Neurons in the anterior olfactory pars externa detect right or left localization of sources

Shu Kikutaa,b,c, Kenichiro Satob, Hideki Kashiwadanib,c, Koichi Tsunodad, Tatsuya Yamasobaa, and Kensaku Morib,c,1

Departments of aOtolaryngology and bPhysiology, Graduate School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan; dDepartment of Artificial Organs and Medical Device Creation, National Institute of Sensory Organs, Tokyo Medical Center, National Hospital Organization, Meguro-ku, Tokyo 152-8902, Japan; and cJapan Science and Technology Agency, Core Research for Evolutional Science and Technology, Tokyo 113-0033, Japan

Edited by John G. Hildebrand, University of Arizona, Tucson, AZ, and approved June 2, 2010 (received for review March 26, 2010) Rodents can localize odor sources by comparing odor inputs to the the ipsilateral OB (8–11). In addition, AON neurons receive in- right and left nostrils. However, the neuronal circuits underlying puts from the contralateral olfactory cortex via the anterior com- such odor localization are not known. We recorded neurons in the missure (12, 13), suggesting that individual AON neurons receive anterior olfactory nucleus (AON) while administering to odor information originating from both ipsilateral and contralat- the ipsilateral or contralateral (ipsi- or contra-) nostril. Neurons in eral OEs. The AON is composed of two separate structures, the the AON pars externa (AONpE) showed respiration phase-locked pars principalis (AONpP) and pars externa (AONpE) (12). excitatory spike responses to ipsinostril-only stimulation with a We report here that AONpE neurons show ipsilateral (ipsi)- category of odorants, and inhibitory responses to contranostril-only nostril excitation and contralateral (contra)-nostril inhibition (E- stimulation with the same odorants. Simultaneous odor stimulation I) responses, and compare the magnitude of responses to ipsino- of the ipsi- and contranostrils elicited significantly smaller responses stril odor inputs with those of contranostril inputs. than ipsinostril-only stimulation, indicating that AONpE neurons subtract the contranostril odor inputs from ipsinostril odor inputs. Results An ipsilateral odor source induced larger responses than a centrally We recorded single-unit spike responses of individual AON neu- located source, whereas an odor source at the contralateral position rons in urethane-anesthetized rats in response to nasal stimulation elicited inhibitory responses. These results indicate that individual with a panel of odors consisting of 10 categories of odorant mol- AONpE neurons can distinguish the right or left position of an odor ecules, each containing five separate odorants (Fig. 1A and Table source by referencing signals from the two nostrils. S1). A thermoplastic external nasal septum that fitted the external shape of the rat nose was used to prevent odors delivered in front olfactory cortex | binasal inputs | odor localization of one nostril from spreading to the contralateral nostril (Fig. 1A), which enabled the selective stimulation of either the ipsilateral or lfactory neuronal circuits translate odor cues into a variety of contralateral (6). We examined spike re- Obehavioral responses that enable rodents to find and locate sponses of individual neurons in the rostral part of the AON (Fig. food, mates, and predators. For the directional localization of 1B) to ipsinostril-only and contranostril-only stimulation with the sound sources, the central auditory system has neuronal circuit panel of odorants (52 cells in 36 rats). AON neurons that showed mechanisms that compare auditory inputs from the right and left excitatory spike responses to ipsinostril stimulation with an odor- cochleas (1). Similarly, rodents can localize odor sources by com- ant category showed three types of responses to contranostril paring odor inputs through the right and left nostrils (2). How- stimulation with the same odorant category: excitatory responses ever, the neuronal circuits and mechanisms subserving the right or (ipsi-excitatory and contraexcitatory response, or an E-E-type left localization of odor sources are not yet known. response), no response (ipsi-excitatory and contranull response, In the auditory system, binaural sound localization relies on E-0-type response), and suppressive responses (ipsi-excitatory and central neuronal mechanisms that compare auditory inputs from contrainhibitory response, E-I-type response) (6). the two ears. Interaural differences in the intensity of the sound pre- Individual AON neurons that showed E-I responses to ipsi- and ssure level arriving at the two ears are important cues used by the contranostril stimulation with one odorant category did not show