Odorant-Evoked Responses of Juxtaglomerular Neurons in the Mammalian Olfactory Bulb

Home , Glomerulus (olfaction), Tufted cell

* Manuscript

Odorant-evoked responses of juxtaglomerular neurons in the mammalian olfactory bulb.

RUNNING TITLE: Organization of juxtaglomer ular odorant responses.

R. Homma2,3, L.B. Cohen2,3,#, A. Konnerth1, O. Garaschuk1,*,# 1Institute of Neuroscience, Technical University Munich, 80802 Munich, Germany, 2Dept. of Physiology, Yale University, New Haven, CT, 06510, USA, 3NeuroImaging Cluster, Marine Biological Laboratory, Woods Hole, MA, 02543, USA.

* Present address: Institute of Physiology Department II, Eberhard Karls Universität Tübingen, 72076 Tübingen, Germany.

#Address for correspondence: Lawrence B. Cohen Dept. of Physiology, Yale University, New Haven, CT, 06510, USA E-mail: [email protected]

#Olga Garaschuk Institute of Physiology Department II, Eberhard Karls Universität Tübingen, Wilhelmstr. 27 72076 Tübingen, Germany. E-mail: [email protected] SUMMARY Anatomical observations suggest that juxtaglomerular neurons play a key role in shaping the input from olfactory receptor neurons in the mammalian olfactory bulb. However, their responses to odorants and the spatial organization of responsive neurons remain unclear. In in vivo recordings we show that individual mouse juxtaglomerular neurons code for many perceptual characteristics of the olfactory stimulus including odor identity, odorant concentration, odorant onset and offset, and odorant accommodation. Three basic response types were recognized, ON, OFF and INHIBITED. Monitoring a population of neurons revealed additional aspects of coding, such as the recruitment of new responding neurons as odorant concentration increased and clustering of neurons with similar response properties. Neurons with an ON response were closely clustered around glomeruli that also had an ON signal. Whereas some juxtaglomeruluar response features are directly triggered by the activity of olfactory receptor neurons, others have to be created by processing within the glomerular neuronal network.

INTRODUCTION The mammalian olfactory epithelium consists of a single layer of non-interacting olfactory receptor neurons that send their axons to glomerular neuropil in the olfactory bulb. It is in the bulb that the first stage of olfactory processing occurs. Extensive information about the time course and amplitude of the odorant evoked input to the dorsal bulb is available from measurements of calcium or synaptopHluorin in the receptor neuron nerve terminals (Wachowiak and Cohen, 2001; Bozza et al, 2004; Wachowiak et al 2004). What is now needed is information about the post-synaptic response in the bulb both at the level of individual neurons and at a glomerular scale. In the glomerular layer there are three major morphologically distinct classes of local interneurons – periglomerular cells, short-axon cells, and external tufted cells – they are collectively referred to as juxtaglomerular neurons. The information about odorants is transmitted from olfactory receptor neurons either directly or indirectly to these juxtaglomerular interneurons and to the principal neurons (mitral/tufted cells). In the mouse each glomerulus is surrounded by several hundred juxtaglomerular neurons with rich synaptic connections with each other and with the mitral/tufted cells. Synaptic interactions occur both within a glomerulus and in surrounding glomeruli (Shepherd et al, 2004). These juxtaglomerular neurons are presumed to play a significant but as yet undefined role in shaping the neural representation of odors. Previous electrophysiological measurements using in vitro slice preparations have revealed basic physiological characteristics and synaptic connections of some of the juxtaglomerular neurons (e.g. McQuiston et al, 2001; Aungst et al, 2003; Hayer et al., 2004). However, very little is known how these juxtaglomerular neurons respond to odorants and where they are located relative to activated glomeruli. Because of their relatively small cell body size, microelectrode recordings of odor-evoked responses have been reported from only four juxtaglomerular neurons (Wellis and Scott, 1990; Scott, 1991). These four were thought to be periglomerular neurons. All four cells responded with bursts of action potentials to the onset of odorant presentation. Adaptation/desensitization of the response to a repeated odorant presentation was observed in three of the four recorded cells. Two of the cells were spontaneously active and one was silent. A recent in vivo study (Petzold et al, 2008) have reported robust increases in the intracellular calcium concentration in juxtaglomerular neurons in response to odorant stimulation emphasizing the important role of these neurons in the olfactory processing. Here we combined the multi-cell bolus-loading technique (MCBL, Stosiek et al, 2003; Garaschuk et al, 2006b) and in vivo two-photon imaging to measure simultaneously the odorant-evoked Ca2+ signals of hundreds of juxtaglomerular neurons in each preparation.

3 Previous in vivo studies have shown that such somatic Ca2+ signals are often directly proportional to the number of action potentials fired by a cell (Kerr et al., 2005; Yaksi et al., 2006; Garaschuk et al, 2006a, but see Lin et al, 2007).

RESULTS In vivo identification of cells and glomeruli Using the multi-cell bolus-loading technique (Fig. 1A, green), we stained the cells and processes within a spherical volume with a diameter of approximately 300 µm. Stained tissue included the cell bodies of the juxtaglomerular neurons and glial cells and the processes of juxtaglomerular neurons, mitral/tufted cells, glial cells and the axons and nerve terminals of both the olfactory receptor neurons and neurons providing centrifugal input from higher olfactory centers (Shepherd et al., 2004). Fig. 1B shows examples of the images obtained in a preparation that was stained with two dyes, bolus loading with Oregon Green 488 BAPTA-1 AM (OGB-1) and nasal staining (Fig. 1A, red; Wachowiak and Cohen, 2001) with Alexa Fluor 594 dextran. OGB-1 staining showed regions with densely packed cell bodies and cell body free oval-shaped regions that correspond to glomeruli (Fig.1B, Oregon Green 488 BAPTA-1 AM). Alexa Fluor 594 dextran injected into the nose selectively stained the axons and terminals of olfactory receptor neurons. As expected, the terminals of Alexa Fluor 594-positive olfactory receptor neurons were found exclusively in the glomerular regions (Fig. 1B, merged image). In an effort to discriminate glial cells from neurons we attempted to label astrocytes with a cortical astrocyte marker, sulforhodamine 101 (Nimmerjahn et al., 2004). Surprisingly, using a protocol which reliably stains cortical astrocytes (Garaschuk et al, 2006b), we were unable to find any cells in the bulb labeled clearly with sulforhodamine 101. Even though sulforhodamine 101 failed to provide a label for the glial cells in the bulb, it seems most likely that the cellular calcium responses shown below originate from neurons because calcium signals generated in glial cells have longer latencies than the signals reported here (Ikegaya et al., 2005; Wang et al, 2006; Petzold et al, 2008; Schummers et al, 2008).

