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Distinct lateral inhibitory circuits drive parallel processing of sensory information in the mammalian olfactory bulb

  1. Matthew A Geramita
  2. Shawn D Burton
  3. Nathan N Urban Is a corresponding author
  1. University of Pittsburgh, United States
  2. Carnegie Mellon University, United States
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Cite as: eLife 2016;5:e16039 doi: 10.7554/eLife.16039

Abstract

Splitting sensory information into parallel pathways is a common strategy in sensory systems. Yet, how circuits in these parallel pathways are composed to maintain or even enhance the encoding of specific stimulus features is poorly understood. Here, we have investigated the parallel pathways formed by mitral and tufted cells of the olfactory system in mice and characterized the emergence of feature selectivity in these cell types via distinct lateral inhibitory circuits. We find differences in activity-dependent lateral inhibition between mitral and tufted cells that likely reflect newly described differences in the activation of deep and superficial granule cells. Simulations show that these circuit-level differences allow mitral and tufted cells to best discriminate odors in separate concentration ranges, indicating that segregating information about different ranges of stimulus intensity may be an important function of these parallel sensory pathways.

https://doi.org/10.7554/eLife.16039.001

eLife digest

The brain often processes different features of sensory information in separate pathways. For example, when seeing an object, information about colour and movement are processed by separate types of neurons in the eye. These neurons in turn relay information to different sets of brain areas, all of which are active at the same time. Such parallel processing was originally not thought to apply to information about smell. This was because in mammals, the two types of neurons in the brain area that processes smell seemed to play the same role. However, more recent work suggests that there are in fact differences in the responses of these two neuron types (called mitral cells and tufted cells) to odors, suggesting that the brain might use parallel processing for information about smells too.

Information travels along neurons in the form of electrical signals, and this activity is often seen in the form of a series of “spikes”. In a process called lateral inhibition, the activity of one neuron can feed back and inhibit the activity of its neighbors. This is important for enhancing contrast; in terms of the sense of smell, lateral inhibition is thought to help distinguish between similar odors.

A technique called optogenetics allows the activity of particular neurons in an animal’s brain to be controlled by shining light onto them. Geramita et al. have now used this technique in mice to investigate whether there are differences in how lateral inhibition works in mitral cells and tufted cells. This revealed that lateral inhibition affects mitral cells only when they are spiking at intermediate firing rates, whereas tufted cells are only affected by lateral inhibition when spiking at low firing rates. Using computer simulations, Geramita et al. show that these different responses mean that mitral cells are best at distinguishing similar smells when they are present at high concentrations, while tufted cells are best at distinguishing similar smells that are present at low concentrations. These differences also mean that, by working together, mitral and tufted cells can distinguish between smells much better than either type of neuron on its own.

These results demonstrate that, as with the other senses, the brain processes information about smell using parallel pathways. Future work is now needed to see what effect switching off the activity of either mitral or tufted cells will have on an animal’s behavior.

https://doi.org/10.7554/eLife.16039.002

Introduction

Brain sensory systems use parallel pathways to encode different components of sensory information. Motion and color are segregated in the visual system (Merigan and Maunsell, 1993Callaway, 2005), sound location and tonal pattern are processed in parallel pathways in the auditory system (Lomber and Malhotra, 2008), and pain and itch are conveyed by distinct pathways in the somatosensory system (Davidson and Giesler, 2010; Ross, 2011). How local circuits support feature selectivity in these parallel streams remains poorly understood. This issue is of particular interest when considering brain areas in which distinct outputs are generated from initially homogeneous sources, such as in the olfactory system.

In the mammalian olfactory bulb, firing of olfactory sensory neurons (OSNs) excites two classes of projection neurons – mitral cells and tufted cells (MCs and TCs). While long viewed as essentially equivalent neuron classes, emerging evidence shows that MCs and TCs exhibit distinct activity in vivo (Nagayama et al., 2004; Fukunaga et al., 2012; Igarashi et al., 2012; Adam et al., 2014; Otazu et al., 2015), suggesting that MCs and TCs encode complementary aspects of olfactory information. For instance, TCs respond to lower odor concentrations than MCs (Igarashi et al., 2012; Kikuta et al., 2013), suggesting that TCs are involved in processing near-threshold stimuli. Consistent with this notion of parallel pathways, MCs and TCs project their axons to many non-overlapping regions (Nagayama et al., 2010; Igarashi et al., 2012). Recent work has begun to explore the circuit-level origins of these functional differences in odor-evoked activity. For instance, TCs are more intrinsically excitable and receive stronger OSN-mediated excitation than MCs (Gire et al., 2012; Burton and Urban, 2014). Whether other elements of the olfactory bulb circuit account for differences in MC vs. TC odor-evoked activity is unknown.

Throughout the brain, lateral inhibitory circuits enhance contrast and facilitate discrimination of similar stimuli by decorrelating neural responses (Hirsch and Gilbert, 1991; Urban, 2002; Gschwend et al., 2015). In the olfactory bulb, lateral inhibition occurs between pairs of MCs or TCs via reciprocal dendrodendritic synapses with inhibitory granule cells (GCs) (Schoppa and Urban, 2003; Egger and Urban, 2006). Previously, we have shown that lateral inhibition most strongly affects MCs firing at intermediate rates because coincident input is required for the activation of GCs (Arevian et al., 2008). This activity-dependent regulation of the strength of lateral inhibition onto MCs decorrelates MC responses to similar input more effectively than subtractive or divisive forms of inhibition (Arevian et al., 2008). Lateral inhibition onto TCs is largely unexplored, but the marked differences in MC and TC odor-evoked activity suggest that lateral inhibition onto TCs may operate differently.

Here, we show that optogenetic activation of a single, gene-targeted glomerulus elicits larger and more asynchronous lateral inhibitory currents in nearby MCs than in TCs. Moreover, this same photostimulation paradigm inhibits spiking differently in MCs and TCs. While MCs are affected by lateral inhibition at intermediate firing rates (~50 Hz), TCs are affected when firing at low firing rates (<25Hz). This difference arises, in part, due to differential recruitment of morphologically distinct classes of GCs by MCs and TCs. Finally, we use simulations to explore how these circuit-level differences between MCs and TCs influence odor discrimination. Specifically, the combination of activity-dependent lateral inhibition at both low and intermediate rates enables TCs and MCs to collectively encode odors better than either population alone and supports novel computations that are unlikely to occur with a single neuron type.

Results

To analyze lateral inhibition, we optically activated M72-expressing OSN axons in acute slices from M72-ChR2-YFP mice (Smear et al., 2013) while recording from MCs or TCs innervating nearby glomeruli (Figure 1a,b). This approach allows specific and selective activation of a single, genetically-identified glomerulus across animals, eliminating an important potential source of variability. MCs and TCs showed reliable lateral inhibition following a 10 ms light pulse (Figure 1c,d), and similar proportions of MCs (10/17–59%) and TCs (9/15–60%) received lateral inhibitory currents. The MCs and TCs recorded were similar distances from the M72 glomerulus, and lateral inhibition did not vary within this limited range of distances in either MCs or TCs (Figure 1—figure supplement 1). To examine early and late components of the inhibitory responses onto MCs and TCs, we separated early (<250 ms after stimulation) and late (>250 ms after photostimulation) components of the inhibitory currents. The peak amplitude of early inhibition was larger in MCs than in TCs (Figure 1e), while the charge transferred of early phase inhibition was not significantly different (Figure 1f). Additionally, both the peak amplitude and charge transferred of late phase inhibition (>250 ms) was significantly larger in MCs than in TCs. MCs also receive a smaller proportion of total inhibition during the early phase than TCs, indicating that inhibition is more asynchronous onto MCs than onto TCs. Collectively, these results demonstrate that lateral inhibitory currents are larger and more asynchronous onto MCs than onto TCs.

Figure 1 with 1 supplement see all
MCs receive stronger lateral inhibition than TCs.

(ab) Schematic and example of recording from a TC that projects to a glomerulus near the M72 glomerulus to measure lateral inhibition in M72-ChR2-YFP mice (GL: glomerular layer, EPL: external plexiform layer, MCL: mitral cell layer). Scale bar –100 µm. (c) Light stimulation evoked reliable inhibitory currents in recorded TC (TOP; 5 trials – grey, average – black) that are abolished by gabazine (MIDDLE) but did not evoke excitatory currents (BOTTOM). (d) Examples of average inhibitory currents in 2 MCs and 2 TCs. Inhibitory responses were grouped into early phase (<250 ms) and late phase (>250 ms). (e) The peak amplitude was significantly larger in MCs (n = 10) than in TC (n = 10) during both the early and late phases of inhibition. (f) Charge transferred was significantly larger in MCs than TCs during the late phase only. (‘x’ indicates cells lacking apical dendrites). (g) MCs and TC recorded sequentially in the same slice (n = 5 slices) show similar differences in inhibition. (hi) Analogous experiment measuring lateral inhibition in OMP-ChR2-YFP mice before and after limiting GC-mediated inhibition by bath applying APV and LY36785. (j,k) Similar differences in the peak amplitude (j) and charge (k) in MCs (n = 5) and TCs (n = 5) were found. (l) Ratio of early and late phase amplitude and charge after and before limiting GC-mediated inhibition. Data are presented as mean ± s.e.m. Statistical tests in e,f,j,k,l were two-tailed, unpaired t tests and tests in g were paired t tests. (*p<0.05, **p<0.01, ***p<0.001)

https://doi.org/10.7554/eLife.16039.003

We also explored two potential causes of cell-to-cell variability in lateral inhibition. The strength of lateral inhibition onto MCs and TCs did not depend on whether the apical dendrite was truncated during the slicing procedure, indicating that lateral inhibition originating in the glomerular layer (Aungst et al., 2003; Liu et al., 2013; Whitesell et al., 2013; Banerjee et al., 2015) did not significantly contribute to the differences in lateral inhibition studied here. Variability from slice-to-slice did contribute to variability in the strength of inhibition. However, for MCs and TCs recorded in the same slice, the same relationships in peak amplitude and charge transferred for both early and late phase inhibition were observed as in the larger data set. Specifically, early and late phase peak amplitude, but only late phase charge transferred, was significantly higher in MCs than in TCs measured in the same slice (Figure 1g).

To confirm that these results are not specific to the M72 glomerulus, we performed an analogous experiment in OMP-ChR2-YFP mice (Smear et al., 2011) by photostimulating a single unidentified glomerulus (100 ms pulses) in the medial olfactory bulb (Figure 1h–i). Previously we have shown that we can limit photostimulation to single glomeruli in OMP-ChR2-YFP mice (Burton and Urban, 2015). Similar to the results obtained using M72-ChR2-YFP mice, we find that only the peak amplitude of the early phase of inhibition is larger in MCs while both the amplitude and charge transferred of the late phase of inhibition are larger in MCs (Figure 1j,k). Together these two experiments indicate that lateral inhibition is larger and more asynchronous onto MCs than onto TCs.

Finally, using photostimulation of a single glomerulus in OMP-ChR2-YFP mice, we tested whether GCs contribute similar proportions of lateral inhibition onto MCs and TCs using a previously described strategy (Najac et al., 2015) to differentiate between GC- vs. non-GC-mediated inhibition onto MCs and TCs. We recorded lateral inhibition before and after limiting GC-mediated inhibition by bath applying NMDAR antagonist APV (25 μM) and mGluR antagonist LY36785 (100 μM). Using this pharmacological approach, we found that GCs contribute similar proportions of lateral inhibition onto MCs and TCs (Figure 1l). Additionally, in MCs and TCs in which the apical dendrite had been truncated, all lateral inhibition was blocked after limiting GC-mediated inhibition (Figure 1i,l). In contrast, small inhibitory currents remained after limiting GC-mediated inhibition in MCs and TCs with intact apical dendrites (Figure 1i,l). Removing cells with cut apical dendrites and redoing the analysis presented in Figure 1l similarly shows that GCs contribute similar proportions of lateral inhibition onto MCs and TCs. These observations indicate that glomerular layer circuits make small and uniform contributions to lateral inhibition in MCs and TCs and thus are not the primary source of differences in lateral inhibition between MCs and TCs.

We next explored how lateral inhibition influences MC and TC spiking. To do this, we stimulated MCs and TCs by step current injection and measured firing rates to construct input-output curves in cells near the M72 glomerulus. On interleaved trials we activated M72 OSN axons via photostimulation (Figures 2,3). We used a duration of step current injection matching the physiological duration of firing observed following in vivo odor delivery (Patterson et al., 2013) or in vitro glomerular activation (Najac et al., 2015) (Figure 2—figure supplement 1). Similar proportions of MCs (16/25–64%) and TCs (12/18–67%) were affected by lateral inhibition (see Materials and methods), and unaffected cells were excluded from further analysis. Additionally, MCs and TCs were similar distances from the M72 glomerulus (Figure 3—figure supplement 1). Similar to our previous results using paired MC recordings (Arevian et al., 2008), MC firing rates were reduced by lateral inhibition selectively when MCs fired at intermediate rates, although inhibition was observed at more than double the rate observed in paired recordings. For example, the MC in Figure 2b,e was affected by lateral inhibition while firing between 27–76 Hz. Likewise, across a population of 16 MCs, rates between 34 ± 11 Hz and 79 ± 22 Hz were affected by lateral inhibition (Figure 3a–e, Figure 3—figure supplement 2a–c). Surprisingly, TCs were influenced by lateral inhibition only when firing at low rates. The example TC in Figure 2c,d,f was affected by lateral inhibition while firing between 5–42 Hz, and across a population of 12 TCs, rates between 10 ± 4 Hz and 43 ± 8 Hz were affected by lateral inhibition (Figure 3a–e, Figure 3—figure supplement 2d–f). Both the lower and upper bounds of the effective activity range of lateral inhibition (i.e. the range of firing rates over which lateral inhibition reduces firing rates) were significantly lower in TCs than in MCs (Figure 3e).