mammalian auditory system to localize higher-frequency sounds E-I responses to the other odorant categories (Fig. 2), suggesting (3). Neurons in the lateral superior olive are sensitive to interaural that their E-I response is specific to a single odorant category. In intensity differences, being excited by stimulation of the ipsilateral addition, these AON neurons did not show E-E-type responses to ear and suppressed by stimulation of the contralateral ear (4). These any of the 10 odorant categories (Fig. 2). We thus classified these neurons are thus referred to as ipsi-excitation and contrainhibition neurons as single-category E-I-type neurons. Because we prefer- (E-I) neurons and play a key role in sound source localization (5). entially searched for E-I-type neurons, we analyzed 31 E-I-type Odorants are inhaled through the two nostrils into two segre- neurons, 13 E-E-type neurons, and 8 E-0-type neurons. Fig. 1C gated nasal passages. Because the two passages are relatively well shows a representative example of a single-category E-I-type AON isolated, odorants inhaled through one nostril activate olfactory neuron. This cell showed high-frequency burst spike responses to sensory neurons only in the ipsilateral olfactory epithelium (6). ipsinostril stimulation with sulfide odorants and suppressive re- Therefore, to detect the right or left localization of odor sources, sponses to contranostril stimulation with the same odorants. Be- the central needs only to compare afferent odor cause single-category E-I AON neurons are candidate neurons signals originating from the right and left olfactory epithelia (OEs). NEUROSCIENCE Olfactory sensory neurons in the epithelium project their to the ipsilateral (OB), and mitral and tufted cells in the Author contributions: S.K. and K.M. designed research; S.K. performed research; S.K. and OB project their axons to the ipsilateral olfactory cortex (7). K.S. analyzed data; and S.K., K.S., H.K., K.T., T.Y., and K.M. wrote the paper. Therefore, odor signals originating from the right and left olfactory The authors declare no conflict of interest. sensory epithelia are largely segregated at the level of the right This article is a PNAS Direct Submission. and left OBs, and their afferents are segregated to the right or Freely available online through the PNAS open access option. left olfactory cortex. 1To whom correspondence should be addressed. E-mail: [email protected]. The anterior olfactory nucleus (AON) is the most rostral region This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. of the olfactory cortex and receives excitatory axonal inputs from 1073/pnas.1003999107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1003999107 PNAS | July 6, 2010 | vol. 107 | no. 27 | 12363–12368 Downloaded by guest on October 2, 2021 A BD E

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Fig. 1. E-I-type neurons in the AON. (A) Experimental procedure used for uninostril odor stimulation. An external nasal septum was used to ensure delivery of odorants selectively to unilateral olfactory epithelium. Single-unit recordings were obtained from AON neurons. (B) Lateral view of a 3D reconstruction of the olfactory peduncle of a rat . AONpE (black); AONpP (green); MOB, main olfactory bulb; AOB, accessory olfactory bulb; NC, . (C) Spike responses of an E-I-type AON neuron to ipsinostril (Upper left trace) and contranostril (Upper right trace) odor stimulation. Resp., the trace of the respiration monitor. The ascending and descending phases of the trace indicate inspiration and expiration, respectively. Raster, raster representation of the spike responses. Each row corresponds to a single odor stimulation. Peristimulus time histograms of the response are shown at the bottom. Bar, duration of odor stimulation (sulfide category, 3 s). F.R., firing rate (Hz). (D) Locations of E-I type neurons in the AONpE. Dr, Di, Dc: rostral, intermediate, and caudal regions of the dorsal AONpE, respectively. Vr, Vi, Vc: rostral, intermediate and caudal regions of the ventral AONpE, respectively. D-R, dorso-rostral; V-C, ventro-caudal. Red and yellow dots represent individual E-I type neurons. Numbered cells correspond to those in Fig. 2. (E) A raster representation of the spike responses (Upper) and peristimulus time histograms (Lower) from a single cell. Black bar, duration of odor stimulation (sulfide category, 3 s). (F) Morphology of the E-I-type AON neuron whose responses are shown in E. The cell was labeled with biotinylated dextran amine by juxtacellular electroporation (SI Materials and Methods). This cell emitted an collateral (single arrowhead) that reached the superficial part of layer II of the AONpP. AC, anterior commissure (double arrowhead).

