to the mean rate before stimulation. The time axis is relative to time of stimulation onset. A clear reduction in spiking activity can be observed during optical stimulation. F, Raster plot and spike histogram of MTC spiking during inhalation of clean air and optical stimulation (blue shaded area). Inhalation-evoked spike rates decreased during optical stimulation. Inset, Spike waveform of the recorded units. G, Plot of inhalation-evoked spontaneous MTC spiking in the 9 s prior (“no stim”) and during light stimulation (“stim”) for all tested units (n = 85, 10 mice). Firing activity of most units was reduced during photostimulation. Open circles represent units that showed significant light-evoked changes in firing activity when tested on a unit-by-unit basis. Blue filled circles represent example units in F. A total of 52 units showed a significant decrease in spikes per sniff; only two units showed an increase. H, Time course of stimulation-evoked changes in inhalation-evoked MTC spiking. Spiking activity is strongly reduced during optical stimulation. I, Optogenetic AON stimulation elicits fast but brief excitatory responses in MTC. Time course of changes in spontaneous firing rate 50 ms before and 50 ms after the start of the optical stimulation during ongoing inhalation (sniff) show that optogenetic AON stimulation elicits fast but brief excitatory responses in MTC. The mean firing rate of all recorded units (n = 85 units, 10 mice) during the 1 ms time bins is depicted as spikes/s (stim – no stim). In the first milliseconds of optical stimulation, MTCs show an increase in spontaneous spiking. J, Sniff-triggered spike histogram of inhalation-evoked MTC spiking aligned to the start of inhalation of clean air before (blue) and during (red) optical stimulation, normalized to the maximum bin in the no-stimulation condition. Bin width, 100 ms. The histogram is compiled from significant units, with firing rate normalized separately for each unit. Optical stimulation strongly reduced peak spike rate. Inset, Sniff-triggered spike histogram normalized to the maximum and minimum bin for the two conditions independently. No change in spiking dynamics after OB stimulation was observed. K, Schematic of experimental approach. (1) 16-channel silicon probe. (2) Optical fiber. Optical stimulation was performed at the level of the OB to selectively target AON axonal projections to this area (for details, see Materials and Methods).

First, to assess the impact of AON fiber stimulation on MTC excitability in the absence of sensory input, we optically activated AON axons without ongoing inhalation (Fig. 6C–E). In the absence of inhalation-derived input, MTCs display irregular firing (Carey and Wachowiak, 2011; Courtiol et al., 2011; Rothermel et al., 2014). Optical stimulation led to a significant reduction in MTC spontaneous spiking from 3.4 ± 3.68 Hz (mean ± SD) before stimulation, to 1.81 ± 2.45 Hz during stimulation (n = 51 units from 8 mice; p = 1.52 × 10⁻⁴, Wilcoxon signed rank test). Only cells in which the stimulation was repeated ≥5 times were included for analysis. This criterion supports a test of significance on each unit; 21 of these units (41%) showed a significant stimulation-evoked change in firing activity when tested on a unit-by-unit basis (Mann–Whitney U test). Of these cells, 19 showed a statistically significant reduction in firing rate, and only 2 showed an increase (Fig. 6D). Among these 19 units, the median firing rate decreased by 2.89 ± 3.06 Hz. In all recorded units, the decrease in spontaneous firing rates persisted for the whole duration of the optical stimulation (10 s). Following the stimulation, an increase in spiking was observed that returned to prestimulus levels within 25 s after stimulation ceased (Fig. 6C,E). Spiking activity was also reduced when exclusively stimulating AON-derived fibers in the contralateral OB in unilaterally injected animals (n = 13 units from 2 mice; 3.16 ± 2.31 Hz before stimulation, 2.16 ± 2.05 Hz during stimulation; p = 0.017, Wilcoxon signed rank test; Fig. 6D, inset). In control mice, the same optical stimulation protocol failed to significantly modulate MTC spontaneous spiking (n = 15 units from 2 mice; 7.43 ± 3.51 Hz before stimulation, 7.51 ± 3.65 during stimulation; p = 0.5, Wilcoxon signed rank test; data not shown). Thus, optogenetic activation of AON fibers leads to a strong reduction of spontaneous spiking of MTCs.

