Odor-evoked responses in the olfactory tubercle
To begin elucidating the potential contributions of the olfactory tubercle to the multimodal convergence of odors and sounds, we recorded from a total of 62 confirmed olfactory tubercle single-units in urethane anesthetized mice (1–3 units/mouse) in response to either odors, tones or both. The majority of olfactory tubercle single-units recorded were spontaneously active, with a 14.1 ± 15Hz (mean±std) spontaneous firing rate across all units (). However, the mean was biased by several very high frequency single-units as most cells recorded from had a relatively low spontaneous firing rate (mode <5Hz, ). Neural activity at both the population (LFP) and single-unit level were generally phasic with respiration – with the phasic component of each response beginning momentarily after inspiration (). Presentation of an odorant altered the LFP and spontaneous firing rate. In the example shown in , the odorant 1,7-octadiene evoked both a transformation of the LFP and two action potentials upon the first inhalation. In this particular example, subsequent inhalations of the odorant evoked LFP oscillations and unit action potentials until two inhalations after odorant offset (likely reflecting lingering of the odorant around the nostrils). The odor-evoked LFP responses were qualitatively similar to those seen elsewhere in the olfactory system (Freeman, 1978
; Cenier et al., 2008
; Kay et al., 2009
) and consist of beta (10–35 Hz) and gamma band (40–70 Hz) high-frequency oscillations riding on the downward crest of an enhanced theta (1–10 Hz) rhythm (e.g., as in `0–100Hz' trace, ). LFP power spectrum analyses (paired t
-tests for 1Hz bins) confirmed that odorant presentation evoked significant changes in theta (t(9)
, beta (t(24)
) and gamma (t(28)
) oscillatory activity (). These results demonstrate, as predicted by both anatomical (Haberly and Price, 1978
; Scott et al., 1980
; Schwob and Price, 1984
) and physiological studies (Murakami et al., 2005
; Chiang and Strowbridge, 2007
; Carriero et al., 2009
), that the olfactory tubercle responds to odors at both the population and single-unit level.
Odorant presentation evokes neural responses in the tubercle
Previous work has demonstrated that mitral-tufted cells in the OB (Wilson and Leon, 1988
; Kay and Laurent, 1999
; Cang and Isaacson, 2003
; Nagayama et al., 2004
; Rinberg et al., 2006
; Davison and Katz, 2007
) and layer II/III pyramidal cells of the PCX (Wilson, 2001
; Rennaker et al., 2007
; Yoshida and Mori, 2007
; Poo and Isaacson, 2009
) are capable of discriminating between odorants. However, whether tubercle units also discriminate odorants is unknown. Therefore, we recorded odor-evoked responses to a panel of 5 monomolecular odorants (see Methods) in 17 units (n
= 11 mice). Odors were presented in a counterbalanced fashion, for a minimum of 4 trials each.
We found that olfactory tubercle single-units show robust responses to odorants. In particular, approximately 64 percent (11/17) of olfactory tubercle units showed significant responses to at least one of the 5 odorants (p
< .05 for each odor/unit, ≥4 trials/odor, 2-tailed t
-test). In the example shown in , the unit on top (unit #6) responds selectively to a single odorant (ethyl propionate). The unit beneath on the other hand (unit #2) has a broader receptive range and significantly responds to 3 of the 5 odorants (). We also presented the same 17 units with overlapping, complex odorant mixtures – all sharing similar components yet with some omitted (Barnes et al., 2008
). These odorant mixtures were presented in a counterbalanced manner along with the odorants used in . Out of 17 olfactory tubercle units, 8 (47%) showed significant responses to at least one of the 3 odorant mixtures (p
< .05 for each mixture/unit, ≥4 trials/mixture). Out of the 8 units which significantly responded to a mixture, only 2 (20%) showed significant responses to all 3 mixtures (). More commonly a single unit responded to just a single mixture () – even though all 3 mixtures shared overlapping components. These data suggest that olfactory tubercle single-units are capable of discretely responding to individual odorants.
Odorant responsivity of tubercle units
Auditory evoked responses in the tubercle
We next examined whether the olfactory tubercle responds to auditory stimuli. Auditory-associated fiber projections into early olfactory processing areas originate within the hippocampus (Deadwyler et al., 1987
), the ventral pallidum (Budinger et al., 2006
) and even the primary auditory cortex itself (Budinger et al., 2008
). To test the hypothesis that olfactory tubercle units are sensitive to auditory input, we presented anesthetized mice with a 2sec auditory stimulus (`tone') via a simple piezo speaker during simultaneous extracellular recordings.