subserving the comparison between ipsinostril and contranostril tion, we examined the odorant category selectivity of individual odor inputs, we focused our analysis on these neurons in this study E-I AON neurons using the 10 different categories of odorant (31 cells in 23 rats). molecules. We performed detailed quantitative analysis of ipsi- nostril and contranostril responses using a computer-controlled Single-Category E-I-Type Neurons Are Located in the Pars Externa of in 16 of the 31 E-I-type neurons. the AON. To examine whether the single-category E-I-type neurons As shown in Fig. 2, AONpE neurons showed ipsinostril-induced were in the AONpP or AONpE, we located the positions of these excitatory responses (colored bars above the line) to either a sin- neurons by depositing a marker dye at the recording sites. All gle odorant category (cells #1–3, #15, #16) or a specific combi- single-category E-I-type AON neurons (n = 17) were localized to nation of odorant categories (#4–14). In contrast, they showed the AONpE (Fig. 1 D and F). In striking contrast, all E-E-type and contranostril-induced inhibitory responses (gray bars below the E-0-type AON neurons (n = 9) were localized to the AONpP (Fig. line) to only a single odorant category. Because most E-I neurons S1). Although our recordings from AONpE neurons were ob- showed spontaneous discharges in the absence of odor stimula- tained mostly in the ventro-caudal part of the AONpE (Fig. 1D), tion, the inhibitory responses were clearly detected and distin- the above results suggest that a majority of AONpE neurons are guished from null responses (Fig. 1C). Among 16 E-I-type AON single-category E-I-type neurons. neurons, all cells showed E-I responses to only a single odorant AONpE neurons receive massive afferent inputs from mitral category (Fig. 2, shown by asterisks). The single-category E-I re- and tufted cells of the ipsilateral OB. Thus, the E-I response might sponse was selective to the sulfide category in 14 neurons, selective be generated at the level of the mitral and tufted cells in the OB to the terpene hydrocarbon category in one neuron (#6), and se- and transmitted to the AONpE neurons. To examine this possi- lective to the aldehyde category in one neuron (#10). Neuron #10 bility, we recorded the response of mitral and tufted cells in the OB was exceptional in that it showed an I-E-type response to sulfides in to ipsinostril-only and contranostril-only stimulation with the 10 addition to the E-I response to the aldehyde category. These odorant categories in rats fitted with the external nasal septum. results suggest that individual E-I-type neurons are specialized to Mitral and tufted cells (n = 21) showed E-0 responses but did not compare the ipsinostril and contranostril inputs within an odorant show E-I-type responses to any of the 10 odorant categories (Fig. category, although we do not rule out the possibility that different S2), suggesting that the synaptic interactions between ipsinostril component odorants within an odorant category induced the and contranostril inputs in the OB do not play a major role in ipsiexcitatory or contrasuppressive responses. generating the E-I-type response of AONpE neurons. Spike Responses of E-I-Type AON Neurons Are Phase-Locked to the Each AONpE Neuron Shows a Selective E-I Response to a Single Respiration Cycle. External odor information is detected inter- Odorant Category. For sound localization, individual neurons in mittently by right and left OEs during the inhalation phase of each the lateral superior olive compare ipsilateral and contralateral respiration cycle. Because of the possible movement of odor sources sound inputs of equivalent frequency (14). Similarly, if AONpE or the animal’s head position during two successive inhalations, neurons are involved in the localization of odor sources, they might single-category E-I neurons in the AONpE might need to compare compute the difference between ipsinostril and contranostril input ipsi- and contranostril inputs within each respiration cycle. In sup- signals of the same odor quality. As a first step to address this ques- port of this hypothesis, the ipsinostril odor responses of all single-