Three basic types of odorant-evoked calcium signals in juxtaglomerular neurons Using frame rates of 5-10 Hz we measured odorant evoked calcium signals in 46 preparations. All of the results come from single trial recordings; signal averaging was not used. The traces are presented such that an increase in calcium concentration is shown as an upward deflection. Because the odorant was presented at random times relative to the respiratory cycle of the mouse, there is a variable latency between the onset of odorant presentation and the response. This occurs both from trial to trial and from presentation to presentation in trials with multiple odorant presentations. In some trials the modulation of odorant responses by individual breaths

4 can be seen. In the absence of odorant presentation the only calcium signals we observed were infrequent events, ~ 1/min; these were always associated with deep breaths and were not studied further. We were unable to detect signals that might have been associated with action potentials in spontaneously active cells. One limitation was the peak-to-peak noise of 5% to 20% in the ∆F/F measured from individual cells. Detection of spontaneous signals will also be compromised if the spontaneous spike rate is high compared to the time for the cell body calcium signal to return to the baseline (tau 300-800 ms; see Stosiek, et al 2003; Kerr, et al, 2005). Presentations of odorants elicited intracellular Ca2+ transients both in neuronal cell bodies (Figs. 2-5) and in glomeruli (Figs. 4, and 6). Fig. 2A and B illustrates recordings from five cells and from adjacent neuropil regions. Three of the five cells responded with an increase in calcium at the onset of each of the three presentations of the odorant (Fig. 2A). Cells with increases in calcium at odorant onset were termed ON cells. ON cells were the most common responding cells. The kinetics of the change in intracellular calcium in ON neurons varied substantially (e.g., cells 1 and 3 in Fig. 4C). Some ON cell responses were prolonged far beyond the odorant offset. The ON signals we measured (Figs. 2-5) were generally somewhat slower than the calcium signals measured with the same technique in principal neurons in the cortex (Stosiek, et al 2003; Kerr, et al, 2005). Increasing the time resolution of the recording to 200 Hz by using line scans (Figure 2D, right panel) did not result in faster signals (rise times 60-1400 ms; n=10 cells, decay time constants in the range of 0.5-9 s; median 2.2 s; n=40 cells). The slower juxtaglomerular time courses may reflect the time course of the spiking activity of these cells or a difference in calcium handling between juxtaglomerular and cortical neurons. In addition to the ON response neurons, we also found cells, termed INHIBITED, that decreased their calcium at the onset of the odorant (Fig. 2D, cell 2). As exemplified in Fig. 2D, INHIBITED signals typically returned to the baseline rapidly. INHIBITED neurons are likely to be spontaneously active neurons whose activity was suppressed by odorant presentation (Lin et al, 2007; Chen et al., 2009). The third and least frequent type of response were cells that increased their calcium level after the offset of the odorant (OFF response; Fig. 2C). Experiments with three different stimulus durations (Fig. 2C, right panels) verified that these cells responded to the offset of odorant rather than to the onset of the odorant with a long delay. We also occasionally (~ 1% of responding neurons) found cells whose signal seemed to be a combination of ON and OFF responses; these were counted as ON cells. The following two results are consistent with the hypothesis that the signals we measured come from the individual cells in the image and not from generalized signals resulting from activity in regions above or below the image plane. First, we found no correlation between cellular and neuropil signals in 16 out of 19 cell-surrounding neuropil pairs examined (n=5 mice, e.g. Fig. 2B). Second, we often found adjacent cells with qualitatively different signals (Figs. 2, 4,

5 5, 6, and 7). In control experiments a local application of TTX (n=3 mice) into the imaged region reversibly blocked all the fluorescence responses showing that the signals depend on firing of action potentials. In several preparations we varied the duration of the odorant presentation from 1 to 4 seconds but were unable to detect a consistent effect of stimulus duration on the time course of ON cell responses (e.g., cell 1 of Fig. 2C). Similarly, the odorant evoked calcium signals from olfactory receptor neuron nerve terminals were also often insensitive to similar changes in stimulus duration (M. Wachowiak, L.B. Cohen, and D. Vucinic, unpublished observations). We tabulated the frequency of occurrence of the three types of odorant responses. First we examined all 46 preparations that we studied to determine the presence or absence of the three response types. All of the preparations had ON cells. 35 out of the 46 preparations had one or more INHIBITED cells; 32 out of 46 preparations had one or more OFF neurons. In addition in 20 locations in 13 preparations where two odorant concentrations were tested we categorized all of the responding neurons. In five of the measurements the odorant was isoamyl acetate, in the remaining 19 measurements it was 2-hexanone. Table 1 shows the results from 24 frames from these 13 preparations. A large majority of the responding neurons were ON neurons. At high odorant concentration (9% of saturated vapor) 312 of the 340 responding neurons were ON, 25 were INHIBITED and 3 were OFF. When the odorant concentration was reduced to 1.5% the proportion of ON neurons increased. Stability of response types. We examined the stability of the response types as a function of odorant concentration in the 24 frames whose results are shown in Table 1. 80% of the ON neurons that responded at 9% odorant concentration had an ON response at 1.5%. There was no detectable signal in the remaining 20% of the ON neurons. None of the ON cells changed its response type as concentration was reduced. In contrast, only 20% of INHIBITED cells at 9% also had an INHIBITED response at 1.5%. One INHIBITED cell changed its response to ON while the remaining neurons with an INHIBITED response at 9% had no detectable signal at 1.5%. The proportion of OFF responding neurons also decreased when the odorant concentration decreased. As a result INHIBITED and OFF cells were most frequently seen at 9% of saturated vapor. Glomerular Signals. Calcium signals in response to odorant presentation were also recorded from regions of interest covering individual glomeruli (e.g., Figs 4A and 6). We examined the recordings from 160 glomeruli in 17 preparations in response to an odorant concentration of 9% of saturated vapor. 112 (70%) of the glomeruli had a detectable response to the odorant. The signals from the responding glomeruli had a similar distribution of response types as the cellular signals; 87% were ON, 11% were INHIBITED, and 2% had OFF responses.

Consistency of response to repeated trials Next we examined the consistency of the odorant-evoked calcium transients in individual neurons. Fig. 3 illustrates one such experiment. In 75% of neurons the responses were quite similar from trial to trial (white circles; response amplitudes differ by less than two standard deviations of the noise band) In other cells larger (red) or smaller (blue) responses were found in trial 2 compared to trial 1. These are cells whose response amplitudes changed by an amount larger than two standard deviations of the noise. The noise in the measurement, taken from the time period prior to the odorant presentation, is indicated by the + symbols near the origin in Fig. 3B. There is considerable spread of the data points about the least squares line (slope =0.98, y-intercept fixed at 0.0) which is near the diagonal (slope = 1). The amplitude of the noise deviations from the line are smaller than the deviations of the odorant responses (p < 0.01, paired t test). Thus the response differences in Fig. 3B are not simply the result of noise in the fluorescence measurements. Similar data were obtained in 13 experiments at two different odorant concentrations. At 1.5 % of the saturated vapor the mean slope of the fitted lines was 0.98 + 0.04 (n=5) and at 9% the mean slope was 0.95 + 0.03 (n=8). Thus, even though the responses of individual cells may vary considerably, the average of all the responding neurons was remarkably constant from trial to trial.