Figure 2 with 1 supplement see all
Examples show a TC and MC affected by lateral inhibition at low and intermediate firing rates, respectively.

(GL: glomerular layer, EPL: external plexiform layer, MCL: mitral cell layer). (a) Fluorescent image of a recorded MC that projects to a glomerulus near the M72 glomerulus. (bf) The impact of lateral inhibition on one example MC (b, e) and one example TC (c, d, f) was assessed by constructing FI curves for each cell via somatic current injections of increasing amplitudes. At each current step, the number of action potentials evoked with and without M72 photostimulation was determined (10 ms pulses at 15 Hz). (bc) Examples of voltage traces in a MC (b) or TC (c) at 2 different firing rates with and without M72 photostimulation. (d) The effect of lateral inhibition is illustrated by comparing the FI curves for the light off (grey) versus light on (red) trials in the TC. (ef) Plots of the percent decrease in firing rate (TOP) or absolute firing rate (BOTTOM) in light on trials for a MC (e) or TC (f). Asterisks signify firing rates that are reduced by more than 10% in at least 2 consecutive light on trials.

https://doi.org/10.7554/eLife.16039.005
Figure 3 with 3 supplements see all
Summary results show that TCs are influenced by lateral inhibition at low rates while MCs are influenced at intermediate rates.

(a) The range of firing rates that are significantly influenced by lateral inhibition is plotted with respect to the firing rate in light off trials. Outer rectangle for each cell indicates the total range of firing rates evoked during light off trials. Inner (shaded) rectangle indicates the range of firing rates that are reduced during light on trials. Asterisks indicate cells that lack apical dendrites. ($ - MC used in Figure 2, # - TC used in Figure 2). (c) Percentage of cells that are significantly inhibited is plotted with respect to the firing rate in light off trials. (cd) Average decrease in firing rate (c – plotted as percent, d – plotted as Hz) across the population of MCs (black) and TCs (red). Grey dotted line in c represents the 10% threshold used to indicate significant inhibition. Shaded areas represent s.d. (e) The lower (LEFT, unpaired t-test, p = 2.8 × 10−7) and upper bound (RIGHT, unpaired t-test, p = 1.7 × 10−5) of the range of rates affected by lateral inhibition are significantly lower in TCs compared to MCs. (f) There is no change in the average decrease in firing rate (Hz) between MCs and TCs (unpaired t test, p = 0.87). In f, decreases in firing rate were calculated as the average decrease across all significantly affected firing rates. Data are presented as mean ± s.e.m.

https://doi.org/10.7554/eLife.16039.007

The average fractional reduction in firing rate was much larger in TCs than in MCs (Figure 3c), though the absolute firing rate decrease was not significantly different (MC: −8.2 ± 2.5 Hz n = 16, vs TC: −8.8 ± 3.2 Hz n = 12) (Figure 3d,f). Similar to the findings reported above, neither the effective activity range of lateral inhibition nor the decrease in absolute firing rate depended on whether the cell had an intact apical dendrite. Additionally, these effects of lateral inhibition on firing rate were present even in short timescales matching a single 4 Hz sniff (Figure 3—figure supplement 3). Collectively, our results demonstrate that lateral inhibition is functionally distinct in MCs and TCs.

This difference in activity-dependent lateral inhibition (ADLI) may arise from differences in the lateral inhibitory circuits engaged by MCs and TCs. Our finding that inhibition is largely unaffected by apical dendrite truncation suggests that differences in ADLI between MCs and TCs most likely involves inhibitory circuitry within the external plexiform layer (EPL). Consistent with MCs and TCs engaging distinct lateral inhibitory circuits within the EPL, classical morphological studies suggest that GCs are subdivided into superficial GCs (sGCs), which innervate the superficial EPL, and deep GCs (dGCs), which innervate the deep EPL (Mori et al., 1983; Orona et al., 1983). This putative morphological subdivision of GCs suggests that sGCs inhibit TC lateral dendrites in the superficial EPL while dGCs inhibit MC lateral dendrites in the deep EPL (Mori et al., 1983; Orona et al., 1984). Therefore, functional differences between sGCs and dGCs may mechanistically underlie the difference in ADLI observed between MCs and TCs. In particular, given our prior results demonstrating that low firing rates in MCs are unaffected by lateral inhibition because many GCs require cooperative inputs from multiple glomeruli in order to be activated (Arevian et al., 2008), we hypothesized that low firing rates in TCs are affected by lateral inhibition because sGCs are more strongly recruited than dGCs following activation of a single glomerulus.

Several previous findings are consistent with this hypothesis. GC soma position correlates with GC subtype (Mori et al., 1983; Orona et al., 1983) and odor-evoked activity in vivo (Wellis and Scott, 1990). Specifically, GCs located in the MCL and upper GCL tend to exhibit sGC morphologies and suprathreshold odor responses while GCs located in the lower GCL tend to exhibit dGC morphologies and subthreshold odor responses (Mori et al., 1983; Orona et al., 1983; Wellis and Scott, 1990). Whether these differences in odor-evoked activity in vivo reflect functional differences between GC subtypes and M/TC connectivity remains unclear, however, as: 1) odors activate multiple glomeruli in distinct spatiotemporal patterns, which can evoke a complex array of convergent excitation and inhibition onto individual GCs (Burton and Urban, 2015), and 2) GC activity in vivo is strongly influenced by centrifugal input (Balu et al., 2007; Boyd et al., 2012; Markopoulos et al., 2012) and anesthesia (Kato et al., 2012; Cazakoff et al., 2014).

Therefore, to more directly test our above hypothesis, we recorded the excitatory synaptic input and spiking response of GCs to activation of single nearby glomeruli, with post-hoc recovery of Neurobiotin-filled cell morphologies. For this experiment, we switched from selective optogenetic stimulation of M72 glomeruli to extracellular stimulation of untagged glomeruli in order to test a large number of GC-glomerulus pairs. While this approach introduces a degree of glomerulus-to-glomerulus variability to our data, we capitalized on the well-established all-or-none nature of glomerular activation at low stimulation intensities (Carlson et al., 2000; Gire and Schoppa, 2009) to enable across-cell comparisons of synaptic input and spiking responses following glomerular activation. In addition, we and others have previously demonstrated that optogenetic photostimulation and extracellular stimulation of OSN axons trigger comparable sensory-evoked input to both M/TCs and GCs (Gire et al., 2012; Burton and Urban, 2014, 2015).

Consistent with previous morphological accounts (Mori et al., 1983; Orona et al., 1983), GCs exhibited distinct sGC or dGC morphologies upon visual inspection (Figure 4a,d; Figure 4—figure supplement 1). Indeed, reconstruction and visual classification of a large subset of recorded GCs and analysis of the spatial distribution of gemmules – the site of reciprocal dendrodendritic synapse formation (Rall et al., 1966) – confirmed that sGCs preferentially innervate the superficial EPL while dGCs preferentially innervate the deep EPL (Figure 4g). Moreover, somatic depth significantly – but incompletely – predicted GC subtype (Figure 4a,d; Figure 4—figure supplement 1; Figure 4—source data 1, Table 1), as previously observed (Mori et al., 1983; Orona et al., 1983). To determine whether the observed morphological differences reflect subtypes of GCs rather than a continuum, we additionally performed unbiased clustering of GCs. Specifically, clustering of GCs by the Euclidean distances among their normalized gemmule distributions (using Ward’s method) and application of the gap statistic method yielded 3 distinct clusters: dGCs, sGCs, and a small group of sGCs with prominent innervation of the deep glomerular layer (Figure 4—figure supplement 1c). Moreover, these clusters closely aligned with our original classification by visual inspection, with 19 of 19 sGCs and 9 of 11 dGCs correctly assigned (Figure 4—figure supplement 1c). Our results therefore quantitatively confirm the morphological subdivision of GCs into distinct subclasses of sGCs and dGCs.

Figure 4 with 2 supplements see all
Synaptic and intrinsic differences regulate sGC vs. dGC recruitment following glomerular activation.

(a) Reconstructed morphologies and distribution of apical dendritic gemmules across the MCL (black bars), EPL (grey bars), and GL (red bars) of 3 representative sGCs. Grey/red ticks represents the midpoint of the EPL/mean of the cell’s gemmule distribution. (b,c) Spiking response (b) and synaptic input (c) of the 3 sGCs shown in a following activation of a single glomerulus superficial to the targeted GC. (df) Same as a-c for 3 representative dGCs. (g) Distribution of apical dendritic gemmules across reconstructed sGCs and dGCs. (h) A greater proportion of sGCs than dGCs fired in response to glomerular activation (Chi-square test, p = 4.2 × 10–3). (i) Excitatory input to sGCs exhibited larger peak currents (rank-sum test, p = 8.5 × 10–3) and charge transferred (rank-sum test, p = 0.046) than excitatory input to dGCs. No difference in excitation latency was observed (6.6 ± 11.6 vs. 9.7 ± 11.4 ms; rank-sum test, p = 0.12). Scalebar: 0.2 s/10 pA (inset: 40 ms/20 pA). (j,k) sGCs and dGCs showed significantly different firing rate-current (FI) curves in response to somatic step current injection (2-way ANOVA, p = 4.1 × 10–3). Individual (j) and mean (k) FI curves shown. Dashed lines show diminished firing due to depolarization block. (l) sGC action potentials exhibited more hyperpolarized thresholds (unpaired t test, p = 4.8 × 10–3), larger amplitudes (unpaired t test, p = 1.1 × 10–4), and faster rising slopes (unpaired t test, p = 2.9 × 10–4) than dGC action potentials. Inset: action potential phase plot. Scalebar: 30mV/100 mVms−1; dashed lines show origin. Shaded regions show mean ± SEM.

https://doi.org/10.7554/eLife.16039.011

In agreement with our hypothesis, a strikingly higher percentage of sGCs than dGCs fired in response to activation of a single glomerulus (Figure 4b,e,h) due, at least partially, to stronger excitatory synaptic input to sGCs than dGCs (Figure 4i). As a caveat, we note that the greater recruitment of sGCs following glomerular activation may arise as an artifact of our acute slice preparation. Specifically, as TC circuitry is closer to any given glomerulus than MC circuitry, TC-mediated input to GCs (likely sGCs) may be better preserved than MC-mediated input to GCs (likely dGCs) in the acute slice, leading to stronger sGC excitation and recruitment following glomerular activation. However, despite the fact that these observations were made in vitro, three lines of evidence support greater feedforward recruitment of sGCs as a physiological feature of the olfactory bulb circuit. First, our in vitro observation of greater sGC firing following glomerular activation (Figure 4b,e,h) corresponds well with the previous in vivo observation of stronger odor-evoked activity in putative sGCs (Wellis and Scott, 1990). Second, examination of GC biophysical properties revealed several intrinsic differences supporting greater recruitment of sGCs than dGCs, including a more hyperpolarized action potential threshold in sGCs (Figure 4l; Figure 4—source data 1, Table 2) and greater intrinsic excitability in sGCs in response to somatic step current injections (Figure 4j,k; Figure 4—source data 1, Table 3), despite equivalent somatodendritic sizes (Figure 4—figure supplement 1d; Figure 4—source data 1, Table 1) and passive membrane properties (Figure 4—source data 1, Table 4) between sGCs and dGCs. Third, analysis of spontaneous synaptic activity revealed no difference in event frequency or amplitude between sGCs and dGCs (Figure 4—source data 1 Table 5). Critically, recordings of spontaneous synaptic activity were performed in the absence of TTX and thus contain some degree of action potential-dependent input, which likely originates from intact presynaptic cells. Therefore, equal spontaneous event frequencies between sGCs and dGCs suggests that their respective presynaptic circuits are comparably intact. Moreover, equal spontaneous event amplitudes suggest a comparable contribution of larger action potential-dependent and smaller action potential-independent events between sGCs and dGCs, again consistent with comparably intact presynaptic circuits. In total, our results thus support the hypothesis sGCs are more strongly recruited than dGCs following activation of a single glomerulus due to stronger excitatory input and greater intrinsic excitability, although we cannot rule out an important role for other mechanisms, especially in the intact system in vivo.

As an additional test of our hypothesis that differences in the excitability of GCs provide one potential mechanism underlying the functional difference in ADLI between MCs and TCs, we measured the effects of lateral inhibition on MC spiking before and after increasing the excitability of GCs with the mGluR agonist (RS)-3,5-dihydroxyphenylglycine (DHPG, 10 µM) (Dong et al., 2007; Heinbockel et al., 2007). At this concentration, DHPG selectively enhances the excitability of GCs (Heinbockel et al., 2004) but not MCs (Figure 5—figure supplement 1). The effective activity range of lateral inhibition in MCs (n=6) fell from 31 (± 9)–73 (± 10) Hz to 16 (± 12)–49 (± 23) Hz after adding DHPG (Figure 5a–d), and the lower and upper bounds of the effective activity range of lateral inhibition were significantly reduced after the addition of DHPG (Figure 5e). Additionally, these effects did not depend on whether apical dendrites were intact (Figure 5d), and therefore does not reflect effects of DHPG on glomerular layer circuitry. Despite these observations, however, we note that increased GC excitability may not be the sole mechanism responsible for these DHPG-induced shifts in the effective range of lateral inhibition. For instance, DHPG-induced increases in the excitability of external tufted cells (Dong and Ennis, 2014) may, in turn, increase the activity in M72-MCs, the activity in GCs and GC-mediated inhibition onto the recorded MC. These data (Figures 45) provide two indirect lines of evidence that suggest that differences in GC populations provide one mechanism for differences in the effective activity range of lateral inhibition onto MCs and TCs.

Figure 5 with 1 supplement see all
Increasing GC excitability shifts the effective activity range of lateral inhibition in MCs to lower frequencies.