12364 | www.pnas.org/cgi/doi/10.1073/pnas.1003999107 Kikuta et al. Downloaded by guest on October 2, 2021 Ipsi-nostril Category 40 Cells Sulfide Ester Terpene Acid Ether AldehydeLactoneAlcohol Ketone Phenol A C sulfide 1sec 1.0 ＊ raster Ipsi 1 Contra 40 Bi-nostril -1.0 1.0 ＊ Ipsi Contra raster 1 -1.0 30 1.0 ＊ resp. 20 Ipsi sulfide 10 Contra B F.R. (Hz) 0 -1.0 0 180° 360° respiration cycle 1.0 ＊ 0.7 Ipsi 0.2 Contra D Ipsi-induced R.P.H. -1.0 Bi-nostril-induced R.P.H. 1.3 1.0 ＊ 80 n=9 Ipsi 0.1 Contra -1.0 40 1.0 1.0 ＊ Ipsi Contra 0 0 -1.0 spike / sec (Hz) -10 -5 50 0 180° 360 respiration cycle ° 1.0 ＊ respiration cycles response ratio / respiration Ipsi 0.3 0.4 Contra -1.0 Fig. 3. Respiration phase-locked spike responses of AONpE neurons. (A) 1.0 ＊ 1.1 0.6 Ipsi Spontaneous spike discharges and spike responses to ipsinostril odor stimu- Contra lation of an AONpE neuron. Resp, respiration. Black bar, duration of odor -1.0 stimulation (sulfide category, 3 s). (B) A raster plot and a respiration-phase 1.0 ＊ 1.1 Ipsi 0.2 histogram of the spike responses of the neuron shown in A. Note the respi- Contra -1.0 ration phase-locked discharges during odor stimulation. (C) Raster represen- -1.8 0.6 1.0 tation and respiration-phase histograms (Bottom) of spike responses of an E-I- Ipsi ＊ Contra type AON neuron to ipsinostril-only stimulation (blue dots and bars) and -1.0 2.1 binostril odor stimulation (red dots and bars). (D) The averaged respiration- 1.0 ＊ 0.9 Ipsi 0.4 0.6 0.2 phase histograms for ipsinostril responses (blue bars) and binostril responses Contra (red bars). R.P.H., respiration phase histogram. The vertical axis indicates re- -1.0 sponse probability at each phase of a respiration cycle. 1.0 ＊ 0.7 Ipsi 0.5 0.1 0.4 0.4 Contra -1.0 1.0 1.1 C D Ipsi ＊ 0.6 0.5 -0.6 0.3 phase (blue, Fig. 3 and and Fig. S3). In addition, simultaneous Contra -1.0 odor inputs to both nostrils caused the suppression of respiration -1.5 C D 1.0＊ -1.1 phase-locked spike responses (red, Fig. 3 and ). These results Ipsi 0.4 Contra suggest that the comparison of inputs from ipsi- and contranostrils -1.0 in the AONpE occurs within a short time window during the ex- 1.0 ＊ -0.8 -0.7 Ipsi piration phase of each respiration cycle. Contra -1.0 1.0 ＊ -0.6 -0.6 -0.5 -0.7 -0.8 -0.5 -0.7 Ipsi -0.2 Computation of Differences Between Ipsinostril and Contranostril Contra -1.0 Odor Inputs. In all single-category E-I neurons examined in detail (n = 9), ipsinostril-only stimulation evoked significantly larger Fig. 2. AONpE neurons showed an E-I-type response to only a single responses than binostril odor stimulation (paired t test between odorant category. The odorant category selectivity of each neuron (#1–16) is the peak of ipsinostril- and binostril-induced responses, P = represented by the columns above and below each line. The colored and 0.0009, n = 9), suggesting that single-category E-I neurons sub- gray bars indicate excitatory and inhibitory responses, respectively, to stim- ulation with individual odorant categories. Bars above and below the line tract the contranostril inputs from ipsinostril inputs of single- indicate responses to ipsinostril and contranostril stimulation, respectively. In category odorants. all of the recorded neurons, only a single odorant category induced an E-I- The external nasal septum enabled us to independently con- type response (shown by asterisks). The numbers indicate the relative mag- trol the odor concentrations used for ipsi- and contranostril stim- nitude of responses. ulation. To examine whether odorant concentration affects the decrease in ipsinostril odor responses of single-category E-I neu- rons caused by simultaneous stimulation of the contranostril, we category E-I neurons were strictly phase-locked to the respiration A B A examined the neuronal responses to simultaneous ipsi- and con- cycle (Fig. 3 and and Fig. S3 ). In addition, we observed that the tranostril stimulation at the following odorant concentration ra- odor-induced respiration phase-locked spike responses ended just tios: 1:0, 1:1, 1:2, 0:1, and 0:2 (ipsinostril:contranostril, n =5 C A B after the cessation of the odor stimulus (Figs. 1 and 3 and ,and cells in five rats) (Fig. 4 A and B). Fig. S4). We also observed that the inhibitory responses of single- Ipsinostril-only stimulation (1:0) with sulfide-category odor- category E-I-type neurons to contranostril-only stimulation ended ants produced excitatory responses in the neuron shown in Fig. just after the cessation of the stimulation (Fig. 1C and Fig. S4). 