Concentration - response relationship of ON neurons In several preparations (n=4) we measured the response to odorant concentrations ranging from 0.1% to 9% of the saturated vapor. One such experiment is illustrated in Fig. 4. Fig 4A, right panel, shows the concentration dependence of the response of a large region containing many juxtaglomerular neurons. There is no detectable average signal at 0.1% of saturated vapor. The average signals at higher concentration show a clear indication of modulation by individual odorant inhalations. Fig. 4C illustrates the responses of three individual neurons whose locations are indicated by arrows in the top frame of Fig. 4B. Cell 1 is very sensitive and has an easily detectable signal at 0.1% of saturated vapor. The concentration dependence of the three cells in Fig. 4C is plotted in Fig. 4D together with the average of all of the cells (red). Cells 1 (green) and 3 (black) were chosen because they represent the cells that required the lowest and highest odorant concentration to give a 50% response. Fig.4B illustrates the concentration dependence of the responses of all the responding neurons in the recorded frame. Red represents the largest signals, fractional changes, ΔF/F, of between 50% and 70%. Blue, ΔF/F < 10%, indicates no detectable response or a very small response. A ΔF/F value of 10% is near the noise level in these trials. Recruitment. In the experiment illustrated in Fig. 4 increasing odorant concentration

7 resulted in a marked recruitment of neurons with detectable responses. Similar data were obtained from five different locations in four preparations (Table 2). There was a twenty-fold increase in the number of active cells detected per frame as the odorant concentration increased. Much of the increase occurred between 0.1% of saturated vapor and 1.5%. Although these five frames were from locations that had relatively sensitive odorant responses, they are unlikely to be the most sensitive glomeruli because only a small fraction of the dorsal glomeruli were examined in each preparation and only two odorants were tested.

Clustering of different desensitizing responses to repeated odorant presentations Three of the four juxtaglomerular neurons monitored electrophysiologically (Wellis and Scott, 1990; Scott, 1991) had declining responses to repeated odorant presentation. We also observed a similar desensitization/accommodation/habituation in some neurons when multiple odorant pulses were presented with an inter-stimulus interval of 10 seconds or less. Fig 5B illustrates the responses of eight neurons to three two-second-long odorant presentations with a nine second interval from the start of one stimulus to the start of the next stimulus. The top four neurons had ON responses that did not desensitize, the bottom four had ON responses that declined with repeated odorant presentations. Many additional neurons in the recorded frame had responses that were similar to the cells shown in Fig. 5B. Cells with desensitizing responses are indicated in red in Fig. 5C, cells with non-desensitizing responses are indicated in green. The desensitizing and non-desensitizing cells are clustered. Similar desensitization and clustering was seen in three additional recordings made at the same location in this preparation and in two additional preparations where cells with differing desensitization properties were found in the same frame. There were no counterexamples where cells with different desensitization rates appeared to be intermingled.

Clustering of cells that are odorant selective In one preparation we found a region containing several glomeruli which selectively responded to only one of two different odorants. This result is illustrated in Fig. 6A, B. Glomerulus 4 had an ON response to isoamyl acetate but no response to 2-hexanone while glomeruli 1 and 2 had ON responses to 2-hexanone but no response to isoamyl acetate. Glomerulus 3 appears to have an INHIBITED response to both odorants. This situation allowed us to determine the spatial relationship of responding glomeruli and responding neurons. Fig. 6C, D shows that the ON cells (red) are clustered near the glomeruli with ON responses. Similar clustering of ON cells around ON glomeruli was seen in all other preparations examined (14 glomeruli in 10 mice). The clustering of INHIBITED cells (blue) in Fig. 6 is less clear in part because there are fewer cells with an INHIBITED response. Some are near the INHIBITED glomerulus but others are relatively

8 distant. We examined the relationship of INHIBITED cells and glomeruli in four preparations with a total of 12 INHIBITED cells; 9 of the 12 INHIBITED neurons were close to INHIBITED glomeruli. Thus, it seems that both ON and INHIBITED cells tend to be located close to glomeruli of the same response type (see also Fig. 7). The result illustrated in Fig. 6 came from measurements at one Z position. We made similar pairs of measurements at an additional seven Z positions each separated by 10 µm to obtain a more complete recording of activity in this glomerular region. Fig. 7 shows a 3-dimensional presentation of the results obtained with the two odorants (viewed from three different viewing angles). The glomeruli are lightly colored according to their response to the odorants; ON = red, INHIBITED = blue, no response = gray. Three additional glomeruli were found in the additional Z-locations. The additional glomerulus at the top right had an ON response to 2-hexanone but not to isoamyl acetate and the glomerulus in the upper middle had an INHIBITED response to 2-hexanone but not to isoamyl acetate. A movie showing the rotation of the two reconstructions is included as Supplemental movie 1. Again, both the ON cells and the INHIBITED cells are located near glomeruli that have similar responses. There tends to be ON cells separating ON and INHIBITED glomeruIi. In Fig. 6 there is only one instance of overlap between the ON cells that respond to 2-hexanone and the ON cells that respond to isoamyl acetate. However, three cells that were INHIBITED by isoamyl acetate were also INHIBITED by 2-hexanone. When this analysis was carried out on all eight z-locations of the preparation illustrated in Figure 7, we found that only 11 neurons had an ON response both to 2-hexanone and isoamyl acetate. They corresponded to 8% of 139 neurons that had an ON response to 2-hexanone and 14% of 77 neurons that had an ON response to isoamyl acetate. In contrast, 12 neurons with an INHIBITED response to both odorants were 71% of 17 neurons that had an INHIBITED response to 2-hexanone and 75% of 16 neurons that had an INHIBITED response to isoamyl acetate. Thus there is only a small overlap in the neurons with ON responses but a large overlap in the neurons with INHIBITED responses. Surprisingly, four of the neurons in Fig. 7 with an INHIBITED response to one odorant switched to an ON response with the second odorant. Thus there are two kinds of odorant specificity: neurons that respond to one odorant but not the other and neurons that switched response type (e.g. from ON to INHIBITED) when the odorant was changed.