(ac) Example of lateral inhibition in one example MC before and after bath applying mGluR agonist, DHPG (10 µM). (a) Example voltage traces from one MC. LEFT: Before application of DHPG, intermediate firing rates are affected by lateral inhibition. RIGHT: After application of DHPG, low firing rates are affected. (bc) The effect of lateral inhibition is illustrated by comparing the FI curves for the light off versus light on trials in a MC before (b) and after (c) applying DHPG. Plot of the percent decrease in firing rate vs. the firing rate of light off trials. Dotted line in b,c represents the 10% threshold used to indicate significant inhibition. Asterisks signify firing rates which are reduced by more than 10% in light on trials. (d) Summary of 6 cells recorded before (black) and after (blue) bath application of DHPG ($ - cell depicted in ac). (e) The lower (LEFT, paired t-test, p = 0.009) and upper bound (RIGHT, paired t-test, p = 0.009) of the effective activity range of lateral inhibition are significantly lower after application of DHPG.

https://doi.org/10.7554/eLife.16039.015

Together these data suggest that distinct odor-evoked activity observed in MCs and TCs in vivo arises, in part, due to differences in how ADLI affects each cell type (Figure 4—figure supplement 2). To begin assessing how these circuit-level differences between MCs and TCs impact their ability to encode olfactory information, we performed simulations of MC and TC networks to determine how differences in ADLI may translate into differences in stimulus encoding.

We simulated an olfactory discrimination task in which a presented odor must be identified from a panel of similar odors. Because the goal of the simulations was to understand how the differences in MC and TC circuit properties described above influence population coding, we used simple firing rate models in which we could directly and independently modify lateral inhibition properties without changing other model features. We therefore made relatively few assumptions in performing our simulations, mostly relating to how odor concentration is encoded (as firing rate differences [Cang and Isaacson, 2003; Fukunaga et al., 2012; Igarashi et al., 2012; Sirotin et al., 2015] – however see [Meredith, 1986]), how lateral inhibitory connectivity is specified (randomly), and how MCs and TCs differ (which was explicitly explored). Each odor was simulated as a pattern of inputs to populations of MCs or TCs, corresponding to activated glomeruli (Figure 6a,b, Figure 6—figure supplement 1). Differences in odor concentration were modeled as changes in the number and intensity of activated glomeruli (Rubin and Katz, 1999; Meister and Bonhoeffer, 2001) (see Materials and methods). Additionally, we added trial-to-trial variability in each presentation of a particular odor (Figure 6b, see Materials and methods) in order to mimic natural fluctuations in the background odors and in the patterns of odor-evoked glomerular activation (Wachowiak et al., 2004).

Figure 6 with 3 supplements see all
MCs and TCs discriminate between odors best in separate concentration ranges.

(a) Odors are defined as the pattern of activated glomeruli (i.e. pixels) in the model. Each glomerulus provides input to only 1 MC/TC. (b) TOP: Pattern of activated glomeruli for 2 odors. BOTTOM: Two separate presentations of Odor 1. (c) Outputs of MCs or TCs are used to train and test a linear classifier to predict which odors are presented on each trial. (de) Lateral inhibition differences between MCs and TCs are modeled using Gaussian distributions centered at different firing rates. Firing rates of MCs (d) and TCs (e) with and without lateral inhibition. (fg) Discrimination accuracy of 8 (f) and 32 (g) odors in MCs (black), TCs (red) and a control population of neurons that lacked any inhibition (purple). Shaded areas represent concentration ranges where TCs (light red) or MCs (grey) discriminate significantly better (see Online methods). (h) Differences in the strength of excitatory inputs and intrinsic excitability between MCs and TCs are modeled using 2 sigmoids to translate glomerular inputs into MC/TC outputs. (i) Discrimination accuracy of 32 odors in MCs (black) and TCs (red) that differ in excitability, strength of glomerular input. (j) Discrimination accuracy of 32 odors in MCs (black) and TCs (red) that differ in excitability, strength of glomerular input and ADLI. (k) Overview of simulations comparing 3 separate output neuron configurations: 2 MCs per glomerulus, 2 TCs per glomerulus or 1 MC and 1TC per glomerulus. (l) Discrimination accuracy of 32 odors for MC-MC (black), TC-TC (red) or MC-TC (blue) networks across a range of concentrations. (m) Average discrimination accuracy across all concentrations plotted in l (*p<1e-4). Width of plots in panels f,g,i,j,I reflect the s.e.m.

https://doi.org/10.7554/eLife.16039.017

In our simulations, we asked how well MC and TC population activity discriminated between similar odors presented at the same odor concentration. While understanding how animals discriminate between odors presented at a variety of concentrations is important, we confined our discriminations to odors presented at the same concentration to more closely match behavioral experiments in mice (Abraham et al., 2010; Lepousez and Lledo, 2013) and to keep from making a number of assumptions about how the representation of individual odors varies with concentration. In this simulation, each MC or TC received excitatory input from one of the 150 glomeruli. On each trial, we randomly presented one odor from the panel to networks comprised entirely of either MCs or TCs. We applied a common decoding algorithm, linear discriminant analysis, to determine the extent to which different odors were discriminable in our simulated MC and TC populations (Figure 6c). While the strategy that downstream brain areas use to decode information contained in MC and TC outputs is unknown, linear discriminant analysis is a simple classification algorithm, has some degree of biological plausibility and has been widely applied in similar contexts (Quiroga et al., 2007; Quian Quiroga and Panzeri, 2009; Giridhar et al., 2011).

We first compared the discrimination accuracy of three models: one in which ADLI affected low rates (i.e. an all TC network), one in which ADLI affected intermediate rates (i.e. an all MC network), and a control population that lacked any inhibition. ADLI was modeled by explicitly adjusting the sensitivity of MC and TC firing rates to inhibition without changing other parameters of the model (Figure 6d–e). When the odor panel consisted of only 8 odors, both MC and TC populations discriminated between odors with an accuracy that did not differ from the control population that lacked inhibition (Figure 6f). However, when we increased the difficulty of the task by increasing the size of the odor panel (to 32 odors), the difference in accuracy between MCs and TCs dramatically increased (Figure 6g). For the set of 32 odors, TCs significantly outperformed MCs and the control population at low odor concentrations (90% accuracy in TCs compared to 70% accuracy in MCs at 30% of maximum concentration) while MCs significantly outperformed TCs and the control population at high concentrations (73% accuracy in TCs compared to 91% accuracy in MCs at 60% of maximum concentration). Therefore, differences in ADLI alone support concentration-dependent differences in odor discrimination.

Next we tested how well different forms of activity-independent inhibition compare to ADLI in their ability to improve discrimination accuracy. Specifically, we compared discrimination accuracy (in panels consisting of 32 odors) between 3 populations of neurons; one with subtractive inhibition (Figure 6—figure supplement 2a), one with divisive inhibition (Figure 6—figure supplement 2b) and a control population that lacked inhibition. We found that populations using subtractive or divisive inhibition performed no better than the control population across all concentrations (Figure 6—figure supplement 2c). These results agree with prior work that showed that ADLI substantially decorrelates MC responses to similar odors while subtractive and divisive forms of lateral inhibition have little effect on MC correlation (Arevian et al., 2008).

Prior work has shown that, in addition to ADLI differences, TCs are more intrinsically excitable (Burton and Urban, 2014) and receive stronger OSN inputs than MCs (Gire et al., 2012; Burton and Urban, 2014). Therefore, we next asked how well these differences between MCs and TCs affect odor discrimination. Differences in the strength of OSN input and intrinsic excitability were modeled by altering sigmoid functions that relate the intensity of glomerular inputs to M/TC outputs (Figure 6h). These differences in MC vs. TC excitability and OSN input strength also allow TCs and MCs to discriminate odors best at low and high concentrations respectively (Figure 6i). Finally we simulated how all three differences between MCs and TCs – ADLI, intrinsic excitability and glomerular input – affected odor concentration. Intriguingly, TCs were best at discriminating between low concentration odors when all three differences were included in the model (Figure 6j). Together, these simulations show that intrinsic and circuit-level differences between MCs and TCs work cooperatively to optimize discrimination between similar odors in separate concentration ranges.

Finally, we asked more generally how sensory systems, including the olfactory system, might benefit from splitting information into multiple channels with distinct ADLI. First, we asked whether models containing a combination of MCs and TCs discriminate odors better than models that have only MCs or TCs. We used a variant of the models presented above: here, each glomerulus provided input to 2 neurons instead of 1. We compared three different output neuron configurations: 2 MCs per glomerulus, 2 TCs per glomerulus, and 1 MC and 1 TC per glomerulus (Figure 6k, see Materials and methods). Similar to the data presented in Figure 6g, TC models discriminated between 32 odors best at low concentrations and MC models were best at high concentrations (Figure 6l). However, MC-TC models significantly outperformed other models at intervening odor concentrations. Interestingly, MC-TC models also exhibited the second best performance at high and low concentrations, and after averaging discrimination accuracy across all concentrations tested, we found that models containing a combination of MCs and TCs performed significantly better than models composed of only MCs or only TCs (Figure 6m). Second, new features might arise in any brain area that implements ADLI and splits information into parallel channels. Using an image processing analogy, we show that if an image is split into parallel channels and then recombined by a downstream population, new information about the image (i.e. higher contrast), absent in either single channel, emerges (Figure 6—figure supplement 3). Therefore systems that split sensory information into multiple pathways can not only outperform single pathway systems at comparable tasks, but are also capable of performing novel computations unlikely to occur in single pathway systems.

Discussion

Parallel pathways are a common feature of many sensory systems. Yet how local circuit activity creates stimulus selectivity in parallel pathways remains poorly understood. Here, we have identified a novel circuit mechanism for generating differential responses across two parallel pathways (formed by MCs and TCs) in the olfactory system and examined the emergence of feature selectivity. We find that differences in ADLI selectively reduce intermediate firing rates in MCs and low firing rates in TCs. We provide evidence that these effects could be caused, in part, by differences in the excitability of subclasses of GCs that preferentially inhibit TCs vs. MCs. Moreover, using simulations, we show that differences in the effective activity range of lateral inhibition, along with other intrinsic and circuit-level differences between MCs and TCs, work cooperatively to enable MCs and TCs to best discriminate between similar odors in separate concentration ranges. Finally, we show that the combination of MCs and TCs facilitates odor discrimination across a wide range of odor concentrations.

The activity-dependence of lateral inhibition depends on three cell populations – the 'presynaptic' M/TCs associated with the M72 glomerulus (which we activate optogenetically via stimulation of OSNs), the inhibitory interneurons and the 'postsynaptic' M/TCs. Our prior work has shown that increasing the firing rate of presynaptic MCs (the M72 MCs in this case) shifts the range of MC firing rates influenced by lateral inhibition (Arevian et al., 2008). Given that TCs fire at higher rates than MCs following glomerular activation (Burton and Urban, 2014), M72 TCs will fire at higher rates than M72 MCs following M72 photoactivation and, in part, contribute to the decreased lower bound of the effective activity range of lateral inhibition in postsynaptic TCs. However, higher firing rates in M72 TCs alone is insufficient to explain the differences in both the lower and upper bound in the effective activity range of lateral inhibition between MCs and TCs.

Our analysis supports the hypothesis that TCs and MCs are preferentially connected to sGCs and dGCs, respectively. Additionally, we provide indirect evidence that differences in the recruitability of sGCs and dGCs may be one potential mechanism underlying the differences in ADLI between MCs and TCs. Our data suggests that sGCs spike with a higher probability than dGCs following the activation of a single glomerulus. Consequently, postsynaptic TCs can effectively recruit lateral inhibition when firing at low rates. dGCs are less excitable and therefore require more input to be activated. Therefore M72 photostimulation alone causes relatively weak activation of dGCs. However, when the postsynaptic MC fires at intermediate rates, additional dGCs will become activated and mediate lateral inhibition. In support of this mechanism, we show that increasing the excitability of GCs by activating mGluRs (Dong et al., 2007; Heinbockel et al., 2007) shifts the effective activity range of lateral inhibition in MCs to lower firing rates (Figure 5). Additionally, these differences between sGCs and dGCs can explain why the upper bound on the effective activity range of lateral inhibition differs between MCs and TCs. Cells firing above this upper bound recruit the maximum amount of recurrent inhibition such that additional inputs to GCs cannot trigger additional GABA release (Urban and Arevian, 2009). Because sGCs are more excitable than dGCs, maximal GABA release, and consequently the upper bound on the effective activity range of lateral inhibition, occurs at lower rates in TCs than in MCs (Figure 4—figure supplement 2).

While preferential connectivity of TCs with sGCs and MCs with dGCs can explain the observed differences in the activity-dependence of lateral inhibition, other circuit mechanisms may also contribute. In particular, EPL interneurons (EPL-INs) can mediate inhibition onto M/TCs (Huang et al., 2013; Kato et al., 2013; Miyamichi et al., 2013), and it remains possible that EPL-INs or distinct subclasses of EPL-INs preferentially inhibit MCs or TCs. However, it is unlikely that EPL-INs predominantly drive the difference in MC vs. TC ADLI given that EPL-INs mediate linear (divisive) but not activity-dependent inhibition of MC/TC outputs (Kato et al., 2013; Uchida et al., 2013). Glomerular layer circuits have also been shown to play a role in M/TC lateral inhibition (Aungst et al., 2003; Liu et al., 2013; Whitesell et al., 2013; Banerjee et al., 2015), however these circuits likely do not play a role in the differences in ADLI reported here because our results were not influenced by apical dendrite truncation. Therefore, distinct GC populations that differ in excitability are the most parsimonious explanation for the observed differences in ADLI.