4A. Simultaneous stimulation of the contranostril at the same In 30 single-category E-I neurons, we generated respiration concentration (1:1) greatly reduced the magnitude of the excit- NEUROSCIENCE phase histograms of spike responses to ipsinostril stimulation. The atory response. The suppression was observed in each respiration ipsinostril-induced spike responses of the cell in Fig. 3 occurred phase-locked response during the simultaneous odor stimulation consistently during the late part of the expiration phase. In a ma- (Fig. 4B). Furthermore, increasing the odorant concentration for jority of single-category E-I neurons (28 of 30 cells), the peak contranostril stimulation (1:2 in Fig. 4A) completely suppressed of ipsinostril odor responses occurred during the expiration phase the excitatory response. This suppression was also observed in (255 ± 9.0°, mean ± SEM) (Fig. S3B). Averaged spike responses to each respiration phase-locked response during odor stimulation ipsinostril odor stimulation in these 30 single-category E-I neurons (Fig. 4B). These results suggest that the degree of suppression also showed that their responses were phase-locked to expiration, of excitatory responses to ipsinostril stimulation is proportional with the peak of responses during the middle part of the expiration to the concentration of odorants used to stimulate the contra-

Kikuta et al. PNAS | July 6, 2010 | vol. 107 | no. 27 | 12365 Downloaded by guest on October 2, 2021 A B tinct positions of the odor source were observed at each respiration cycle (Fig. 4D) and in all of the E-I neurons examined (n = 5 cells). 1:0 1:1 1:2 0:1 0:2 1st resp. 2nd 3rd 4th These results indicate that single-category E-I neurons differen- ＊＊ ＊＊ ＊＊ ＊＊ ＊＊ 15 15 ＊＊ ＊ ＊＊ ＊ tiate between odor sources at different positions with reference to ＊ 10 10 ＊＊ the right and left nostrils, suggesting that E-I neurons participate in ＊＊ 5 5 the right or left localization of odor sources. 0 0 Discussion -5 -5

spikes / sec (Hz) The present results revealed that individual AONpE neurons -10 -10 ＊ ＊ ＊＊ Bi-nostril stim. ＊ showed an E-I-type response to only a single category among 10 -15 Ipsi. stim. Contra. stim. -15 categories of odorant molecules. Each AONpE neuron was strongly activated by ipsinostril and inhibited by contranostril stim- C D 1st resp. ulation. Among all of the AONpE neurons examined, simulta- 15 a c ＊ neous odor stimulation of ipsi- and contranostrils elicited a sig- 15 fi

2nd 3rd 4th ni cantly smaller response than that elicited by ipsinostril-only

＊＊ b ＊＊ 10 ＊＊ 10 ＊＊ ＊＊ stimulation. Increasing the odor concentration of contranostril stimulation caused a larger suppression of the ipsinostril response. 5 5 These results indicate that AONpE neurons detect differences in 0 0 the concentration of odorants of a single category between ipsi- spikes / sec (Hz) ab and contranostril inputs. -5 Center stim. c -5 ＊＊ By analogy with the auditory E-I neurons in the lateral superior Ipsi. stim. Contra. stim. ＊＊ ＊＊ olive (3), the above results suggest that AONpE neurons partici- Fig. 4. AONpE neurons compare ipsinostril odor inputs with contranostril pate in the localization of odor sources to the right or left side. Our inputs. (A) Responses of an E-I neuron to different ratios of odor concen- results without the external nasal septum support this hypothesis. tration for ipsi- and contranostril stimulation. Upper diagrams indicate the AONpE neurons were activated most strongly when the odor ratio (1:0, 1:1, 1:2, 0:1, and 0:2) of odor concentration (sulfide category) source was placed at the ipsilateral position, were activated mod- between ipsi- and contranostril stimulation. Bars above the zero line indicate erately when the odor source was at the central position, and were excitatory responses (mean ± SEM). Bars below the zero line indicate in- hibitory responses (ANOVA among the different ratios, significant differ- inhibited when the odor source was placed at the contralateral ences, five of five cells; posthoc analysis with Student-Newman-Keuls, *P < position (Fig. 4). Thus, the closer the position of the odor source 0.05; **P < 0.01). (B) Differential responses to stimulation of ipsi- and con- is to the