DISCUSSION Using in vivo two-photon Ca2+ imaging we were able to characterize the odor-evoked responses of a large number of juxtaglomerular neurons in the mouse main olfactory bulb. Our data revealed three functionally distinct types of juxtaglomerular cells – ON, OFF, and INHIBITED. ON

9 neurons represented the majority of odor responsive cells. The ON cells had different response kinetics as well as desensitization/accommodation properties. Neurons with the same response type were frequently clustered. In many cases, the clusters had the same response type as a nearby glomerulus. Moreover, our data show that the response type of neurons (ON, INHIBITED or OFF) can be stimulus-dependent and code for (i) the identity of the odorant as well as (ii) its concentration (Figs. 4, 6, 7, Table 1, Table 2). We were unable to distinguish the three major juxtaglomerular cell types or any of the periglomerular subtypes (Antal et al., 2006; Aungst et al, 2003; Kosaka and Kosaka, 2005, 2007; Puopolo and Belluzzi, 1998; Panzanelli et al., 2007; Parrish-Aungst et al., 2007) in our recordings in part because bulk loading does not reveal the processes of neurons. In the future it may be possible to correlate response type and cell type using transgenic mice that express GFPs in individual cell types (Pignatelli et al., 2005; Garaschuk et al., 2006a; Sohya et al., 2007, Yaksi and Friedrich, 2006).

INHIBITED responses in cells and glomeruli While the existence of ON responses in the juxtaglomerular neurons could have being anticipated based on electrophysiological data (Wellis and Scott, 1990; Scott, 1991), the existence of INHIBITED and OFF responses was largely unexpected. In INHIBITED cells and glomeruli there was a decline in the calcium level upon odorant presentation. We presume that we detect INHIBITED responses in juxtaglomerular neurons that maintain an elevated calcium level as a result of ongoing spontaneous activity and this activity is suppressed in response to odorant presentation. In addition to the s pontaneous activity reported in vivo by Wellis and Scott (1990), in vitro measurements show that both external tufted cells and dopaminergic periglomerular neurons are spontaneously active (Hayer et al. 2004; Pignatelli et al, 2005; Puopolo et al., 2005, Liu & Shipley 2008). Furthermore, Lin et al, (2007) observed an odor-evoked decline of intracellular calcium levels that was accompanied by a suppression of spontaneous firing of mitral and granule cells in Xenopus tadpoles. INHIBITED neurons are likely to be substantially underrepresented in our recordings because inhibition will be difficult to detect in neurons that are not spontaneously active. From in vitro measurements it is roughly estimated that only 20% of juxtaglomerular neurons are spontaneously active (Parrish-Aungst et al., 2007; Hayer et al., 2004; Pignatelli et al., 2005; Puopolo et al., 2005). If all juxtaglomerular cell types receive a similar inhibitory input, then inhibition could be underrepresented by as much as five fold. Underrepresentation of inhibition will also mean an underrepresentation of the number of neurons that switched from ON to INHIBITED when the odorant was changed in the experiment illustrated in Fig. 7. We observed a net odor-evoked INHIBITED response in glomerular regions in 11% of

10 the glomeruli with a detectable response. This inhibitory signal is likely to be from the processes of spontaneously active juxtaglomerular and mitral/tufted cells because inhibitory responses have not been observed in the calcium recordings from olfactory receptor neuron axon terminals (Wachowiak and Cohen, 2001, 2003; Wachowiak et al, 2004; Vucinic et al, 2006). Thus we conclude that a non-trivial fraction of the glomerular signal comes from the dendritic processes of juxtaglomerular and mitral/tufted neurons (see also Chen et al., 2009). The inhibitory responses in glomeruli and in juxtaglomerular cells may not indicate inhibition of the glomerular output. Because many of the juxtaglomerular neurons are themselves inhibitory, inhibiting an inhibitory cell can result in disinhibition and thereby lead to an increase in glomerular output. Chaignau et al. (2007) did not report inhibitory responses in their measurement of odor-evoked glomerular calcium signals using two-photon microscopy and G-CaMP2 mice (Diez-Garcia et al., 2005) that express genetically encoded calcium indicator in mitral cells and some juxtaglomerular neurons. One possibility is that the cell types that contribute to the inhibitory response we observed and cell types that express G-CaMP2 are different.

Comparisons with electrophysiological recordings in vivo Because of their relatively small cell body size, odorant responses have been recorded in only four juxtaglomerular neurons (likely to be periglomerular cells) using microelectrodes (Wellis and Scott, 1990; Scott, 1991). These intracellular recordings revealed that (i) some cells show non-desensitizing bursts in response to odorant presentation and others show a desensitizing response (ii) some cells fire spontaneously at various rates and others don’t. We also found cells showing desensitization at different rates as well as cells showing no desensitization and we presume that INHIBITED neurons are spontaneously active. Because we found that INHIBITED and OFF responses are seen much less frequently than ON responses (Table 1), it is not surprising that INHIBITED or OFF cells were not seen in the earlier study. We found many cells that responded to only one of two odorants we tested (e.g. Figures 6 and 7) even at relatively high odorant concentrations and a few that responded oppositely to the two odorants. Thus, juxtaglomerular cells can have striking odorant specificity. Because our odorant set was limited, we have no insight about the tuning width of juxtaglomerular neurons.

Clustering In both the basic ON response and in desensitization types, neurons with similar responses were usually clustered and, further, it was often observed that the clustered cells were near a glomerulus with the same response type. The clustering around a glomerulus suggests that the response of ON juxtaglomerular neurons is dominated by intraglomerular relative to

11 interglomeular interactions. Several different synapses may be involved. First, many periglomerular neurons receive direct excitatory input from olfactory receptor neurons. Second, Hayer et al. (2004) found that the short-axon cells and the majority of periglomerular neurons receive excitatory input from external tufted cells in the same glomerulus. Third, Murphy et al. (2005) showed that a single mitral/tufted cell can excite an ensemble of periglomerular cells in the same glomerulus. In some instances we found clusters of cells of similar response type with no related glomerulus in the X-Y plane that was imaged. In these cases it is possible that a related glomerulus was present at adjacent depths. Only a small fraction of ON cells are located at a considerable distance from an ON glomerulus e.g. Fig. 6 & 7. Consistent with this observation we found that there was a only a 10% overlap in the ON cells that responded to 2-hexanone and isoamyl acetate in Figure 7. In contrast, both odorants inhibited a common glomerulus (number 3 in Figure 6a) and the overlap of INHIBITED cells responding to the two odorants was large, about 70%. The INHIBITED cells and glomeruli tend to be located outside of the ring of ON cells that surround ON glomeruli (Fig. 6-7). We speculate that if a single glomerulus were activated, INHIBITED cells and glomeruli would form a second ring surrounding the ON cells. Such an inhibitory surround could result from interglomerular inhibition of juxtaglomerular neurons (e.g. INHIBITED (i), Figure 8). Using multi-cell bolus-loading technique in cat visual cortex, Ohki et al. (2005, 2006) demonstrated that neurons were arranged with a single cell precision according to their stimulus preference. The clusters of ON juxtaglomerular neurons in the mouse olfactory bulb often appeared to be as precise as clusters of direction-selective cells in the cat cortex (Fig. 5 - 7) although the anatomical structures that support these functional units are very different.