Differential connectivity of MCs and TCs with distinct GC subclasses has long been predicted based on the putative morphological subdivisions of GC apical dendritic morphologies (Mori et al., 1983; Orona et al., 1983) and separation of MC and TC lateral dendrites in the deep and superficial EPL (Mori et al., 1983; Orona et al., 1984), respectively. However, distributed M/TC-GC connectivity along exceedingly long M/TC lateral dendrites has, thus far, precluded a direct demonstration of differential connectivity of MCs and TCs with distinct GC subclasses. Here, we provide quantitative evidence that GCs indeed form two distinct morphological subclasses – sGCs and dGCs – rather than a continuum of morphologies. Moreover, our results provide – to our knowledge – the first functional evidence that MCs and TCs engage distinct lateral inhibitory circuits, likely via differential connectivity with dGCs and sGCs, respectively. Future experiments involving selective manipulation of MCs vs. TCs and dGCs vs. sGCs will ultimately be needed in order to directly demonstrate whether (and the degree to which) these lateral inhibitory circuits overlap.

Our data describe local, circuit-level mechanisms that can account for several differences between MC and TC odor-evoked responses observed in vivo. Weaker lateral inhibitory currents may contribute to the finding that TCs are less frequently inhibited by odors (Nagayama et al., 2004). Additionally, the finding that intermediate and high firing rates are unaffected by lateral inhibition in TCs may help explain why TCs show odor-evoked responses that are more highly correlated to OSN input (Adam et al., 2014) and less dependent on concentration (Fukunaga et al., 2012; Igarashi et al., 2012). Additionally, TCs firing above the upper bound of the effective activity range of lateral inhibition are shielded, not only from lateral inhibition, but other sources of GC-mediated inhibition as well, such as inhibition triggered by cortical feedback (Boyd et al., 2012; Markopoulos et al., 2012), Consequently, the circuit-level differences we describe may explain why MCs but not TCs are decorrelated by cortical feedback (Otazu et al., 2015).

These circuit-level differences between MCs and TCs likely affect olfactory discrimination (Figure 6). ADLI, but not subtractive or divisive inhibition, allows TCs and MCs to best discriminate between odors at low and high concentrations, respectively. Yet how does this improvement in discrimination occur? Prior work has shown that activity in M/TC populations becomes decorrelated over time (Bathellier et al., 2008; Cury and Uchida, 2010) and that this decorrelation is driven by GCs (Gschwend et al., 2015). Additionally, GC-mediated M/TC decorrelation improves odor discrimination (Gschwend et al., 2015). Consequently, ADLI likely improves odor discrimination by driving decorrelation of M/TC firing in ranges that engage lateral inhibition (Arevian et al., 2008). M/TCs firing at rates outside the effective range of lateral inhibition may be encoding complementary olfactory information beyond odor identity.

Performing these experiments in acute slices provides the best opportunity to explore the causes and consequences of differences in lateral inhibition between MCs and TCs. Given the novel features and mechanisms of ADLI, using a reduced and carefully controlled system in which a single glomerulus can be reliably activated is an important first step. Moreover, our in vitro approach allowed us to apply pharmacological manipulations that were vital in identifying differences in GC excitability (Figure 5) rather than differences in glomerular layer circuitry (Figure 1l) as the main mechanism supporting differences in MC vs. TC ADLI.

We note, however, that our in vitro approach also has certain limitations. In particular, the slicing procedure may introduce artifacts that could confound our conclusions. Importantly, however, our findings that MCs receive stronger lateral inhibitory currents and are affected at different ranges of firing rates than TCs are unlikely to reflect slicing artifacts. Due to slicing, some MCs and TCs that project to the M72 glomerulus will be truncated, and cells that reside farther from the M72 glomerulus have a higher probability of being truncated. However, our findings do not depend on the cell’s distance from the M72 glomerulus (Figure 1—figure supplement 1; Figure 3—figure supplement 1). Additionally, the lateral dendrites of MCs and TCs innervate distinct strata of the EPL and these differences could result in differences in the fraction of original connections left intact following slicing. However, the effect of ADLI on MCs that are observed here following activation of an entire glomerulus closely mirrors the effects that were observed when activating a single presynaptic MC in our previous work (Arevian et al., 2008). That is, only the probability of finding connections and the absolute magnitude of the effect are increased by activating all of the cells associated with a glomerulus (compared to the prior study when only a single presynaptic cell was activated). This suggests that the critical feature of the effect (maximal lateral inhibition at intermediate firing rates) is a property of single connections rather than one that depends on having a full repertoire of connections intact. Moreover, the close correspondence of our GC data to previous in vivo recordings of odor-evoked GC activity (Wellis and Scott, 1990), as well as the equivalent spontaneous synaptic activity – a proxy for circuit intactness – observed between sGCs and dGCs suggests that slicing artifacts cannot explain the differences in excitatory input or intrinsic excitability observed between sGCs and dGCs.

Our in vitro approach additionally cannot address multiple important factors that are only present in the intact animal. For instance, respiration and centrifugal feedback likely modulate ADLI in vivo. Addressing the extent to which ADLI affects odor-evoked responses in MCs and TCs in vivo is thus an important future direction.

Finally, what are the benefits of parallel processing? In other sensory systems, different pathways may arise from functional differences at the initial stages of stimulus detection, which are then maintained through selective feedforward connectivity. In the olfactory system there is no evidence that MCs and TCs are targeted by distinct subsets of OSNs. Rather, we show that differences in bulbar circuitry are sufficient to generate important differences in response properties. Here we show that MCs and TCs perform odor discriminations best at separate concentration ranges and that a combination of MCs and TCs discriminates similar odors better than either population alone. These results suggests that parallel processing may offer similar benefits in other sensory systems in which stimulus intensities can vary over many orders of magnitude. For instance, rods and cones in the retina function best at different ranges of light intensity. Other similarities between the olfactory and visual systems suggest that the mechanisms behind parallel processing may be shared across sensory systems. In the retina, feature selectivity in each ganglion cell type emerges due to its connectivity to specific bipolar and amacrine cell types (Masland, 2012). Similarly, differences in odor-evoked responses in MCs and TCs emerge due to differences in connectivity to OSNs, external tufted cells (Najac et al., 2011; Gire et al., 2012) and GCs. Therefore, in these systems, feature selectivity is not inherited but emerges via differential connectivity with distinct neuron types. Similarly, recombining the specific features encoded in individual channels allows new features to emerge. For instance, if populations of neurons in higher order sensory areas integrate inputs from parallel sensory channels, such as the anterior piriform cortex (Nagayama et al., 2010; Igarashi et al., 2012), and perform simple computations, such as averaging, new features may emerge (Figure 6—figure supplement 3). More complex integration mechanisms may allow the calculation of other complex stimulus features using the different information encoded in multiple parallel sensory channels.

Materials and methods

Ethical approval

All experiments were completed in compliance with the guidelines established by the Institutional Animal Care and Use Committee of Carnegie Mellon University (IACUC # AS15-010) and University of Pittsburgh (IACUC # 15116582).

Slice preparation

For MC and TC recordings, postnatal day 16-23 M72-ChR2-YFP (Smear et al., 2013) mice were anaesthetized with isoflurane and decapitated into ice-cold oxygenated dissection solution containing (in mM): 125 NaCl, 25 glucose, 2.5 KCl, 25 NaHCO3, 1.25 NaH2PO4, 7 MgCl2 and 0.5 CaCl2. Sagittal slices (280 μm thick) of the MOB were prepared using a vibratome (VT1200S; Leica, Nussloch, Germany). Slices recovered for 15–30 min in 37°C oxygenated Ringer solution that was identical to the dissection solution except for lower Mg2+ concentrations (1 mM MgCl2) and higher Ca2+ concentrations (2 mM CaCl2). Slices were then stored in room temperature oxygenated Ringer solution until recording. For MC and TC recordings in OMP-ChR2-YFP mice, horizontal slices (280 μm thick) were prepared from postnatal day 17–21 mice. For GC recordings, equivalent methods were used to prepare horizontal slices (310 μm) of the MOB from postnatal day 18–28 C57BL/6, Thy1-YFP-G (Feng et al., 2000) albino C57BL/6J, and heterozygous OMP-ChR2-YFP (Smear et al., 2011) mice using a vibratome (5000mz-2, Campden).

Cell identification and morphological analyses

TCs were identified as those cells with large somas (>10 μm in diameter) that reside completely in the EPL. Cell bodies resided in the superficial half of the EPL. All TCs included in our final dataset had at least 1 lateral dendrite and did not display the rhythmic bursting characteristic of external tufted cells (Hayar et al., 2004; Antal et al., 2006; Liu and Shipley, 2008). MCs were identified as large cells located in the mitral cells layer (MCL). ‘Displaced MCs (Mori et al., 1983)’ or ‘internal TCs (Igarashi et al., 2012)’, those cells with somata that only partially reside in the mitral cell layer were excluded from analysis due to their ambiguous identity as MCs or TCs. GCs located in the MCL or GC layer were distinguished from other cell types and classified as sGCs or dGCs as previously described (Burton and Urban, 2015). Specifically, GCs were classified as dGCs if their apical dendritic gemmules were visibly concentrated in the deep half of the EPL, while GCs were classified as sGCs if their apical dendritic gemmules were visibly concentrated in the superficial half of the EPL. Cell morphologies were reconstructed under a 100X oil-immersion objective and analyzed with Neurolucida (MBF Bioscience). Anatomical positions of GC apical dendritic gemmules were manually identified from 3D reconstructions using custom software written in Matlab (Mathworks).

Electrophysiology

Cells were visualized using infrared differential interference contrast video microscopy. For MC and TC recordings, slices were continuously superfused with 37°C oxygenated Ringer solution that contained 0.2 mM Mg2+ unless otherwise noted. Current clamp recordings were made from individual cells using electrodes filled with (in mM) 120 potassium gluconate, 2 KCl, 10 Hepes, 10 sodium phosphocreatine, 4 Mg-ATP, 0.3 Na3GTP, 0.2 EGTA, 0–0.025 Alexa Fluor 594 (Life Technologies, Carlsbad, CA) and 0.2% Neurobiotin (Vector Labs, Burlingame, CA). Voltage clamp recordings were made using electrodes filled with (in mM): 140 Cs-gluconate, 10 HEPES, 2 KCl, 10 sodium phosphocreatine, 3 Mg-ATP, and 0.3 Na3GTP.

M72 photostimulation was provided by a 250 μm multimode optical fiber (Thorlabs) coupled to a high-intensity light emitting diode (M470F1; Thorlabs) and driver (DC2100: Thorlabs) controlled by TTL pulses. For photostimulation in OMP-ChR2-YFP, slices were illuminated with 100 ms light pulses by a xenon arc lamp directed through an YFP filter set and 60x water-immersion objective centered on a single glomerulus. Photostimulation was confined to single glomeruli by closing the field stop as previously described (Burton and Urban, 2015). All data were low-pass filtered at 4 kHz and digitized at 10 kHz using a MultiClamp 700A amplifier (Molecular Devices, Sunnyvale, CA, USA) and an ITC-18 acquisition board (Instrutech, Mineola, NY, USA) controlled by custom software written in Igor Pro (WaveMetrics, Lake Oswego, OR, USA).

For GC recordings, slices were continuously superfused with warmed oxygenated Ringer’s solution (temperature measured in bath: 32°C) containing 1 mM Mg2+ and 2 mM Ca2+. Current clamp recordings were made as described above. Voltage clamp recordings were made using electrodes filled with either the Cs-based solution supplemented with 10 mM QX−314 and 0.2% Neurobiotin or the K-based solution. To examine GC activity following activation of a single glomerulus, extracellular stimulation of olfactory sensory neuron fibers within a single glomerulus was performed as previously described (Burton and Urban, 2015).

Data analysis

Lateral inhibitory currents were measured in 5 trials at a holding potential of +10 mV. Analysis of IPSCs was performed using custom Matlab (Mathworks) analysis software. The presence or absence of IPSCs was calculated by taking the average trace of 5 trials and finding the mean and standard deviation of the trace during the second prior to photostimulation. Then the baseline current (mean of the second prior to photostimulation) was subtracted from each trace. IPSCs evoked through lateral inhibition were present if positive deflections of the current trace exceeded 3*s.d. for longer than 10 ms in the 500 ms time window following M72 photostimulation.

Lateral inhibitory currents were split into early and late phases and the peak current amplitude and charge transfer were calculated in each. Charge transfer was calculated as the integral of the current trace in either the early phase (0–250 ms) or late phase (250–1500 ms) following photostimulation.

The effect of lateral inhibition on spiking was measured by performing FI curves in MCs and TCs via somatic current injection of increasing amplitudes. At each current step (500 ms), we measured the number of action potentials evoked with and without M72 photostimulation (10 ms pulses at 15 Hz). Two full FI curves (a full FI curve is defined as having current steps with and without photostimulation) were performed on each cell and the average change in firing rate at each current step was calculated and used for the presented analysis. Lateral inhibition was defined to have a significant effect on a cell if there was a greater than 10% decrease in firing rate in at least 2 consecutive current steps.

Intrinsic biophysical properties of GCs, including passive membrane, action potential, and spike train properties were calculated as previously described (Burton and Urban, 2015).

Computational model

Models of odors and olfactory bulb circuitry were developed in Matlab (Mathworks) (see Source code). The scheme for generating odor panels and individual odor trials is depicted in Figure 6—figure supplement 1. Each odor is represented by the spatial pattern of a 15 × 10 array of pixels (i.e. glomeruli). For each odor presented at a particular concentration (Ipresentation), we first made Odor 1. To do this, we randomly sampled 1/3 of pixels to represent non-activatable (NA) pixels that are not responsive to odors, regardless of concentration. Next a concentration threshold (Ti) was sampled from a uniform distribution between 0 and 1000 for each activatable pixel (Ti = U([0,1000]). Activatable pixels were then divided into ON and OFF pixels. ON pixels were defined as having Ti < Ipresentation, while OFF pixels are ones where Ti > Ipresentation. Each ON pixel then got a mean activation intensity sampled from a normal distribution (Ibase = N(Ipresentation, 50)).