ipsilateral nostril and the more distant from the con- tranostrils with different concentration ratios were observed at each respi- tralateral nostril, the stronger the response of AONpE neurons. ration cycle (ANOVA, significant differences for each respiration cycle, 20 of However, behavioral studies with selective inactivation of AONpE 20 respiration cycles in five cells; posthoc analysis with Student-Newman- using lesions or pharmacological blockers are necessary to de- Keuls, *P < 0.05; **P < 0.01). (C) E-I-type neurons differentiate the odor termine whether AONpE is essential for the directional localiza- source at three different positions relative to the two nostrils. Upper dia- tion of odor sources. The right and left nostrils are very close to gram indicates the three positions for odor stimulation: a, an ipsilateral each other and thus might not be suitable for the ipsi- and con- position; b, a central position; c, a contralateral position. Each bar indicates ± tranostril comparison of odor concentration. However, the ana- the magnitude of the responses (mean SEM) to odor stimulation at the fi corresponding positions (a–c; ANOVA across the three positions, significant tomical con guration of the rat external nose produces laterally differences, five of five cells; posthoc analysis with Student-Newman-Keuls, directed respiratory airflows, such that lateral inhalation might **P < 0.01). (D) The differential responses to distinct positions of odor source produce a functional widening of the internostril distance (15). were observed at each respiration cycle (ANOVA, significant differences for The lateral inhalation may thus help AONpE neurons to localize each respiration cycle, 19 of 20 respirations in five cells; posthoc analysis with odor sources. Student-Newman-Keuls *P < 0.05; **P < 0.01). Most of the AONpE neurons recorded in the present study were located in the ventro-caudal region of the AONpE (Fig. 1D) and showed E-I-type responses selectively to sulfide-category nostril. We also noted that the concentration of odorants for odorants, suggesting that computation of the directional locali- contranostril-only stimulation was proportional to the amount of zation of sulfide odors is performed by neurons in the ventro- suppression of spontaneous spike discharges in E-I neurons (Fig. caudal region. In agreement with this finding, ipsinostril-only A 4 , 0:1 and 0:2). The suppression was again observed in each stimulation with sulfide odorants resulted in c-fos expression in respiration phase-locked response during the contranostril odor the ventro-caudal region of the AONpE (Fig. S5). These results B stimulation (Fig. 4 ). Thus, the single-category E-I neurons ap- suggest that distinct parts of the AONpE might be specialized for pear to subtract the contranostril inputs from the ipsinostril in- E-I-type responses to different categories of odorants, although puts or ongoing activity during each respiration cycle. we do not rule out the possibility that AONpE neurons are tuned The external nasal septum might produce artificial nasal airflow to other groupings of odorants, or even to one odorant in a cate- on both sides. To address whether the single-category E-I neurons gory. Further experiments are necessary to record from neurons in detect the right or left localization of odor sources in natural the rostral and intermediate regions of the AONpE and examine conditions without the external nasal septum, we examined the their odorant specificity. responses of these neurons when the odor source was placed at an AONpE receives topographical axonal projections from the ipsilateral, central, or contralateral position with regard to the two ipsilateral OB (11, 16–18), raising the possibility that the odorant nostrils (five cells in four rats) (Fig. 4C). We first identified a single- category tuning of ipsinostril input to individual AONpE neu- category E-I-type neuron with the external nasal septum in place. rons is a result of the topography of afferent projection from the We then removed the external nasal septum and presented odor- ipsilateral OB. In accordance with this hypothesis, Johnson et al. ants of the same category at the above three positions. The single- demonstrated that sulfide-category odorants activate glomeruli at category E-I-type neuron shown in Fig. 4C showed an excitatory the ventro-caudal region in the ventral zone of the OB (19), and response to nasal stimulation from the ipsilateral position and mitral/tufted cells in these regions project axons to the ventro- a smaller excitatory response to stimulation from the central po- caudal region of the AONpE (11, 18). sition. Odor stimulation from the contralateral position elicited Fig. 5 A and B illustrates candidate neuronal circuits that could inhibitory responses (Fig. 4C). The differential responses to dis- be responsible for the odorant category tuning of inhibitory re-