Possible glomerular circuit models In Fig. 8, we present several circuit models that can account for the observed ON, INHIBITED and OFF responses. Based on in vitro physiological measurements (Aungst et al, 2003; Hayer et al, 2004; Murphy et al, 2005; for review, see Wachowiak and Shipley, 2006) the sources of excitatory synaptic signals for the ON response could be inputs directly from olfactory receptor neurons, disynaptic inputs from external tufted cells and mitral/tufted cells, or polysynaptic inputs via interglomerular connections of short-axon cells. Neither the OFF response nor the INHIBITED response are likely to simply echo the input from olfactory receptor neurons in the same glomerulus because neither OFF nor INHIBITED responses have been reported in calcium imaging of activity in receptor neuron axon terminals (Wachowiak and Cohen, 2001, 2003; Wachowiak et al, 2004; Vucinic et al, 2006). Furthermore, the INHIBITED response requires

12 inhibitory inputs whereas all olfactory receptor neurons are thought to be glutamatergic and excitatory (Shepherd et al, 2004). For the INHIBITED responses, we assume that the neurons are spontaneously active and receive inhibitory synaptic inputs. Both external tufted cells and dopaminergic periglomerular cells are spontaneously active. Inhibitory inputs could be provided by GABAergic periglomerular cells intraglomerularly or interglomerularly. Another possibility is that the spontaneous firing of dopaminergic periglomerular cells is suppressed by acetylcholine, the neurotransmitter used by centrifugal input to olfactory bulb (Pignatelli and Belluzzi 2008). A possible model for OFF cells is the following: if a neuron receives approximately balanced inhibitory and excitatory inputs during the odorant presentation and the excitatory inputs are prolonged while the inhibitory inputs turn off quickly (Fig. 2 ) the result will be a net excitation beginning at odorant offset. Fig. 8 shows three different combinations of inputs that could lead to such an OFF response. At present several different models can explain the three response types and other, more complex, models are also possible. To further refine the proposed model the following strategies could be used: (i) recording from identified genetically-labeled cell types, (ii) using dual color calcium recordings from pre- and postsynaptic neurons, and (iii) by pharmacological manipulations. For example, blocking cholinergic receptors would test the role of centrifugal inputs.

Functional and organizational implications Our findings show that individual juxtaglomerular neurons code for several perceptual characteristics of the olfactory stimulus such as (i) odor quality, (ii) odorant concentration, (iii) odorant onset and (iv) offset. The activity of individual neurons also carries information about repeated odorant presentations like those that an animal might encounter when following an odorant plume. Some juxtaglomerular cells respond equally to repeated presentations (useful in recognizing a plume) while others rapidly desensitize (useful for recognizing a new odorant). Although all of the ON cells code for the onset of the odorant, the time course of ON responses can vary substantially from neuron to neuron (e.g., cells 1 and 3 in Fig. 4). The different time courses could be used by the nervous system to generate time-dependent aspects of odor perception such as accommodation and persistence. The strength of the stimulus (odorant concentration) is coded both on the level of single cells (increasing amplitude of Ca2+ transients, Fig. 4) and on a population level (recruitment of new responding neurons; Fig. 4 and Table 2). The amplitude of the response of an individual juxtaglomerular neuron sometimes varied substantially from trial to trial (Fig. 3). Noisy responses of individual neurons are expected and are not unique to mouse juxtaglomerular neurons (e.g., Drosophila (Bhandawat et al 2007), Aplysia (Wu et al, 1994), and cat and monkey visual cortex (Heggelund and Albus, 1978;

13 Tolhurst et al, 1983)). One possible mechanism for generating consistent behavioral responses from noisy individual neurons would be to have many noisy neurons synapse onto post-synaptic cells that are closer to the behavioral output (e.g., Bhandawat et al 2007). Among juxtaglomerular neurons the response of individual neurons was noisy but the averaged amplitude of all of the responding neurons was relatively stable; the mean trial-to-trial change in the averaged response was only 2-5%. In an analogous situation in primate motor cortex Georgopolous et al., (1986, 1988) found that the average of the activity of many neurons was a good predictor of movement direction even though the activity of individual neurons was a relatively poor predictor. Although inhibitory responses are seen in ~5% of individual mammalian olfactory receptor neurons (Duchamp-Viret, et al, 1999), neither INHIBITED nor OFF responses have been reported in the averaged input to individual glomeruli (Wachowiak and Cohen, 2001, 2003; Wachowiak et al, 2004; Vucinic et al, 2006), In contrast, mitral/tufted cell activity (the bulb output) can show very complex time courses that include inhibitory phases (Wellis et al., 1989; Luo and Katz, 2001; Nagayama et al, 2004). The complexity of the functional responses of juxtaglomerular cells (ON, OFF and INHIBITED) suggests that they may play a significant role in shaping the odor-evoked response of the mitral/tufted cells. INHIBITED responses were seen more frequently at higher odorant concentration. This result suggests that one role of the juxtaglomerular network might be to adjust the strength of odorant responses to increase the range of odorant concentrations over which the mitral/tufted cells can respond to odorants.

EXPERIMENTAL PROCEDURES Animal preparation BALB/c mice, 22-35 days old at the time of recording, were anesthetized by intraperitoneal injection of a mixture of Ketamine/Xylazine (80/8 or 80/4 µg/g of bodyweight for the initial injection). Anesthetic depth was monitored by toe pinch and additional Ketamine/Xylazine was injected occasionally to maintain the depth of anesthesia. The animal breathed freely throughout the experiment. The respiration rate was monitored using a pressure sensor attached to the body of animal and we attempted to keep it between 160 and 200 cycles per minute. Pure oxygen gas was supplied to maintain the animal’s physiological state when the respiration rate of animal fell below 100 per minute. The rectal temperature was kept between 37.0°C to 38.5°C. The skin covering the dorsal part of the skull was trimmed following a subcutaneous injection of local anesthetics (2% lidocaine or 0.5% bupivacaine) and a chamber with a hole in the center (Garaschuk et al., 2006b) was attached with cyanoacrylic superglue to the skull so that the hole was over the recording site in the olfactory bulb. The microscope stage held the

14 chamber firmly during the recordings. The skull above the dorsal bulb was thinned using a dental drill and then a craniotomy (typical size about 1mm x 0.5mm) was made using a 30 gauge syringe needle. Immediately after the craniotomy, perfusion of extracellular solution (in mM: 125

NaCl, 4.5 KCl, 26 NaHCO3, 1.25 NaH2PO4, 2 CaCl2, 1 MgCl2, and 20 glucose, pH 7.4, bubbled with 95%O2 and 5%CO2) warmed to 38°C was started inside the chamber covering the exposed dura with the solution. The dura was left intact. The chamber opening was then covered by 2% agarose to suppress the movement of brain during the recordings. The experimental protocols were approved by the Institutional Animal Care and Use Committees of the Marine Biological Laboratory, Yale University, and the Technical University of Munich.