The next step in the construction of the odor panel was to make an arbitrary number of other odors that are 90% similar to Odor 1. To make these odors, we enforced a set of rules.

  1. Each pixel in Odor 1 had a 10% probability of changing.

  2. For each NA pixel that was chosen to change:

    • P(NA – NA) = 1/3

    • P(NA – ON) = 2/3

    • If a NA pixel became an ON pixel, it received an activation intensity. Ibase=N(Ipresentation, 50)

  3. For each ON pixel that was chosen to change:

    • P(ON – NA) = 1/3

    • P(ON – ON) = 2/3

    • if an ON pixel remained ON, it received a new activation intensity. Ibase=N(Ipresentation, 50)

  4. For each OFF pixel that was chosen to change:

    • P(OFF – OFF) = 1

The last step in odor panel construction was to add noise to create trial-to-trial variability. To do this, we enforced a set of rules for sampling the activation strength of each pixel on each trial.

  1. The strength of activation of NA and OFF pixels: Itrial = U([0,1000])

  2. The strength of activation of ON pixels: Itrial = N(Ibase, 5)

These odors became the glomerular input for olfactory bulbs composed of either MC or TCs. Each MC/TC was represented by a continuous firing rate variable, v. Each neuron received a leak current, an inhibitory current and an excitatory current input from 1 pixel (Equation 1). The excitatory current was calculated by passing the pixel activation strength through a sigmoid. Two differences between MCs and TCs, higher excitability and increased excitatory input are reflected by increased slope (0.007 for MCs and 0.01 for TCs) and reduced midpoint (500 for MCs and 350 for TCs) of the TC sigmoid. MCs/TCs were randomly connected to 75/60% of other MC/TCs, reflecting the shorter extent of lateral dendrites in TCs compared with MCs (Igarashi et al., 2012; Burton and Urban, 2014). Inhibition was calculated as the product of the sum of network activity, u(t) (Equation 5), and a Gaussian function that determines which range of firing rates are influenced by lateral inhibition (Equations 3,4). The center and width of the MC and TC Gaussian distribution are based on differences in the range of rates influenced by lateral inhibition in our data.

(1) tdvdt=stim(t) leak(t) inhib(t)
(2) leak=gl(v(t)El)
(3) inhibtufted(t)=gi  u(t) e(v(t)μt)σt2, μt=10, σt=350
(4) inhibmitral(t)=gi  u(t)  e(v(t)μm)σm2, μm=50, σm=500
(5) inhibsubtractive(t) = gi  u(t)
(6) inhibdivisive(t) = gi  v(t)
(7) u(t)= n=1Tvn(t), where T is the number of connected MCs or TCs
(8) stim(t)= f(Itrial), where f is a sigmoid

One of N odors from the panel was presented to the MC or TC bulb on each of 2000 trials. The MC or TC outputs from half of the trials are used to train a naïve Bayes classifier which was then used to predict which odor is being presented on the remaining half of trials. The percent correct is used as the discrimination accuracy for that odor panel. For simulations, we constructed 100 odor panels at each concentration. Concentrations in Figures 6 are plotted as the percent of maximum concentration. The maximum concentration was defined as the concentration that evokes maximum firing rates in MCs and TCs, which in our models is set at 100 Hz – a rate often observed in vivo. A sigmoidal transfer function was used to translate glomerular inputs into MC/TC outputs so that odor concentrations could be defined in terms of MC/TC firing rates while not make any explicit comparisons to actual odor concentrations. Significance in discrimination accuracy between output neuron configurations was determined in 2 steps. First, we conducted one-way ANOVA tests (corrected for multiple comparisons – i.e. the number of odor concentrations tested) on the discrimination accuracy of the three output neuron configurations at each odor concentration. At concentrations with significant ANOVA tests, we performed post-hoc t-tests to determine whether one particular output neuron configuration was significantly better than the other two. The results of the post-hoc t-tests determine the highlighted areas of Figure 6.

The simulations used to generate the data in Figure 6k–m, in which 2 output neurons receive inputs from 1 glomerulus required additional connectivity rules. For bulbs containing only MCs or TCs, each of the two cell that input from the same glomerulus receive inhibition from a random set of other neurons. In bulbs containing both MCs and TCs, inhibition remained segregated, that is TCs/MCs only received inhibition from a random set of other TCs/MCs. Additionally, for all 3 output neuron configurations, neurons projecting to the same glomerulus did not inhibit one another. The simulations used to generate the data in Figure 6—figure supplement 3, in which one input image served as input to 2 separate populations that only differed in the range of ADLI. For each population, each pixel in the image provided into to one neuron.

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Decision letter

  1. Naoshige Uchida
    Reviewing Editor; Harvard University, United States

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "Distinct lateral inhibitory circuits drive parallel processing of sensory information in the mammalian olfactory bulb" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Naoshige Uchida as the Reviewing Editor and Gary Westbrook as the Senior Editor. The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

The authors report that tufted cells are inhibited by lateral inhibition from neighboring glomeruli at lower firing rate ranges than for mitral cells. That is, activity-dependent lateral inhibition (ADLI) occurs at different firing rate ranges in tufted versus mitral cells. The authors also present evidence suggesting that the difference in ADLI is caused by differences in excitation of two sub-classes of granule cells. Finally, the authors used computational modeling to show that differential ADLIs between mitral and tufted cells help improve discrimination of odors at wider concertation ranges.

All the reviewers thought the main finding were very interesting. During discussion between the reviewers, however, there were different opinions whether it was necessary to determine whether differential ADLIs occurs in vivo. Although such an experiment would greatly strengthen the significance of this work, it is eLife's policy not to suggest experiments that will take more than 2 months. Reviewers agreed that the experiments in reduced preparations (slice) have certain advantages. However, the reviewers had other concerns that were considered substantive and therefore the authors must address these concerns before publication.

Essential revisions:

1) It is not convincing that the difference in ADLIs between mitral and tufted cells is not a result of tissue slicing. Furthermore, it is unclear whether the different properties of superficial versus deep granule cells that might have caused the differences in ADLIs are a side effect of slicing. The authors have to provide more convincing, quantitative arguments to exclude artifacts associated with slicing.

2) The authors examined the significance of differential ADLI between mitral and tufted cells using modeling. However, mitral and tufted cells are different not only in their ADLI properties but also their intrinsic and synaptic input properties. In the modeling, all of these are changed at the same time, and therefore, it is unclear whether improved odor discriminability at different odor concentrations is due to ADLI alone, differences in intrinsic/synaptic properties alone, or a combination of all factors. Please clarify this issue with additional analysis.

3) In Figure 3, the authors mention that "decreases in firing rate were calculated as the average decrease across all significantly affected trials". Selecting trials based on "significantly affected trials" seems inappropriate. Exclusion of such trials in tufted cells would have resulted in an artificially large value for the percent change in spike rate due to ADLI.

4) in vivo experiments would greatly increase the significance of the present study as lateral inhibition may be sensitive to subtle details of the network properties, spontaneous activity and breathing etc. that can be seen only in an intact network. If the authors decide not to perform in vivo experiments, potential caveats of their in vitro experiments must be explicitly discussed.

Reviewer #1:

This study explores differences in the olfactory bulb's output mitral cells (MCs) and tufted cells (TCs) in a previously-described phenomenon known as activity dependent lateral inhibition (ADLI). Evidence is provided based on slice recordings that MCs and TCs differ in the kinetics of lateral inhibition, the spike frequency range over which ADLI is observed, as well was the relative effect of ADLI on spiking. Also, recordings from granule cells (GCs) suggest that differences in ADLI in MCs versus TCs could reflect differences in excitation of two sub-classes of GCs. Finally, computational modeling is used to make the case that differences in ADLI between MCs and TCs can confer differences in the concentration ranges over which the two neuron types best discriminate odors and may also expand the concentration ranges over which odors can be effectively discriminated by the bulb.

Overall, this is an interesting study that addresses a broadly relevant question: How can two parallel sensory processing paths (in this case, mediated by MCs and TCs) enhance sensory discrimination? Much of the study also employs a powerful experimental paradigm involving optogenetic stimulation of a single, molecularly-defined glomerulus (M72) as a means to drive lateral inhibition. This strategy should eliminate ambiguities about whether only one glomerulus is being stimulated and, also, should reduce variabilities across experiments. Some of the results are also basically convincing, for example that MCs and TCs have different kinetics for lateral inhibition and also differ in the frequency range over which ADLI occurs. However, I have a number of concerns with both the experimental and modeling sections of the paper.

1) The analysis methods used raise questions about results suggesting that TCs display a larger percent decrease in spike rate due to ADLI than MCs (Figure 3C). The legend of Figure 3 indicates that changes in firing rate due to ADLI were calculated based only on "significantly affected trials". Because ADLI in TCs occurred at low spike frequencies, it seems quite possible that many of the trials in which small effects were actually occurring may have been missed by a per-trial statistical analysis (due to a low total number of spikes). Exclusion of such trials in TCs would have resulted in an artificially large value for the percent change in spike rate due to ADLI, as they calculated it. Such exclusions would be less common in MCs that displayed ADLI at higher spike frequencies.

2) That differences in the properties of superficial versus deep GCs (sGCs and dGCs) contribute to differences in ADLI between MCs and TCs is not completely convincing. The argument is based on the parallel observations that TCs display ADLI at lower spike frequencies than MCs and also that sGCs (which may preferentially contact TCs) are more responsive to glomerular stimulation. However, the authors need to exclude better the possibility that the greater spike probability and evoked currents in sGCs is not a slicing artifact. sGCs, which are closer to glomeruli, may naturally be expected to have more of their input circuitry intact in slices. The fact that sGCs appear to have greater intrinsic excitability helps their argument, but it is not sufficient. Comparing ADLI and the responsiveness of GCs is also complicated by the fact that the experiments assaying these two features appear to have been conducted in differing magnesium concentrations (0.2 mM versus 1 mM).

The authors also use the fact that the mGluR agonist DHPG reduces the frequency range for ADLI in MCs to argue that differences in ADLI between MCs and TCs are due to GC properties. This is based on prior observations that DHPG can enhance GC excitability. DHPG appears not alter MC spiking due to direct current injection (Figure 5—figure supplement 1), yet other circuit effects of DHPG need to be better controlled. For example, DHPG could enhance stimulus-evoked lateral inhibition by altering glomerular layer neurons. This, in turn, could cause the same frequency shifts in ADLI in MCs as increases in GC excitability.

3) The computational results suggesting that differences in ADLI between MCs and TCs enable these cells to perform odor discrimination at different concentrations are not completely convincing. The most important results are shown in Figure 6L, which displays the output of a model that incorporates differences in the level of sensory neuron input onto TCs versus MCs, differences in intrinsic excitability, as well as differences in ADLI. This shows that TCs perform better discrimination at lower odor concentration than MCs. However, it is unclear how much differences in ADLI between MCs versus TCs are contributing to these discrimination curves. The fact that TCs discriminate better at lower concentrations may mainly reflect their greater responsiveness to low concentrations of odor. The authors need to test whether removing inhibition substantially impacts the discrimination curves in Figure 6L, as well as Figure 6N, as they did for their simpler model in Figure 6G.

Reviewer #2:

Geramita, Burton & Urban report through a series of elegant experiments in acute olfactory bulb preparations that the two types of bulb principal neurons, tufted and mitral cells, receive different levels of inhibition from granule cells (stronger and more asynchronous to mitral cells compared to tufted cells). The authors show through a combination of patch-clamp recordings, current injections and optogenetic stimulation of genetically labeled glomeruli that activity dependent lateral inhibition (ADLI) occurs at low firing rate regimes in tufted cells and intermediate firing rates in mitral cells. They further propose as underlying substrate at play for the different ADLI regimes two distinct populations of superficial and deep granule cells (sGCs, dGCs). Anatomical reconstructions and clustering suggest that sGCs and dGCs contact differentially the lateral dendrites of TC and respectively MCs. Further functional analysis reveals differences in intrinsic excitability and threshold for action potential firing between sGCs and dGCs consistent with their proposed roles. In my opinion, the manuscript adds important novel understanding by documenting that functional differences between mitral and tufted cells emerge not only due to differences in intrinsic excitability, but also in their local connectivity with inhibitory interneurons. These findings bring timely mechanistic insight that parsimoniously explains recently published differences in TCs and MCs responses to odors, across concentrations, and to modulation of cortical feedback. Therefore, I consider the manuscript fit for publication in eLife should the authors address several concerns listed below.

Importantly, I have doubts regarding the necessity of the computational model presented and some of its underlying assumptions. Given the temporal complexity of active sampling (sniffing) in vivo, relevant time window for odor identification (200-300 ms, Uchida & Mainen, 2003), possible effects of the slice preparation on the functional connectivity of the bulb, and lack of top-down regulation (glutamatergic, GABA-ergic and neuromodulatory input) of mitral and tufted cell activity in the slice preparation, I suggest focusing the manuscript on the experimental results with fewer incursions into the simulations.

1) The authors present in Figure 6K two sigmoids that govern the responses of MC and TCs to increasing concentrations. MCs have been reported to display a wide range of concentration response curves that are not necessarily monotonic (Meredith, 1986), and thus at odds with the chosen transfer function.

2) What is the functional relevance of the concentration regime sampled? What does 20% to 80% of maximal concentration translate to in terms of absolute odor concentration? Are such concentrations ever encountered by the animal?

3) Why is the classifier trained independently for each of the concentrations sampled? This seems an unrealistic regime compared to processes that may occur in the brain.

4) Differences in functional properties of sGCs and dGCs can be in principle heavily modulated by top-down signals that are absent in the slice. The current version of the manuscript does not discuss this possibility.