Tones evoked responses at both the population (LFP) and single-unit level in the olfactory tubercle (). In the example shown in , a tone (onset timed to the respiration cycle as in & ) evoked an increase in spike rate across multiple trials. We screened a total of 26 isolated single-units for tone-evoked responses. 5 of the 26 units (>19%) showed significant responses to tone presentation (; p < .05 for each unit, ≥4 tone trials/unit, 2-tailed t-test). Further, LFP power spectrum analysis revealed that whereas tone presentation did not significantly increase theta (t(9) = −1.36, p = .20) oscillations, beta (t(24) = −11.0, p < .0001) and gamma (t(28) = −7.36, p < .0001) oscillations were both enhanced with tone (). While we limited our analysis of auditory stimuli to a simple tone for the purposes of this study, we also informally observed that alternative auditory stimuli (i.e., manual clapping, electronic buzzer) also evoked unit-level changes in activity (data not shown). These data demonstrate the functional input of auditory information into the olfactory tubercle.
Tubercle units respond to an auditory stimulus
Olfactory-Auditory modulation in the olfactory tubercle
The above data show, for the first time, that single-units in the olfactory tubercle respond to both olfactory and auditory input. Therefore, finally, to provide a test of olfactory-auditory modulation in the olfactory tubercle, we presented a subset of units (n=17) with a temporally overlapping odor and tone (odor+tone) in order to ask whether the presence of a stimulus in one modality affected the response to the other. The onset of both stimuli were timed off of the animal's respiratory signal as done previously for each stimulus (–)(see Methods). LFP and unit traces for an odor, tone and the overlapping odor+tone are shown in . As reported in and , odor presentation evoked responses phasic with each respiration cycle. Also, similar as displayed in , a tone elicited a brief burst of action potentials (, middle). Finally, in this example simultaneous presentation of odor+tone resulted, on average, in a modest supra-additive effect ( (lower trace) and ). As displayed in , across all 17 units, the average evoked response for the odor was greater than that of the tone ((t(352) = −2.64, p < .01), n=353 total trials). Odor+tone presentation on average elicited responses greater than that of the tone ((t(353) = −2.97, p < .005), n=355 total trials) but not of odor ((t(363) = −1.49, p > .05), n=365 total trials) alone. Similar to that of the odor presentation alone (see ), odor+tone elicited a significant change in beta (t(24) = −8.19, p < .0001) and gamma (t(28) = −8.10, p < .0001) oscillatory activity compared to the 2sec pre stimulus (). No changes in theta oscillations were observed in response to odor+tone (t(9) = −1.98, p = .07).
Olfactory-auditory interactions in olfactory tubercle single-units
As shown in , olfactory tubercle single-units possessed diverse responses towards not only the odor or tone alone, but also to the odor+tone stimulus. It is likely that averaging across all units, as done in , presents a less-than-representative image of the cross-modal functions within the olfactory tubercle. Looking at the response of individual units, a subpopulation (>29%) of olfactory tubercle single-units displayed cross-modal modulation in response to odor+tone. For example, one unit showed a significant response to tone, but not odor and showed response-suppression to odor+tone (, `#'). In another example, a unit (, `*') failed to show significant responses to either tone or odor alone, but was significantly excited by odor+tone. Thus, particular sub-populations of units in the olfactory tubercle functionally display olfactory-auditory convergence and cross-modal modulation.
Subpopulations of tubercle single-units show olfactory-auditory cross-modal modulation
Absence of olfactory-auditory convergence in upstream OB neurons
The data so far suggest that a population of olfactory tubercle units are capable of olfactory-auditory convergence. This effect may be due to the direct convergence of both olfactory and auditory input at the level of the tubercle. Alternatively, convergence may arrive upstream (within the OB) which is simply reflected within the olfactory tubercle. Therefore, finally, we examined whether upstream MTs within the OB show responses to a tone.
We performed extracellular MT single and/or multi-unit recordings in an additional set of experiments (n=5 mice, 20 units). Similar to that done for olfactory tubercle recordings ( & ), MT unit responses were assessed in the presence of either a tone, odor and an odor+tone stimulus (n=5–15 trials/stimulus/unit; see Methods). As shown in , individual MTs showed responses to the odor stimulus (t(214) = −10.47, p < .0001), and to the odor+tone stimulus (t(214) = −9.89, p < .0001), yet failed to respond to the tone alone (t(214) = −1.49, p > .05). In fact, 0/20 of the MT units showed a significant response to tone (p > .05 for each unit, ≥5 tone trials/unit, 2-tailed t-test). Further, the evoked responses of odor+tone stimuli did not significantly differ from that of odor alone (; (t(214) = −.02, p > .05)). This was true even at the single-unit (vs. group) level. Indeed, unlike in the tubercle, no individual MT units showed significant differences in evoked-responses between odor and odor+tone (p > .05 for each unit, ≥5 trials/unit, 2-tailed t-test; data not shown). Thus, while units within the OB and the olfactory tubercle both represent odors ( & ), olfactory tubercle units respond to both olfactory and auditory information.
Olfactory-auditory convergence is not detected upstream of the olfactory tubercle