Next, we investigated the effect of AON axon activation on MTC responses during artificial inhalation of clean air. Inhalation-linked spiking patterns could be observed in 85 units from 10 mice, most likely reflecting weak sensory-evoked responses (Grosmaitre et al., 2007; Carey et al., 2009; Rothermel et al., 2014). Optical stimulation of AON axons strongly diminished inhalation-linked spiking of MTCs (Fig. 6F). However, an initial but fast decaying (within 30 ms) excitatory stimulation effect (Fig. 6I), similar to that reported by Markopoulos et al. (2012), could be observed by adjusting the bin size from 100 to 1 ms. Light-induced reduction of spiking activity was highly significant across the population of presumptive MTCs, with median spike rate decreasing from 3.07 ± 2.4 Hz to 1.66 ± 2.39 Hz during optical stimulation (p = 7.45 × 10⁻¹¹, Wilcoxon signed rank test). When tested on a unit-by-unit basis, 54 of 85 recorded units (62.35%) showed a significant optical stimulation-evoked change in spiking (Fig. 6G); 52 units showed a significant decrease in spikes per sniff. Inhalation-evoked spiking of these 52 cells decreased by 2.2 ± 1.69 spikes/sniff/s (mean ± SD). Only 2 units showed an increase in spikes per sniff. Sniff-triggered spike histograms depicting MTC spiking within the course of one inhalation/sniff showed that, although AON axon stimulation in the OB strongly reduced peak spike rate, it did not alter the temporal pattern of MTC responses relative to inhalation (Fig. 6J); that is, the time bin of peak firing did not change across the population of recorded units (p = 0.16, paired t test comparing time bin of the peak of inhalation-evoked firing rate for baseline vs optical stimulation). Across the population of all recorded units, the decrease in inhalation-evoked spike rates persisted for the duration of the 10 s optical stimulation (Fig. 6H) and was not significantly different from the reduction in spike rate observed on spontaneous MTC firing (decrease by 1.59 ± 2.94 spontaneous MTC spiking condition, 1.41 ± 1.82 inhalation evoked spiking condition, p = 0.46, Mann–Whitney U test). Following stimulation, spike rate increased above baseline levels for <25 s before returning to prestimulus levels (Fig. 6F, Unit 2, Fig. 6H). Thus, transient activation of AON axons in the OB leads to a reduction in inhalation-linked MTC activity without grossly reorganizing temporal patterns of sensory input. This inhibition has a fast onset and persists for the duration of the optical stimulation.

Optogenetic activation of AON axons in the OB suppresses odorant-evoked MTC spiking

Since odor detection was strongly suppressed in our behavioral data, we next evaluated the impact of bulbar AON modulation on odorant responses by comparing MTC odorant responses with and without optogenetic AON activation (Fig. 7A). Across the population of recorded presumptive MTCs (n = 55 units, from 6 mice), AON axon stimulation significantly decreased MTC odor activity (Fig. 7A–C), with a decrease from 4.02 ± 2.37 spikes/sniff/s (median ± SD) during odorant presentation alone to 2.95 ± 2.96 spikes/sniff/s during odorant paired with light (p = 1.55 × 10⁻⁴, Wilcoxon signed rank test). Optical stimulation also decreased the odorant-evoked component of the MTC response when evaluated on a single-cell level (measured as Δ spikes/sniff/s relative to pre-odor presentation; Fig. 7B). Tested on a unit-by-unit basis, 19 of the 55 recorded cells (35%) showed a significant change in odor-evoked spiking; 17 of those cells showed a significant decrease, only 2 showed an increase. The median decrease in spike rate, across the 17 cells found to be significantly inhibited, was 2.64 spikes/sniff/s (from 3.42 ± 1.84 to 0.78 ± 1.15). As with effects on spontaneous spiking, the reduction of odorant-evoked MTC responses occurred rapidly and persisted for the duration of optical stimulation. The light-mediated inhibition was followed by an increase in spiking that lasted for <25 s after the end of optical stimulation (Fig. 7A, Units 1 and 2; Fig. 7C). As with inhalation-evoked MTC responses, no change in the temporal component of MTC odor responses could be observed (Fig. 7D, inset; p = 0.6, paired t test comparing time bin of the peak of odorant-evoked firing rate for baseline versus optical stimulation; n = 55 units from 6 mice). Optical stimulation resulted in an uniform spike reduction across the sniff cycle (Fig. 7D). Overall, this shows that activating AON axons strongly reduces not only MTC spontaneous activity but also sensory-evoked responses.

• Download figure
• Open in new tab
• Download powerpoint

Figure 7.