5) MC and TCs have been reported to project with differential biases to downstream target areas. How do such biases contribute to the ability of the decoder to incorporated odor information carried by both TC and MC?

In Figure 1L to assess in an unbiased manner the contribution of GC-mediated vs. glomerular layer mediated lateral inhibition on MCs vs. TCs, the analysis (before vs. after drug cocktail application) should be restricted only onto those cells that have the apical dendrite intact. Otherwise, by construction, the contribution of the glomerular layer lateral inputs is reduced/absent in cells with apical dendrite cut. In the current version, 1 out of 5 MCs, and respectively 2 out of 5 TCs have their apical dendrite cut.

Reviewer #3:

The authors present a detailed in vitro analysis of differences in lateral inhibition experienced by MCs and TCs. They attribute these differences to different innervation of different populations of GCs and use a simplified model to illustrate that the differential inhibition can lead to improved discriminability overall (with TCs aiding to low-concentration and MCs to high-concentration discrimination).

In a previous submission (to another journal) there were numerous criticisms that the authors largely address well. One key concern that was reiterated by all reviewers was that the findings of differential inhibition were exclusively based on in vitro experiments (and simulations) with little direct link to how sensory information might be processed. Here I think the current manuscript is still lacking. While the authors argue – rightly so – that they perform an in depth mechanistic analysis (which is difficult in vivo) I have to say I tend to agree in that the relevance / role of the differential inhibition for odor processing is still unclear. If indeed – as stated in the response to prior reviewer 1 – the variability / signal-to-noise will mask the effect in vivo one might question how dominant it could be in a behaving context. I surely don't argue for repeating all experiments in an awake behaving animal but key experiments (parts of Figures 1 and 4) at least in an anesthetized (if not in an awake) animal would convince the reader that the effects studied are indeed shaping odor processing in vivo.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Distinct lateral inhibitory circuits drive parallel processing of sensory information in the mammalian olfactory bulb" for further consideration at eLife. Your revised article has been favorably evaluated by Gary Westbrook (Senior editor), a Reviewing editor, and three reviewers.

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance as agreed upon by the reviewers in discussion following their original reviews that are appended below. Specifically, while all the referees agreed that the manuscript has been greatly improved, there are two remaining concerns. First, the reviewers thought that it remains unclear whether the authors can completely exclude the possibility that slicing biased the contribution of superficial versus deep granule cells in activity-dependent lateral inhibition (ADLI) (point # 1 of reviewer 1). Furthermore, the interpretation of the experiment using DHPG should be revised so that the possibility of the network effect (point #2, reviewer 1) is appreciated. We think that both points can be addressed with revisions to the text as outlined by reviewer 1. For more details, please see the reviewers' original reviews below.

Reviewer #1:

The Reviewers have adequately addressed most of my concerns. Especially helpful are their new modeling results showing that differences in ADLI between MCs and TCs contribute to odor discrimination across a wider range of odor concentrations even in the presence of other important differences between MCs and TCs. Some concerns about slice artifacts and their interpretation of differences in excitation of sGCs and dGCs however remain, as do concerns about their DHPG experiments (both brought up in Major Point 2 of my prior review). These, which are explained below, however will just require changes in the text. Their overall story is quite interesting, and I would be satisfied if they simply backed off on some of their claims pertaining to GCs.

1) That the greater evoked responses in sGCs versus dGCs is due to greater excitatory input and intrinsic excitability rather than slice artifacts is still not completely convincing. In response to this concern, raised previously, the authors provided three lines of evidence in the rebuttal letter and Results section, which I will address individually (authors' text is in parentheses).

A) (Our in vitro observation of greater sGC firing following glomerular activation (Figure 4B,E, H) corresponds well with the previous in vivo observation of stronger odor-evoked activity in putative sGCs (Wellis and Scott, 1990).)

This argument is not very helpful because the stronger odor-evoked activity in sGCs in vivo could simply be due to the well-described greater odor-responsiveness of tufted cells (to which the sGCs may preferentially connect). The results do not support that sGCs have synaptic and intrinsic properties that make them more responsive.

B) (Examination of GC biophysical properties revealed several intrinsic differences supporting greater recruitment of sGCs than dGCs, including a more hyperpolarized action potential threshold in sGCs (Figure 4L; Figure 4—figure supplement 2, Table 2) and greater intrinsic excitability in sGCs in response to somatic step current injections (Figure 4J, K; Figure 4—figure supplement 2, Table 3), despite equivalent somatodendritic sizes (Figure 4—figure supplement 1D; Figure 4—figure supplement 2, Table 1) and passive membrane properties (Figure 4—figure supplement 2, Table 4) between sGCs and dGCs. Collectively these observations provide an independent line of evidence that supports our conclusions about differences between sGCs and dGCs.)

These data are somewhat helpful but they do not directly address whether the differences in the evoked responses in sGCs versus dGCs are not mainly a slice artifact.

C) (Analysis of spontaneous synaptic activity revealed no difference in event frequency or amplitude between sGCs and dGCs….Critically, recordings of spontaneous synaptic activity were performed in the absence of TTX and thus contain some degree of action potential-dependent input, which likely originates from intact presynaptic cells. Therefore, equal spontaneous event frequencies between sGCs and dGCs suggests that their respective presynaptic circuits are comparably intact. Moreover, equal spontaneous event amplitudes suggest a comparable contribution of larger action potential-dependent and smaller action potential-independent events between sGCs and dGCs, again consistent with comparably intact presynaptic circuits.)

These data are not especially helpful for excluding slice artifacts because the spontaneous events in GCs (sEPSCs) could reflect spontaneous release of glutamate from pieces of M/T lateral dendrites that remain in the slice but are not associated with a cell body. This differs from responses evoked by stimulation of a single glomerulus, which requires intact cell bodies so that action potentials can pass to the lateral dendrites. That the experiments were conducted in the absence of TTX is not very supportive of there being intact cell bodies, since no evidence is provided to suggest that a major portion of the sEPSCs are being driven by action potentials. They could be mEPSCs (and, notably, the mean amplitudes of the sEPSCs are quite small). In addition, it cannot be excluded that there are spontaneous action potentials in pieces of lateral dendrites that are dissociated from cell bodies. Full-sized action potentials can propagate down mitral cell lateral dendrites, suggesting the presence of sodium channels in these dendrites. To use the spontaneous events as an argument against slice artifacts, the authors would need to perform additional controls (e.g., recordings of miniature events in TTX).

Taking these points together, I still am not convinced that the differences in evoked responses in dGCs and sGCs do not mainly reflect a slice artifact. However, at this point, I would be satisfied if the authors make changes in the text and reduce their emphasis on differences in the synaptic and intrinsic properties of the two GC sub-types being causal to the different ADLI properties of MCs versus TCs.

2) Some concern also remains about the DHPG experiments, which the authors use to argue that the requirement for higher spike frequencies in MCs to obtain ADLI reflects the excitatory properties of GCs. I previously raised the issue that the DHPG-induced shifts in the effective range of lateral inhibition could reflect drug-effects on glomerular layer neurons rather than GCs. In response, the authors point out that 2 MCs with cut apical dendrites (that do not receive inputs from glomerular layer cells) show similar DHPG-induced shifts in the effective frequency range for ADLI as those MCs with intact apical dendrites. However, these data do not exclude the following plausible network effect involving DHPG-induced excitation of external tufted cells in the glomerular layer (Dong and Ennis, 2014): DHPG-induced excitation of eTCs causes greater feedforward excitation of a population of MCs at a glomerulus; this in turn leads to greater excitation of GCs and GC-mediated inhibition. Such an effect, involving GC inputs onto MC lateral dendrites, would not depend on a test MC having intact apical dendrites.

Despite this caveat, their results showing that the 2 MCs with cut apical dendrites show similar DHPG-induced shifts in the effective range of lateral inhibition as MCs are helpful. They at least support the conclusion that changes in the degree of GC excitation (either direct or indirect) can control the frequency range over which the ADLI occurs, which is itself interesting.

Reviewer #2:

The authors addressed the concerns raised and, in my opinion, the manuscript is fit for publication at this point.

Reviewer #3:

The authors are discussing my key concern (whether the findings hold true in vivo) very well in the rebuttal and following their logic it is indeed very likely that most of the key findings will hold true. I find the well-structured and expanded (compared to the Discussion) overview given in the rebuttal very helpful and would suggest incorporating this version into the Discussion.

https://doi.org/10.7554/eLife.16039.021

Author response

Essential revisions:

1) It is not convincing that the difference in ADLIs between mitral and tufted cells is not a result of tissue slicing. Furthermore, it is unclear whether the different properties of superficial versus deep granule cells that might have caused the differences in ADLIs are a side effect of slicing. The authors have to provide more convincing, quantitative arguments to exclude artifacts associated with slicing.

We agree with the reviewers that the issue of tissue slicing is important. However, we believe that the differences between MCs and TCs and between sGCs and dGCs reported here are not slicing artifacts, but true features of the olfactory bulb circuit. Below, we provide rationale for why each of the three main experimental findings are not artifacts of the slicing procedure. We additionally state how our revised manuscript presents these rationale.

Main finding 1: Lateral inhibitory currents are larger and more asynchronous onto MCs than TCs (Figure 1)

Due to slicing, some MCs and TCs that project to the M72 glomerulus may be truncated, and cells that reside farther from the M72 glomerulus will be expected to have a higher probability of being truncated. Therefore, we are likely to lose more M72 MCs than M72 TCs with the slicing procedure. Additionally, prior work (Mori, et al., J Comp Neurol,1983; Scott, et al., J Comp Neurol, 1983) shows that lateral dendrites of MCs and TCs project radially in all directions. Therefore, the lateral dendrites of the remaining MCs and TCs still connecting to the M72 glomerulus are likely to be affected approximately equally by the slicing procedure. Consequently, we would expect that slicing would be more likely to reduce M72-mediated lateral inhibition onto MCs than TCs. However, in Figure 1, we find that MCs have stronger lateral inhibition than TCs. Therefore, the differences in lateral inhibition that we report are likely an underestimation of the actual differences between MCs and TCs. Additionally, Figure 1—figure supplement 1 shows that the strength of lateral inhibitory currents does not vary with distance from the M72 in either MCs or TCs.

Main finding 2: Lateral inhibition affects intermediate firing rates in MCs and low firing rates in TCs (Figures 2, 3)

Similar to the above argument, we include data in Figure 3—figure supplement 1 that shows that the effect of lateral inhibition on spiking does not vary with distance from the M72 glomerulus in either MCs or TCs. Additionally, we have added panels (E-J) to Figure 3—figure supplement 1 that show that neither the lower nor upper bound of the effective range of lateral inhibition depend on the distance from the M72 glomerulus in either MCs or TCs.

Finally, the lateral dendrites of MCs and TCs innervate distinct strata of the external plexiform layer and these differences could result in differences in the fraction of the original connections left intact following slicing. However, the effect of lateral inhibition on MCs that are observed here following activation of an entire glomerulus closely mirrors the effects that were observed when activating a single presynaptic MC in our previous work (Arevian et al., Nat Neuro, 2008). That is, only the probability of finding connections and the absolute magnitude of the effect are increased by activating all of the cells associated with a glomerulus (compared to the prior study when only a single presynaptic cell was activated). This suggests that the critical feature of the effect (maximal lateral inhibition at intermediate firing rates) is a property of single connections rather than one that depends on having a full repertoire of connections intact.

Main finding 3: Compared with deep GCs, superficial GCs spike with higher probability and receive stronger excitatory currents following glomerular stimulation (Figure 4)

We agree with the reviewers that the greater recruitment of sGCs following glomerular activation may be a consequence of our use of an acute slice preparation. Specifically, as TC circuitry is closer to any given glomerulus than MC circuitry, TC-mediated input to GCs (likely sGCs) may be better preserved than MC-mediated input to GCs (likely dGCs) in the acute slice, leading to stronger sGC excitation and recruitment following glomerular activation. We explicitly note this as a caveat in the Results section of our revised manuscript.

We note, however, that three lines of evidence argue against this possibility, and support greater feedforward recruitment of sGCs as a physiological feature of the olfactory bulb circuit.

A) Our in vitro observation of greater sGC firing following glomerular activation (Figure 4B, E, H) corresponds well with the previous in vivoobservation of stronger odor-evoked activity in putative sGCs (Wellis and Scott, 1990). In the Results section of our revised manuscript, we now explicitly discuss this prior in vivostudy in the context of our current results.

B) Examination of GC biophysical properties revealed several intrinsic differences supporting greater recruitment of sGCs than dGCs, including a more hyperpolarized action potential threshold in sGCs (Figure 4L; Figure 4—figure supplement 2, Table 2) and greater intrinsic excitability in sGCs in response to somatic step current injections (Figure 4J, K; Figure 4—figure supplement 2, Table 3), despite equivalent somatodendritic sizes (Figure 4—figure supplement 1D; Figure 4—figure supplement 2, Table 1) and passive membrane properties (Figure 4—figure supplement 2, Table 4) between sGCs and dGCs. Collectively these observations provide an independent line of evidence that supports our conclusions about differences between sGCs and dGCs.

C) Analysis of spontaneous synaptic activity revealed no difference in event frequency or amplitude between sGCs and dGCs. We have included this new analysis in Figure 4—figure supplement 2, Table 5 of the revised manuscript. Critically, recordings of spontaneous synaptic activity were performed in the absence of TTX and thus contain some degree of action potential-dependent input, which likely originates from intact presynaptic cells. Therefore, equal spontaneous event frequencies between sGCs and dGCs suggests that their respective presynaptic circuits are comparably intact. Moreover, equal spontaneous event amplitudes suggest a comparable contribution of larger action potential-dependent and smaller action potential-independent events between sGCs and dGCs, again consistent with comparably intact presynaptic circuits.