Multi-cell bolus-loading The general procedure of multi-cell bolus-loading was described previously (Stosiek et al., 2003; Garaschuk et al., 2006b). To prepare the dye solution, 10 mM Oregon Green 488 BAPTA-1 AM (Invitrogen, Carlsbad, CA) or Fura PE-3 AM (TEFLAB, Austin, TX) was prepared by dissolving the dye powder in 20%w/v Pluronic F-127 (Invitrogen) in DMSO. Then the mixed solution was diluted 10-50 times with calcium and magnesium free pipette buffer solution (in mM: 150 NaCl, 2.5 KCl and 10 HEPES). The dye solution was injected into the brain tissue through a patch pipette using a Picospritzer II (8-10 psi for 1-2 min; General Valve, Fairfield, NJ). We injected dyes at one to four sites in order to cover most of the cranial window. We could record calcium signals for up to 8 hours. In several experiments, the dye solution also contained the cortical astrocyte marker sulforhodamine 101 (Sigma-Aldrich, St. Louis, MO, 0.023 %w/v final concentration).

Nose-loading with dextran dye The olfactory receptor neurons were stained with the fluorescent dye Alexa Fluor 594-dextran (Mw 10,000, Invitrogen) following the methods described in Wachowiak and Cohen, 2001. The animal was anesthetized by intraperitoneal injection of a mixture of Ketamine/Xylazine (80/8 µg/g of bodyweight). The dye solution was composed of 4%w/v dye and 0.06%v/v Triton X-100 dissolved in distilled water. 10 µl of dye solution was slowly injected in two portions a few minutes apart through a microloader pipette tip (Eppendorf, Westbury, NY) inserted into the nostril. The animal was used for the two-photon recording four or five days after the nose injection. We subsequently found that this loading protocol was not optimal for reliable staining. Better staining was obtained with the following: an injection of 2µl of dye solution, 4% dye and 0.25% Triton X-100, was followed a minute later by another injection of 2µl of dye solution alone.

Two-photon imaging

15 The two-photon imaging system was an Olympus FluoView FV-300 laser-scanning system combined with an Olympus BX51 fluorescence microscope using a 16x N.A. 0.80 (Nikon, Melville, NY), a 20x N.A. 0.80 (Zeiss, Thornwood, NY), a 40x N.A. 0.80 (Nikon), or a 60x N.A. 1.0 (Nikon) water-immersion objective. For calcium signal imaging, the bi-directional scanning mode was used with a zoom factor of either 2 or 4. We first used the lower magnification objective to search regions showing a large odor-evoked response and then recorded from those regions with the higher magnification objectives. A mode-locked Ti:sapphire pulsed-laser (MaiTai, Spectra-Physics, Mountain View, CA) was used for excitation. Most measurements were made at 800 nm. In the majority of experiments, a device for the compensation of the group velocity dispersion (Femto Control, APE, Berlin) was used to compensate for the lengthening of the laser pulse which occurred through the light path. In one commonly used setting, a combination of 40x objective and zoom factor of 2, the frame dimension ranged from 176 x 66 µm2 (512 x 192 pixels) to 176 x 134 µm2 (512 x 388 pixels), where the pixel size was 0.35 x 0.35 µm2. Scanning rates were 5-10 Hz depending on the height of the frame. To achieve higher temporal resolution recordings, we also used line scanning at 200Hz.

TTX application For the local application of TTX, a patch pipette filled with 1-2µM TTX (Alomone labs, Jerusalem, Israel) in the pipette buffer solution was moved to the region of interest. TTX was applied by application of a pressure pulse (10psi, 20sec) provided by the Picospritzer II. The pipette solution also contained the fluorescent dye, Alexa Fluor 594 free salt, 20µM, (Invitrogen) used to visualize the ejected solution.

Odorant delivery In an individual trial, from one to five pulses of odorant were applied through a custom-made flow-dilution olfactometer (Vucinic et al, 2006). Trial durations were 10-50 seconds. The timing of the odorant presentations was controlled by pulses from a stimulator. The trials were separated by at least 60 sec to avoid adaptation. The typical duration of odorant pulses was 2 seconds and the inter-stimulus intervals were 8 sec when a three pulse per trial protocol was used. The odorants were monomolecular compounds that were known to evoke activity in the dorsal glomeruli (Wachowiak and Cohen, 2001; Vucinic et al, 2006). All the odorants were purchased from Sigma-Aldrich and of highest commercially available purity.

Data analysis Data inspection during the experiment was carried out using Fluoview (Olympus, Tokyo, Japan), and NeuroPlex (RedShirtImaging LLC, Decatur, GA) software. Detailed data analysis

16 was performed using a combination of Fluoview, NeuroPlex, ImageJ (http://rsb.info.nih.gov/ij/) with WCIF plug-in (Wright Cell Imaging Facility, Toronto, Canada), MetaMorph (Molecular Devices, West Chester PA) and Excel (Microsoft, Redmond, WA), as well as custom made routines written for Labview (National Instruments, Austin, TX), Igor (WaveMetrics, Portland, OR), or IDL (ITT, Boulder, CO). Two complementary methods of initial data analysis were used. In the first method, responsive cells were found using a frame subtraction procedure. Ten to 20 consecutive frames (1 to 2 seconds in actual duration) corresponding to the peak of odor-evoked calcium responses were averaged. The same number of frames taken from the period just before the odorant presentation were averaged and then subtracted from the average during the response. The resulting subtracted image highlights the pixels showing a change in fluorescence (either negative or positive) in response to an odorant. To search for OFF responses, the frame subtraction was performed between the periods just after and just before the stimulus offset. The group of responding pixels with the size of a cell or glomerulus was identified as a responsive cell or glomerulus and time courses of fluorescence intensity from those structures were used for further analysis. Frame subtractions were performed with NeuroPlex software. This method was used for generating the data reported in Tables 1 and 2. In the second method, the locations of all the cell bodies were extracted manually from a high quality image made by averaging all the frames in a trial. The fluorescence recordings from the regions of interest containing the light from each of cells were then examined for odorant responses. In the manual extraction all identifiable cells and glomeruli were delineated using ImageJ. The pixel values over the delineated area of each cell or glomerulus was retrieved from each frame. Reasonably consistent results were obtained when a data set was analyzed with the two methods. The frame subtraction identified the responsive cells rapidly and occasionally identified cells that were difficult to discern from the high quality image. On the other hand, it failed to extract a small population of cells with small signals and it does not provide information about the number of non-responsive cells. To calculate a fractional fluorescence change, ∆F/F, the fluorescence, F, was first background-corrected by subtracting the intensity value measured in large blood vessels in the imaging frame. Then the changes in the fluorescence intensity were divided by the corrected fluorescence intensity. Temporal and spatial filtering. In some experiments the time course data was temporally smoothed with a binomial (1-2-1) low-pass filter. A spatial low-pass filter that replaced each pixel with the mean of a 3x3 pixel region surrounding the pixel was used to produce the frame subtraction images. Sophisticated algorithms have been developed for reconstructing spike trains from neuronal