Optogenetic activation of AON axons in the OB inhibits odor-evoked MTC spiking. A, Odorant-evoked MTC spiking is suppressed by optical OB stimulation for neurons that show an excitation (Unit 1, top) or suppression (Unit 2, bottom) of firing rate in response to odorant presentation. Inset, Spike waveform of the recorded units. B, Plot of odorant-evoked changes in MTC spiking activity (Δ spikes/sniff) in the absence (“no stim”) and during (“stim”) photostimulation (n = 55, 6 mice). Open circles represent units that showed significant light-evoked changes in firing activity when tested on a unit-by-unit basis. Seventeen of those cells showed a significant decrease; only two showed an increase. C, Time course of the effect of AON fiber activation on odor-evoked MTC spiking, averaged across all units. Shaded area represents the variance (±SEM) around mean. Blue bar represents the time of optical stimulation and simultaneous odorant presentation. Photostimulation leads to a strong reduction of odorant-evoked firing activity. Inset, Changes in odor (ET) evoked spiking with (light blue) and without optical stimulation (brown). D, Sniff-triggered spike histogram of odorant-evoked MTC spiking during odorant presentation in baseline conditions (“no stim,” blue) and during (“stim,” red) optical OB stimulation. Inset, Sniff-triggered spike histogram normalized to the maximum and minimum bin for the two conditions independently. E, Light-evoked changes of odorant-evoked MTC activity for all tested odorants. F, Effect of OB optical stimulation on odorant response spectrum for an example MTC tested with five odorants. Blue represents baseline response. Red represents response during optical stimulation. Odorants are ordered with the strongest excitatory response in the baseline condition in the middle of the abscissa. The effect of optical stimulation varies with odorant but is always inhibitory. Circles represent firing rates for each trial. Lines connect median responses across all tested trials. G, Effect of OB optical stimulation on odorant response spectrum averaged across all recorded MTCs. Blue represents baseline response. Red represents response during optical stimulation. Odorants are ordered separately for each unit, with the strongest excitatory response in the baseline condition in the middle of the abscissa. Odorant-evoked spike rates uniformly decreased during light stimulation. Lines connect median responses. Shaded areas represent the variance (SEM). H, Odorant response magnitudes (Δ spikes/s) plotted for baseline (blue) and optical stimulation (red) as a function of cell identity, sorted in order of magnitude of excitatory response in baseline conditions. Most units show a decrease in odorant-evoked excitation, including those that are suppressed during odorant presentation.

Finally, we tested how AON modulates MTC activity across multiple odorants. Thus, in a separate set of experiments, we measured responses of the same MTC to multiple odorants (for odorant panel, see Materials and Methods) with and without optogenetic activation of AON bulbar input (75 units, 3 animals, 375 cell-odor pairs). Odorant-evoked spike rates uniformly decreased during light stimulation for the example unit (Fig. 7F) as well as for the average across all recorded units (Fig. 7G). AON-mediated reduction of MTC sensory responses was homogeneous across the different odorants tested (Fig. 7E; p = 0.22, Kruskal–Wallis test); 68% of cell-odor pairs (255 cell-odor pairs) even exhibited an inhibition below pre-odor baseline firing rates. Only 2.6% (10 of the 375 cell-odor pairs) showed an increase (>1 Δspike/s) in odorant-evoked spike rates during AON activation (Fig. 7H). Thus, the AON strongly inhibits sensory-evoked responses across odorants similar to what we observed in the behavioral experiments.

Optogenetic AON activation reduces odorant-evoked MTC activity in awake mice

To further elucidate the mechanism behind the suppression of licking response observed in the behavioral experiments, we conducted multitetrode recordings and optical AON stimulation in awake mice. MTAs with 32 recording channels were chronically implanted in the OB of 3 mice injected with AAV-ChR2-EYFP into the AON (Fig. 8A). Odorants were either presented alone or in combination with AON photostimulation. While odorant responses across the population of recorded presumptive MTCs (n = 61 units, from 3 mice, 10 sessions total) were generally sparse, AON stimulation significantly decreased sensory-evoked MTC activity (Fig. 8C; decrease from 9.22 ± 7.61 spikes/s [median ± SD] during odorant presentation alone to 5.73 ± 6.43 spikes/s during odorant paired with light, p = 1.05 × 10⁻⁵, Wilcoxon signed rank test). Optical stimulation also reduced MTC activity when evaluated on a single-cell level (measured as Δ spikes/s relative to pre-odor presentation) (Fig. 8D). Tested on a unit-by-unit basis, 35 of the 61 recorded units (57%) showed a significant decrease in odor-evoked spiking.

• Download figure
• Open in new tab
• Download powerpoint

Figure 8.

Optogenetic AON activation decreases odor-evoked MTC activity in awake mice. A, Schematic of the experimental approach. A MTA was implanted into the OB of AON AAV-ChR2-EYFP-injected Chrna7-Cre mice, and an optical fiber was implanted into the AON. B, Top, Exemplary waveforms from a presumptive MTC recorded in the awake mouse. Middle, Spike waveforms. Bottom, ISI distribution of a well-isolated unit. C, Raster plot and spike histogram of MTC spiking during odor presentation alone (top) and in combination with AON stimulation (bottom). A decrease in odorant-evoked MTC spiking was observed during optical stimulation. D, Plot of odorant-evoked changes in MTC spiking activity (Δ spikes/sniff) in the absence (“no stim”) and during (“stim”) photostimulation (n = 61 units, 3 mice). Open circles represent units that showed a significant decrease in firing activity when tested on a unit-by-unit basis.

In summary, our results point to AON-mediated MTC suppression as at least one mechanism underlying the strong behavioral response elicited by optogenetic AON stimulation.