In view of these three lines of evidence, we believe that our results strongly suggest that sGCs are more strongly recruited than dGCs following activation of a single glomerulus due to stronger excitatory input and greater intrinsic excitability.

In addition to the specific revisions to our manuscript described above, we have also broadly summarized this rationale in the following section of our revised Discussion:

“Performing these experiments in acute slices provides the best opportunity to explore the causes and consequences of differences in lateral inhibition between MCs and TCs. […]Addressing the extent to which ADLI affects odor-evoked responses in MCs and TCs in vivois thus an important future direction.”

2) The authors examined the significance of differential ADLI between mitral and tufted cells using modeling. However, mitral and tufted cells are different not only in their ADLI properties but also their intrinsic and synaptic input properties. In the modeling, all of these are changed at the same time, and therefore, it is unclear whether improved odor discriminability at different odor concentrations is due to ADLI alone, differences in intrinsic/synaptic properties alone, or a combination of all factors. Please clarify this issue with additional analysis.

We agree with the reviewer that understanding how each of the differences between MCs and TCs contribute to concentration-dependent differences in odor discrimination is important and have updated Figure 6 to explore the contributions of these specific differences. First, in Figure 6G, we show that MC-like and TC-like ADLI is alone sufficient to allow otherwise identical cells to best discriminate between high and low concentration odors, respectively. Second, in Figure 6H, I, we show that differences in intrinsic excitability and glomerular input strength between MCs and TCs (in the absence of ADLI differences) also allow MCs and TCs to best discriminate between high and low concentration odors, respectively. Finally, we perform the simulation in which all three factors differ between MCs and TCs (Figure 6J) and find that together these three factors work additively to allow MCs and TCs to best discriminate between high and low concentration odors, respectively.

We make two conclusions from these simulations. First, each of three experimentally determined differences between MCs and TCs (intrinsic excitability, glomerular input strength, and ADLI) alone is sufficient to allow TCs perform better at low concentration. Second, TCs are best at performing low concentration odor discrimination and MCs are best at performing high concentration discriminations when all three differences are present. Therefore, because these intrinsic and circuit-level differences between MCs and TCs all lead to the same functional difference between MCs and TCs, this functional difference is likely to be an important feature of processing of odor-evoked information.

We have updated the Results, Figure 6 and the Figure 6 legend, to reflect these new analyses and conclusions.

3) In Figure 3, the authors mention that "decreases in firing rate were calculated as the average decrease across all significantly affected trials". Selecting trials based on "significantly affected trials" seems inappropriate. Exclusion of such trials in tufted cells would have resulted in an artificially large value for the percent change in spike rate due to ADLI.

We apologize for the confusion caused by the Figure 3 legend. The statement “decreases in firing rate were calculated as the average decrease across all significantly affected trials” only refers to Figure 3F, in which distinct trials corresponded to distinct firing rates. In Figure 3B-D, data from all trials (so that none were excluded) were used, and these data demonstrate that lateral inhibition reduces the absolute firing rate by similar amounts in both MCs and TCs.

To reduce confusion, we have changed the Figure 1 legend to state, “In F, decreases in firing rate were calculated as the average decrease across all significantly affected firing rates.”

4) in vivo experiments would greatly increase the significance of the present study as lateral inhibition may be sensitive to subtle details of the network properties, spontaneous activity and breathing etc. that can be seen only in an intact network. If the authors decide not to perform in vivo experiments, potential caveats of their in vitro experiments must be explicitly discussed.

We agree that exploring how ADLI influences odor-evoked responses in MCs and TCs in vivowill be important to the field. However, we believe that the reduced slice preparation that we use in our work provides several advantages for exploring the novel features and mechanisms of ADLI in MCs and TCs. Therefore, we have added an additional section to our Discussion that more explicitly addresses the advantages and limitations of performing our experiments in vitro. We refer the reviewer to this section and to our response to Essential Revision #1 above.

Reviewer #1:

Overall, this is an interesting study that addresses a broadly relevant question: How can two parallel sensory processing paths (in this case, mediated by MCs and TCs) enhance sensory discrimination? Much of the study also employs a powerful experimental paradigm involving optogenetic stimulation of a single, molecularly-defined glomerulus (M72) as a means to drive lateral inhibition. This strategy should eliminate ambiguities about whether only one glomerulus is being stimulated and, also, should reduce variabilities across experiments. Some of the results are also basically convincing, for example that MCs and TCs have different kinetics for lateral inhibition and also differ in the frequency range over which ADLI occurs. However, I have a number of concerns with both the experimental and modeling sections of the paper.

1) The analysis methods used raise questions about results suggesting that TCs display a larger percent decrease in spike rate due to ADLI than MCs (Figure 3C). The legend of Figure 3 indicates that changes in firing rate due to ADLI were calculated based only on "significantly affected trials". Because ADLI in TCs occurred at low spike frequencies, it seems quite possible that many of the trials in which small effects were actually occurring may have been missed by a per-trial statistical analysis (due to a low total number of spikes). Exclusion of such trials in TCs would have resulted in an artificially large value for the percent change in spike rate due to ADLI, as they calculated it. Such exclusions would be less common in MCs that displayed ADLI at higher spike frequencies.

We apologize for the confusion caused by the Figure 3 legend and refer the reviewer to our response to Essential Revision #3 above.

2) That differences in the properties of superficial versus deep GCs (sGCs and dGCs) contribute to differences in ADLI between MCs and TCs is not completely convincing. The argument is based on the parallel observations that TCs display ADLI at lower spike frequencies than MCs and also that sGCs (which may preferentially contact TCs) are more responsive to glomerular stimulation. However, the authors need to exclude better the possibility that the greater spike probability and evoked currents in sGCs is not a slicing artifact. sGCs, which are closer to glomeruli, may naturally be expected to have more of their input circuitry intact in slices. The fact that sGCs appear to have greater intrinsic excitability helps their argument, but it is not sufficient. Comparing ADLI and the responsiveness of GCs is also complicated by the fact that the experiments assaying these two features appear to have been conducted in differing magnesium concentrations (0.2 mM versus 1 mM).

We agree that understanding how the slicing may contribute to our reported findings is vital and have addressed this issue above under Essential Revision #1.

The authors also use the fact that the mGluR agonist DHPG reduces the frequency range for ADLI in MCs to argue that differences in ADLI between MCs and TCs are due to GC properties. This is based on prior observations that DHPG can enhance GC excitability. DHPG appears not alter MC spiking due to direct current injection (Figure 5—figure supplement 1), yet other circuit effects of DHPG need to be better controlled. For example, DHPG could enhance stimulus-evoked lateral inhibition by altering glomerular layer neurons. This, in turn, could cause the same frequency shifts in ADLI in MCs as increases in GC excitability.

The reviewer brings up a good point that DHPG could enhance lateral inhibition mediated by glomerular layer circuits. However, we have indicated in Figure 5D whether MCs have intact apical dendrites. We find that the 2 MCs with cut apical dendrites show similar shifts in the effective range of lateral inhibition as those MCs with intact apical dendrites – that DHPG decreases the range of rates influenced by lateral inhibition. Therefore, we conclude that the effects of DHPG on glomerular layer interneurons are insufficient to explain the shifts in ADLI. We have clarified this point in the Results section of our revised manuscript.

3) The computational results suggesting that differences in ADLI between MCs and TCs enable these cells to perform odor discrimination at different concentrations are not completely convincing. The most important results are shown in Figure 6L, which displays the output of a model that incorporates differences in the level of sensory neuron input onto TCs versus MCs, differences in intrinsic excitability, as well as differences in ADLI. This shows that TCs perform better discrimination at lower odor concentration than MCs. However, it is unclear how much differences in ADLI between MCs versus TCs are contributing to these discrimination curves. The fact that TCs discriminate better at lower concentrations may mainly reflect their greater responsiveness to low concentrations of odor. The authors need to test whether removing inhibition substantially impacts the discrimination curves in Figure 6L, as well as Figure 6N, as they did for their simpler model in Figure 6G.

We have made Figure 6 clearer and refer the reviewer to our response to Essential Revision #2 above.

Reviewer #2:

Geramita, Burton & Urban report through a series of elegant experiments in acute olfactory bulb preparations that the two types of bulb principal neurons, tufted and mitral cells, receive different levels of inhibition from granule cells (stronger and more asynchronous to mitral cells compared to tufted cells). The authors show through a combination of patch-clamp recordings, current injections and optogenetic stimulation of genetically labeled glomeruli that activity dependent lateral inhibition (ADLI) occurs at low firing rate regimes in tufted cells and intermediate firing rates in mitral cells. They further propose as underlying substrate at play for the different ADLI regimes two distinct populations of superficial and deep granule cells (sGCs, dGCs). Anatomical reconstructions and clustering suggest that sGCs and dGCs contact differentially the lateral dendrites of TC and respectively MCs. Further functional analysis reveals differences in intrinsic excitability and threshold for action potential firing between sGCs and dGCs consistent with their proposed roles. In my opinion, the manuscript adds important novel understanding by documenting that functional differences between mitral and tufted cells emerge not only due to differences in intrinsic excitability, but also in their local connectivity with inhibitory interneurons. These findings bring timely mechanistic insight that parsimoniously explains recently published differences in TCs and MCs responses to odors, across concentrations, and to modulation of cortical feedback. Therefore, I consider the manuscript fit for publication in eLife should the authors address several concerns listed below.

Importantly, I have doubts regarding the necessity of the computational model presented and some of its underlying assumptions. Given the temporal complexity of active sampling (sniffing) in vivo, relevant time window for odor identification (200-300 ms, Uchida & Mainen, 2003), possible effects of the slice preparation on the functional connectivity of the bulb, and lack of top-down regulation (glutamatergic, GABA-ergic and neuromodulatory input) of mitral and tufted cell activity in the slice preparation, I suggest focusing the manuscript on the experimental results with fewer incursions into the simulations.

1) The authors present in Figure 6K two sigmoids that govern the responses of MC and TCs to increasing concentrations. MCs have been reported to display a wide range of concentration response curves that are not necessarily monotonic (Meredith, 1986), and thus at odds with the chosen transfer function.

We thank the reviewer for bringing this study to our attention. In our updated manuscript (Results, fourteenth paragraph), we have added this reference as a caveat to our assumption that MCs and TCs encode odor concentration as firing rate changes.

While the Meredith work indicates that some MC/TCs may display concentration response curves that are not monotonic, the other cited studies show that firing rate is one of several properties that consistently vary with changes in concentration. Even if some MCs and TCs do not display monotonic concentration response curves, the results of our simulations will still be important for understanding how ADLI may influence the representations encoded in large numbers of MCs and TCs.

2) What is the functional relevance of the concentration regime sampled? What does 20% to 80% of maximal concentration translate to in terms of absolute odor concentration? Are such concentrations ever encountered by the animal?

The x-axis in the Figure 6F,G,H,I,J,L plots the “percent maximum concentration”. The maximum concentration is defined as the concentration that evokes maximum firing rates in MCs and TCs, which in our models is set at 100 Hz – a rate often observed in vivo. We use a sigmoidal transfer function to translate glomerular inputs into MC/TC outputs so that we can define the odor concentrations in terms of MC/TC firing rates while not making any explicit comparisons to actual odor concentrations. Given that odor concentration is defined in terms of MC/TC firing rates, 20-80% of maximal concentration refer to odors that evoke firing rates in MCs and TCs that are 20-80% of their maximum firing rates, which are known to occur in vivo.

We have included an explanation of our rationale for using this quantification metric in the updated Methods (subsection “Computational model”, fifth paragraph).

3) Why is the classifier trained independently for each of the concentrations sampled? This seems an unrealistic regime compared to processes that may occur in the brain.

The reviewer’s question is an important one. We chose to train the classifier independently for each concentration to mimic typical behavioral experiments where animals are trained to discriminate between different odors presented at similar odor concentrations. We believe that in order to train the classifier on odors presented at multiple concentrations, we would need to introduce assumptions about how the same odor is represented at different concentrations. Indeed, this question of how MC/TC representations of odor identity remain invariant across a range of concentrations is important but ultimately one that cannot be adequately addressed with our model.

In the manuscript (Results), we have added the following caveat. “While understanding how animals discriminate between odors presented at a variety of concentrations is important, we confined our discriminations to odors presented at the same concentration to more closely match behavioral experiments in mice (Abraham et al., Neuron, 2010; Lepousez et al., Neuron, 2010) and to keep from making a number of assumptions about how the representation of individual odors varies across concentration.”

4) Differences in functional properties of sGCs and dGCs can be in principle heavily modulated by top-down signals that are absent in the slice. The current version of the manuscript does not discuss this possibility.

We agree with the reviewer that both superficial and deep GCs may be heavily influenced by top-down modulation from a variety of brain areas and have addressed this caveat in the updated manuscript. Our response to this point as well as our discussion of other differences that may occur in vivocan be found under Essential Revisions #1 and #4 above.

5) MC and TCs have been reported to project with differential biases to downstream target areas. How do such biases contribute to the ability of the decoder to incorporated odor information carried by both TC and MC?

The reviewer brings up a good point that MCs and TCs project to separate sets of downstream areas. However, current data suggest that there are areas, such as the anterior piriform cortex, that receive input from both MCs and TCs. The simulation that generated the data in Figure 6K relies on a decoder that incorporates information from both MCs and TCs. This model assumes that neurons in the piriform cortex are performing some kind of clustering/categorization of both MC and TC outputs. This is a relatively common assumption and is supported by a variety of data that suggest the anterior piriform cortex is functioning as an associative memory system performing pattern completion (Haberly, Trends in Neuro, 1989; Bekkers, Trends in Neuro, 2013). In the updated manuscript (Discussion, last paragraph), we have included an explicit reference to the anterior piriform cortex as an area that might be integrating input from both MCs and TCs.