12366 | www.pnas.org/cgi/doi/10.1073/pnas.1003999107 Kikuta et al. Downloaded by guest on October 2, 2021 A B ferior colliculus (25). Similarly, we speculate that information Contra-nostril inputs Ipsi-nostril inputs Contra-nostril inputs Ipsi-nostril inputs about the right and left localization of odor sources detected by single-category E-I neurons in the AONpE may be transmitted to

Contra-OE Ipsi-OE Contra-OE Ipsi-OE higher olfactory centers, in addition to the feedback connection to the contralateral OB. In agreement with this idea, behavioral studies M/T M/T M/T G M/T have shown that rats can properly respond to odor localization cues Contra-OB Ipsi-OB Contra-OB Ipsi-OB Ѝ Ѝ (2, 26). Single-cell labeling revealed that AONpE neurons send an axon collateral to AONpP (Fig. 1F), raising the possibility that AONpE neurons send information to specific subsets of AONpP

AC AC neurons via their axon collaterals, which in turn project their axons to Contra-AONpE Ipsi-AONpE Contra-AONpE Ipsi-AONpE higher olfactory centers. In contrast to the extensive research on odor Fig. 5. Candidate neuronal pathways underlying the right or left localiza- localization using bilateral comparisons of odors at antennae tion of odor sources. (A) One candidate pathway in which E-I-type responses (27, 28), relatively little is known about the neuronal mecha- are first generated in mitral/tufted (M/T) cells in the ipsilateral OB (ipsi-OB) nisms for orientation to an odor source in (2). Fur- and then transmitted to ipsilateral AONpE. G, granule cells; AC, anterior ther studies on AONpE neurons and their connection to higher commissure. (B) Another candidate pathway in which E-I-type responses are olfactory centers will provide additional clues for understand- generated by neuronal circuits within the AON. See text for details. ing the role of stereo-olfaction in odor source localization in the mammalian brain. The olfactory cortex contains many regions including the sponses of ipsilateral AONpE neurons to contranostril inputs. In AONpE, AONpP, anterior , posterior piriform one candidate pathway (Fig. 5A), contralateral AONpE neurons cortex, , and cortical amygdaloid nucleus. This send topographically organized commissural projections to the fi ipsilateral OB (17, 18, 20, 21) and form excitatory synaptic inputs study is unique in providing evidence that a speci c region of ol- on granule cells (“G” in Fig. 5A) that inhibit mitral and tufted factory cortex, AONpE, is involved in the right and left localization cells (22). Ipsinostril excitatory responses and contranostril inhib- of odor sources. Further analysis of each region of the olfactory itory responses (E-I-type responses) are first generated in mitral/ cortex may elucidate the functional differentiation between dif- tufted cells (M/T cells in Fig. 5A) through neuronal circuits in ferent regions of the olfactory cortex. the ipsilateral OB including granule cells, and the E-I-type Materials and Methods responses of mitral/tufted cells are then transmitted to ipsilateral AONpE neurons via the topographic afferent connection. How- Animals. All experiments were performed in accordance with the guidelines of the Physiological Society of Japan and were approved by the Experi- ever, the mitral/tufted cells did not show an E-I-type response to mental Animal Research Committee of the University of Tokyo. Wistar rats any of the 10 odorant categories. The results thus argue against (male, 280–350 g; Japan SLC) were anesthetized with urethane (1.2 g/kg) this model. and placed in a stereotaxic apparatus (SR-6R; Narishige) (29). Respiration Another candidate neuronal pathway underlying the E-I re- was monitored with a strain gauge to measure chest movement (TR-651T; sponses of ipsilateral AONpE neurons is the projection of con- Nihon Kohden). tralateral AONpE neurons to as-yet unidentified inhibitory neu- rons in the ipsilateral AON via the anterior commissure (Fig. 5B) Odor Delivery. For uninostril odor stimulation, thermoplastic material was (10). The activated inhibitory neurons then inhibit the ipsilateral used to construct an external nasal septum that fit the external shape of the AONpE neurons. Further intracellular recordings from AONpE rat snout. To examine the odorant category selectivity of individual AON neurons are necessary to examine whether they receive in- neurons, we used the method of placing test tubes containing diluted hibitory synaptic inputs following contranostril odor stimulation. odorants (5% in odorless mineral oil) in front of a nostril at a distance of 2 cm. It is also necessary to identify the inhibitory neurons that would To examine the response magnitude of individual AON neurons, we used a custom air-dilution olfactometer fitted with Teflon tubing, controlled by be responsible for the synaptic inhibition. AONpE neurons have a program written in Labview software (National Instruments). Ipsinostril- substantial reciprocal connections with the contralateral AONpP only stimulation (ipsi:contra, 1:0), contranostril-only stimulation (0:1), and pars dorsalis and receive additional input from the contralat- binostril stimulation (1:1) were delivered in pseudorandom order. Differ- eral AONpP pars ventralis (23), suggesting close synaptic inter- ential concentrations of odorants were produced by differential airflow di- actions between AONpE and AONpP. Thus, a possible source lution using a pair of mass flow controllers. The outlet of the Teflon tube from of the inhibitory input might be local inhibitory neurons within the olfactometer was placed 2 cm in front of the ipsinostril (recorded side) or the AONpP. the contranostril. An exhaust pipe was placed over the head of the rat to A characteristic feature of the olfactory system is that sampling remove any stray odorants. of external odor information is intermittent. Consistent with the respiration phase-dependent intermittent sampling, the spike re- Odorants. Ten categories of odorant molecules (sulfide, ester, terpene hydro- sponses of AONpE neurons to ipsinostril odor stimulation were carbon, acid, ether, ketone, aldehyde, alcohol, lactone, and phenol) were fi strictly phase-locked to the respiration cycle. No AONpE neurons used (Table S1). A mixture of ve representative odorants was used for stimulation with each category. showed sustained spike responses that outlasted the odor stimulus for more than a few respiration cycles. These results suggest that Electrophysiology. A glass micropipette (10–20 MΩ) filled with 2% Chicago Sky AONpE neurons compare ipsi- and contranostril odor inputs with- Blue 6B (Tocris Bioscience) in 0.5 M sodium acetate was inserted vertically into in each respiration cycle. In addition, the intermittent sampling μ the AON. After single-unit recordings, a negative current (20 A) was applied NEUROSCIENCE may help reduce sensory adaptation of olfactory sensory neurons for 5 min so that the recorded sites could be marked by the dye. The dye- and central olfactory circuits, thus maximizing the amount of ipsi- marked sites were examined histologically (SI Materials and Methods). The and contranostril odor information available to the AONpE for signals of single-unit activity were amplified (AB-610J; Nihon Kohden), fil- comparison at each successive respiration (24), which may enable tered (150–10 kHz; EW-610J; Nihon Kohden), and stored on a computer. the AONpE neurons to temporally follow changes in the position of odor sources. ACKNOWLEDGMENTS. We thank Drs. M. Yamaguchi and H. Nagao for critically reading the manuscript, and members of the Departments of Oto- In the auditory system, information detected by the lateral su- laryngology and Physiology for useful discussion. This work was supported perior olive concerning differences in the interaural intensity of by grants-in-aid for scientific research from Japan Science and Technology sound is transmitted to higher auditory centers, including the in- Agency, Core Research for Evolutional Science and Technology (to K.M.).

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