17 calcium signals (Kerr et al, 2005; Yaksi et al. 2006; Sato et al, 2007). However, because the time course of the fluorescence changes we measured are relatively smooth and responses to individual spikes have not been detected, we did not attempt to use these reconstruction methods. The rise times of odor-evoked calcium transients were determined as the time over which the signal rose from 10% to 90% of its maximal amplitude. Measured values are given as mean ± SEM.

ACKNOWLEDGMENTS

We thank Avrum Cohen for preparing Figure 7 and the supplementary movies. Supported by NIH grants DC05259 and NS42739, Deutsche Forschungsgemeinschaft (GA654 and SFB 596) and the Bundesministerium für Bildung und Forschung (NGFN-2).

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FIGURE LEGENDS Figure 1. Functional calcium imaging of the juxtaglomerular neuronal network. (A) Schematic drawing of the a multi-cell bolus loading technique. We loaded cells in the glomerular layer nonspecifically with a calcium indicator Oregon Green 488 BAPTA-1 AM (green) or Fura-PE3 AM. In a few experiments we also injected a fluorescent marker Alexa Fluor 594 dextran (red) into the nose and selectively stained the olfactory receptor neurons. We allowed four days for the dye to travel to the nerve terminals in the glomeruli. (B) Images from the two-photon microscope. Left panel, axon terminals loaded with Alexa Fluor 594 dextran. Middle panel, cell bodies and processes non-specifically stained with Oregon Green 488 BAPTA-1 AM. Glomeruli rarely had internal cell bodies. Right panel, merged image. Axon terminals are seen only in the glomerular region.

Figure 2. Three distinct odorant response types. (A) An image of the recorded area on the left. Five representative cells were chosen and the time courses of their calcium dye fluorescence is shown in the right. Three odorant presentations are indicated as thick black bars below the bottom trace. Some cells (1, 3, and 5) increased their calcium level in response to the onset of odorant presentation (ON cells). (B) A subregion of (A) indicated by a dashed rectangle. Two regions of interest were chosen so that they are adjacent to but outside of cells 1 and 5 in panel (A). The fluorescence from those two regions is shown below the image. The neuropil in those regions did not respond to odorant whereas the two cells did respond. (C) Two cells from another preparation. The left and center columns show that while Cell 1 responded to the onset of odorant, Cell 2 responded to the offset of odorant. The overlaid traces are shown in the right column. The changes in odorant duration changed the latency of the OFF response, showing that the cell responded to the offset of odorant rather than the onset of odorant with a delay. (D)The intracellular calcium concentration in Cell 1 (ON) increased in response to the onset of stimulus while the intracellular calcium concentration in Cell 2 (INHIBITED) decreased suggesting that the spike activity of the cell was reduced. The right column in (D) shows time course measurements in a subsequent trial with a 200 Hz temporal resolution using a line scan. Here and below the calcium indicator dye was Oregon Green 488 BAPTA-1 AM unless otherwise indicated.

Figure 3. Consistency of odorant-evoked response with repeated trials. (A) and (B) show the locations and response amplitudes for cells which had larger (red) or smaller (blue) responses in trial 2 compared to trial 1. These are cells whose response amplitudes changed by an amount larger than two standard deviations of the noise in the

24 measurement. Cells with amplitude changes of less than two standard deviations are indicated in white. The noise in the measurements, taken from the time period prior to odorant presentation, is indicated by the + symbols near the origin in B. The noise was obtained using the difference between the light intensity averaged over two time windows of one second taken from the pre-stimulus period. The standard deviation of the noise (+ symbols) was calculated along the axis perpendicular to the diagonal line. The odorant was 9% of saturated vapor of 2-hexanone.

Figure 4. Concentration dependence of odor-evoked calcium signals. (A) A region of interest was selected from a glomerulus (GL) and a second region of interest was selected from an area containing many juxtaglomerular neurons (JG). The time courses of the odor-evoked calcium signals averaged over each of the regions are shown in the right hand traces. Here and below the thick black bar under the bottom trace indicates the timing of the odorant presentation. For all traces, the odorant was 2-hexanone with the concentration indicated at the left of the traces. (B) Peak response amplitude of individual cells is shown using a color code that is presented in the bottom panel. The four images correspond to four different concentrations of 2-hexanone. The highest concentration (top panel) recruited more neurons and larger signals than the lowest concentration (bottom panel). The rate of increase in signal size as a function of odorant concentration differed from neuron by neuron. (C) Time courses of odor-evoked response to all four concentrations are presented for three representative neurons indicated with arrows in the top panel of (B). (D) Concentration-response relationships are plotted for the three representative neurons and the mean response across all 34 analyzed neurons. The curves represent results of curve fitting with the Hill equation. Note that the juxtaglomerular neurons have different odorant sensitivities and maximum response amplitudes.

Figure 5. Neurons with similar desensitization properties are clustered. The time courses of the responses (B) to three sequential odorant presentations for eight representative neurons marked with respective numbers in (A). Gray shadings indicate the timing of odorant presentation. The top four traces show ON cells with little or no desensitization; the bottom four cells have desensitizing responses. One pass of the binomial low-pass temporal filter was used (see Experimental Procedures). In (C) the responses of additional neurons in the image are presented using colors which represent their type of desensitization. The non-desensitizing cells (green) and the desensitizing cells (red) are, with some exceptions, clustered spatially.

Figure 6. ON cells are found near ON glomeruli. (A, B) Fluorescence traces illustrating responses of the four adjacent glomeruli (marked with

25 corresponding numbers in (A)) to 9% 2-hexanone (right) and 9% isoamyl acetate (left). Gray shadings indicate the timing of odorant presentations. Glomerulus 4 had a ON response to isoamyl acetate but no detectable response to 2-hexanone. In contrast glomeruli 1 and 2 had an ON response to 2-hexanone but no detectable response to isoamyl acetate. Glomerulus 3 had an INHIBITED response to both odorants. (C, D) Images showing the location of cells with an ON response (red) or an INHIBITED response (blue) to isoamyl acetate (C) and to 2-hexanone (D). Red and blue shadings delineate the glomeruli with ON and INHIBITED responses, respectively. Almost all of the ON neurons are found near ON response glomeruli. There is very little overlap in the ON neurons but three neurons with an INHIBITED response to isoamyl acetate are also inhibited by 2-hexanone. The calcium indicator dye was Fura PE-3 AM.

Figure 7. Distribution of ON, OFF, and INHIBITED neurons and glomeruli in a 3-D volume. The pair of measurements illustrated in Figure 6 was repeated at eight different depths separated by 10 µm. Three images from two 3-D volume reconstructions using the two odorants, isoamyl acetate (top row) and 2-hexanone (bottom row) are shown. Small spheres with high saturation colors indicate ON (red), OFF (green), and INHIBITED (blue) neurons and the larger translucent shapes with low saturation colors indicate ON (red), INHIBITED (blue) and non-responsive (gray) glomeruli. For both odorants ON neurons tend to be near ON responding glomeruli whereas INHIBITED neurons tend to be located relatively far from ON glomeruli. The three images show the same volume from different viewing angles. The readers see the volume from the outside of the olfactory bulb; the front in the volume corresponds to the superficial part of glomerular layer and the back is closer to the center of olfactory bulb. The calcium indicator dye was Fura PE-3 AM.

Figure 8. Simple circuit models that are consistent with the three types of response. Circuit models that explain ON, INHIBITED, and OFF responses. ON responses could be echoes of receptor neuron input mediated by one of several possible monosynaptic or polysynaptic pathways. The INHIBITED neurons are presumed to be spontaneously active and thus are presumed to be external tufted cells or dopaminergic periglomerular neurons. Three models are proposed for the INHIBITED response. (i) inhibitory synaptic inputs from axons of periglomerular cells in a neighboring glomeruli excited through an intraglomerular circuit (left). (ii) Acetylcholine inputs from axons of centrifugal cholinergic cells (center). (iii) inhibitory dendro-dendritic input from periglomerular cells that are activated by short-axon cells in the distant glomeruli (right). For the OFF response, we assume that prolonged excitatory inputs are balanced by inhibitory inputs during odorant presentation and that the prolonged excitation becomes dominant after odorant presentation ends. (i) excitatory inputs from distant short-axon cells and inhibitory inputs from

26 neighboring periglomerular cells (left). (ii) excitatory inputs from receptor neurons and inhibitory inputs from neighboring periglomerular cells (center). (iii) excitatory input from receptor neurons and inhibitory inputs from the mitral-granule-mitral pathway (right). The glomeruli have the same color-coding as the cells.

27 Table

TABLES (Homma et al) TABLE 1 The number of neurons of each type. Concentration ON INHIBITED OFF 9% 312 (91.8%) 25 (7.4%) 3 (0.9%) 1.5% 246 (97.6%) 5 (2.0%) 1 (0.4%)

TABLE 2 Recruitment of cells as odorant concentration increases Concentration ON INHIBITED OFF active cells/frame 9% 117 1 2 24.0 1.5% 101 0 1 20.4 0.3% 61 0 0 12.2 0.1% 6 0 0 1.2

Figure Click here to download high resolution image Figure

Fig. 2. Three distinct odorant response types

A B

25% ∆F/F ROI 1 1 ROI 2 Cell 1 2 1 5 Cell 2

2 3 Cell 3

Cell 4 25% ∆F/F ROI 1 Cell 5 4 ROI 2 10 µm 9% 2-hexanone 5 s 9% 2-hexanone 5 s

C ON Neuron (cell 1) OFF Neuron (cell 2) Overlay 20% ∆F/F

10 µm 2 s 9% isoamyl acetate 9% isoamyl acetate 9% isoamyl acetate

ON Neuron (cell 1) 20% ∆F/F Cell 1 20% ∆F/F

INHIBITED Neuron (cell 2) Cell 2

1 2

10 µm 9% 2-hexanone 5 s 9% 2-hexanone 2 s Figure Fig. 3.Consistencyofodorant-evokedresponsewithrepeatedtrials A B Trial 2 (% ∆F/F) -10 10 20 30 40 50 0 -10 0 Trial 1(%∆F/F) 9 %2-hexanone 10 20 30 40 10 µm 50 Figure Fig. 4. Concentration dependence of odor-evoked calcium signals A B C

20% F/F 10 µm 10% ∆F/F 9% 2-hex. Cell 1 ∆ GL 9%

JG 9% GL JG 1 9% 3 1.5%

0.3% 0.3% 2 0.1% 0.1% 1.5% 2-hex. 2-hexanone 2 s 2-hexanone

D Cell 2 80 - cell 1 - cell 2 - cell 3 9% - Mean of 34 cells

60 1.5% 0.3% 2-hex. 0.3%

0.1% 40 2-hexanone

Cell 3

20 0.1% 2-hex. % ∆F/F 70 9% 50 1.5% 2+ 40 Amplitude of the Ca transient ( % ∆ F/F) 30 0 0.3% 20 -4 -3 -2 -1 0 10 0.1%

Odorant concentration (log of saturated vapor) 0 2-hexanone 2 s Figure Click here to download high resolution image Figure Click here to download high resolution image Figure

. Figure 7. Distribution of ON, OFF, and INHIBITED neurons and glomeruli in a 3-D volume.

A isoamyl-acetate

B 2-hexanone

ON OFF INHIBITED

OB120806AA-QQ movies/OB120806 4/Fig.7 movie stills r.ai

. Figure Figure 8 Simple circuit models that are consistent with the three types of response. ON Directly from ORN (ET and PG) Via ET (PG and SA) ORN olfactory receptor neuron Via M (PG) - no Mitral to SA PG perilomeruar neuron Via distant SA (PG and ET) PGTH dopaminergic PG cell SA short-axon cell ET external tufted cell M mitral cell G granule cell

PG excitatory input PG SA PG PG PG inhibitory input ET SA ET ON resonse cell excitatory input after stim offset INH response cell M electrical coupling OFF response cell INHIBITED (i) From PG (ii) Centrifugal cholinergic (iii) SA activates inhibitory activity PG.

PG ET/PGTH PGTH ET SA PG ACh ET/PGTH OFF (i) Excitation from distant SA; (ii) Intraglomerular excitation; (iii) Intraglomerular excitation; inhibition from PG inhibition from PG inhibition via mitral circuit

ET PG PG ET SA ET PG ET PG PG M M G