Our simulations do not address how other areas such as the anterior olfactory tubercle, which receives stronger input from TCs than MCs, might be integrating or decoding information transmitted in both channels. Therefore, we have refrained from drawing conclusions about how information is integrated in these other downstream areas.

In Figure 1L to assess in an unbiased manner the contribution of GC-mediated vs. glomerular layer mediated lateral inhibition on MCs vs. TCs, the analysis (before vs. after drug cocktail application) should be restricted only onto those cells that have the apical dendrite intact. Otherwise, by construction, the contribution of the glomerular layer lateral inputs is reduced/absent in cells with apical dendrite cut. In the current version, 1 out of 5 MCs, and respectively 2 out of 5 TCs have their apical dendrite cut.

We agree that in order to compare GC-mediated and glomerular layer mediated inhibition between MCs and TCs, only cells with intact apical dendrites should be analyzed. However, after removing cells with cut apical dendrites and redoing the statistical analysis done in Figure 1L, we find no statistical difference between MCs and TCs. Therefore, in the revised manuscript, we have kept cells with cut apical dendrites in Figure 1L in order to emphasize the point that lateral inhibitory currents are eliminated after reducing GC-mediated inhibition. However, in the manuscript we have added, “Removing cells with cut apical dendrites and redoing the analysis in Figure 1L similarly shows that GCs contribute similar proportions of lateral inhibition onto MCs and TCs.”

Reviewer #3:

The authors present a detailed in vitro analysis of differences in lateral inhibition experienced by MCs and TCs. They attribute these differences to different innervation of different populations of GCs and use a simplified model to illustrate that the differential inhibition can lead to improved discriminability overall (with TCs aiding to low-concentration and MCs to high-concentration discrimination).

In a previous submission (to another journal) there were numerous criticisms that the authors largely address well. One key concern that was reiterated by all reviewers was that the findings of differential inhibition were exclusively based on in vitro experiments (and simulations) with little direct link to how sensory information might be processed. Here I think the current manuscript is still lacking. While the authors argue – rightly so – that they perform an in depth mechanistic analysis (which is difficult in vivo) I have to say I tend to agree in that the relevance / role of the differential inhibition for odor processing is still unclear. If indeed – as stated in the response to prior reviewer 1 – the variability / signal-to-noise will mask the effect in vivo one might question how dominant it could be in a behaving context. I surely don't argue for repeating all experiments in an awake behaving animal but key experiments (parts of Figures 1 and 4) at least in an anesthetized (if not in an awake) animal would convince the reader that the effects studied are indeed shaping odor processing in vivo.

We thank the reviewer for his comments. We agree that understanding how lateral inhibition affects odor-evoked responses in MCs and TCs in vivois critical. We have added a paragraph to our Discussion that addresses the limitations of our in vitroapproach. It can be found above under Essential Revision #1.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance as agreed upon by the reviewers in discussion following their original reviews that are appended below. Specifically, while all the referees agreed that the manuscript has been greatly improved, there are two remaining concerns. First, the reviewers thought that it remains unclear whether the authors can completely exclude the possibility that slicing biased the contribution of superficial versus deep granule cells in activity-dependent lateral inhibition (ADLI) (point # 1 of reviewer 1). Furthermore, the interpretation of the experiment using DHPG should be revised so that the possibility of the network effect (point #2, reviewer 1) is appreciated. We think that both points can be addressed with revisions to the text as outlined by reviewer 1. For more details, please see the reviewers' original reviews below.

Reviewer #1:

The Reviewers have adequately addressed most of my concerns. Especially helpful are their new modeling results showing that differences in ADLI between MCs and TCs contribute to odor discrimination across a wider range of odor concentrations even in the presence of other important differences between MCs and TCs. Some concerns about slice artifacts and their interpretation of differences in excitation of sGCs and dGCs however remain, as do concerns about their DHPG experiments (both brought up in Major Point 2 of my prior review). These, which are explained below, however will just require changes in the text. Their overall story is quite interesting, and I would be satisfied if they simply backed off on some of their claims pertaining to GCs.

1) That the greater evoked responses in sGCs versus dGCs is due to greater excitatory input and intrinsic excitability rather than slice artifacts is still not completely convincing. In response to this concern, raised previously, the authors provided three lines of evidence in the rebuttal letter and Results section, which I will address individually (authors' text is in parentheses).

A) (Our in vitro observation of greater sGC firing following glomerular activation (Figure 4B, E, H) corresponds well with the previous in vivo observation of stronger odor-evoked activity in putative sGCs (Wellis and Scott, 1990).)

This argument is not very helpful because the stronger odor-evoked activity in sGCs in vivo could simply be due to the well-described greater odor-responsiveness of tufted cells (to which the sGCs may preferentially connect). The results do not support that sGCs have synaptic and intrinsic properties that make them more responsive.

B) (Examination of GC biophysical properties revealed several intrinsic differences supporting greater recruitment of sGCs than dGCs, including a more hyperpolarized action potential threshold in sGCs (Figure 4L; Figure 4—figure supplement 2, Table 2) and greater intrinsic excitability in sGCs in response to somatic step current injections (Figure 4J, K; Figure 4—figure supplement 2, Table 3), despite equivalent somatodendritic sizes (Figure 4—figure supplement 1D; Figure 4—figure supplement 2, Table 1) and passive membrane properties (Figure 4—figure supplement 2, Table 4) between sGCs and dGCs. Collectively these observations provide an independent line of evidence that supports our conclusions about differences between sGCs and dGCs.)

These data are somewhat helpful but they do not directly address whether the differences in the evoked responses in sGCs versus dGCs are not mainly a slice artifact.

C) (Analysis of spontaneous synaptic activity revealed no difference in event frequency or amplitude between sGCs and dGCs….Critically, recordings of spontaneous synaptic activity were performed in the absence of TTX and thus contain some degree of action potential-dependent input, which likely originates from intact presynaptic cells. Therefore, equal spontaneous event frequencies between sGCs and dGCs suggests that their respective presynaptic circuits are comparably intact. Moreover, equal spontaneous event amplitudes suggest a comparable contribution of larger action potential-dependent and smaller action potential-independent events between sGCs and dGCs, again consistent with comparably intact presynaptic circuits.)

These data are not especially helpful for excluding slice artifacts because the spontaneous events in GCs (sEPSCs) could reflect spontaneous release of glutamate from pieces of M/T lateral dendrites that remain in the slice but are not associated with a cell body. This differs from responses evoked by stimulation of a single glomerulus, which requires intact cell bodies so that action potentials can pass to the lateral dendrites. That the experiments were conducted in the absence of TTX is not very supportive of there being intact cell bodies, since no evidence is provided to suggest that a major portion of the sEPSCs are being driven by action potentials. They could be mEPSCs (and, notably, the mean amplitudes of the sEPSCs are quite small). In addition, it cannot be excluded that there are spontaneous action potentials in pieces of lateral dendrites that are dissociated from cell bodies. Full-sized action potentials can propagate down mitral cell lateral dendrites, suggesting the presence of sodium channels in these dendrites. To use the spontaneous events as an argument against slice artifacts, the authors would need to perform additional controls (e.g., recordings of miniature events in TTX).

Taking these points together, I still am not convinced that the differences in evoked responses in dGCs and sGCs do not mainly reflect a slice artifact. However, at this point, I would be satisfied if the authors make changes in the text and reduce their emphasis on differences in the synaptic and intrinsic properties of the two GC sub-types being causal to the different ADLI properties of MCs versus TCs.

We thank the reviewer for these comments and agree that we cannot completely rule out the possibility that the differences between evoked responses in sGCs and dGCs may be due to slice effects. We also agree that we have not provided direct evidence that differences between dGCs and sGCs cause differences in ADLI between MCs and TCs. However, we believe that we do provide evidence that differences in ADLI are not caused of glomerular layer lateral inhibitory circuits and are thus due to external plexiform layer circuits. Additionally, the data presented in Figures 4 and 5 constitute two independent lines of indirect evidence that support the hypothesis that differences in GCs are one mechanism responsible for the observed differences in ADLI between MCs and TCs. However, we cannot rule out the possibility that other mechanisms also contribute to the observed differences in ADLI.

The revised manuscript reflects this shift in tone in several areas listed below:

A) “Several previous findings are consistent with this hypothesis. GC soma position correlates with GC subtype…”

B) “However, despite the fact that these observations were made in vitro, three lines of evidence support greater feedforward recruitment of sGCs as a physiological feature of the olfactory bulb circuit. First, our in vitroobservation of…”

C) “In total, our results thus support the hypothesis that sGCs are more strongly recruited than dGCs following activation of a single glomerulus due to stronger excitatory input and greater intrinsic excitability, although we cannot rule out an important role for other mechanisms, especially in the intact system in vivo.”

D) “As an additional test of our hypothesis that differences in the excitability of GCs provide one potential mechanism underlying thefunctional difference in ADLI between MCs and TCs, we measured the effects of lateral inhibition on MC spiking before and after increasing the excitability of GCs with the mGluR agonist, DHPG.”

E) “Despite these observations, however, we note that increased GC excitability may not be the sole mechanism responsible for these DHPG-induced shifts in the effective range of lateral inhibition. For instance, DHPG-induced increases in the excitability of external tufted(Dong and Ennis 2014) cells may, in turn, increase the activity in M72-MCs, the activity in GCs and GC-mediated inhibition onto the recorded MC.”

F) “These data (Figures 45) provide two indirect lines of evidence that suggest that differences in GC populations provide one mechanism for differences in the effective activity range of lateral inhibition onto MCs and TCs.”

G) “We provide evidence that these effects could be caused, in part, by differences in the excitability of subclasses of GCs that preferentially inhibit TCs vs. MCs.”

H) “Additionally, we provide indirect evidence that differences in the recruitability of sGCs and dGCs may be one potential mechanism underlying of the differences in ADLI between MCs and TCs. Our data suggests that sGCs spike with a higher probability than dGCs following the activation of a single glomerulus.”

I) “Additionally, these differences between sGCs and dGCs can explain why the upper bound on the effective activity range of lateral inhibition differs between MCs and TCs.”

2) Some concern also remains about the DHPG experiments, which the authors use to argue that the requirement for higher spike frequencies in MCs to obtain ADLI reflects the excitatory properties of GCs. I previously raised the issue that the DHPG-induced shifts in the effective range of lateral inhibition could reflect drug-effects on glomerular layer neurons rather than GCs. In response, the authors point out that 2 MCs with cut apical dendrites (that do not receive inputs from glomerular layer cells) show similar DHPG-induced shifts in the effective frequency range for ADLI as those MCs with intact apical dendrites. However, these data do not exclude the following plausible network effect involving DHPG-induced excitation of external tufted cells in the glomerular layer (Dong and Ennis, 2014): DHPG-induced excitation of eTCs causes greater feedforward excitation of a population of MCs at a glomerulus; this in turn leads to greater excitation of GCs and GC-mediated inhibition. Such an effect, involving GC inputs onto MC lateral dendrites, would not depend on a test MC having intact apical dendrites.

Despite this caveat, their results showing that the 2 MCs with cut apical dendrites show similar DHPG-induced shifts in the effective range of lateral inhibition as MCs are helpful. They at least support the conclusion that changes in the degree of GC excitation (either direct or indirect) can control the frequency range over which the ADLI occurs, which is itself interesting.

We agree with the reviewer that DHPG-induced shifts in the effective range of lateral inhibition onto MCs can, in part, be explained by increases in GC excitability, based on the 2 MCs that lack apical dendrites. However, we agree that we cannot completely exclude the possibility that DHPG also increases the excitability of ETCs, which, in turn, can increase the activity in M72 MCs, the activity of GCs, and the amount of GC-mediated inhibition.

Therefore, in the updated manuscript we have added the following sentence: “As a caveat, however, we note that increased GC excitability may not be the sole mechanism responsible for these DHPG-induced shifts in the effective range of lateral inhibition. For instance, DHPG-induced increases in the excitability of external tufted cells (Dong and Ennis, 2014) may, in turn, increase the activity in M72-MCs, the activity in GCs and GC-mediated inhibition onto the recorded MC.”

Reviewer #3:

The authors are discussing my key concern (whether the findings hold true in vivo) very well in the rebuttal and following their logic it is indeed very likely that most of the key findings will hold true. I find the well-structured and expanded (compared to the Discussion) overview given in the rebuttal very helpful and would suggest incorporating this version into the Discussion.

We thank the reviewer and agree that it is useful to add more of the detailed logic from our first response to reviewers. In addition to the changes above, several of which address this issue and reiterate the logic from the previous rebuttal letter, we have added the following to our Discussion:

“Additionally, the lateral dendrites of MCs and TCs innervate distinct strata of the external plexiform layer and these differences could result in differences in the fraction of original connections left intact following slicing. […] This suggests that the critical feature of the effect (maximal lateral inhibition at intermediate firing rates) is a property of single connections rather than one that depends on having a full repertoire of connections intact.”

https://doi.org/10.7554/eLife.16039.022

Article and author information

Author details

  1. Matthew A Geramita

    1. Department of Neurobiology, University of Pittsburgh, Pittsburgh, United States
    2. Center for the Neural Basis of Cognition, University of Pittsburgh, Pittsburgh, United States
    Contribution
    MAG, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents
    Competing interests
    The authors declare that no competing interests exist.
  2. Shawn D Burton

    1. Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, United States
    2. Center for the Neural Basis of Cognition, Carnegie Mellon University, Pittsburgh, United States
    Contribution
    SDB, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon 0000-0002-8907-6487
  3. Nathan N Urban

    1. Department of Neurobiology, University of Pittsburgh, Pittsburgh, United States
    2. Center for the Neural Basis of Cognition, University of Pittsburgh, Pittsburgh, United States
    Contribution
    NNU, Conception and design, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents
    For correspondence
    nurban@pitt.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon 0000-0002-0365-9068

Funding

National Institute on Deafness and Other Communication Disorders (RO1-DC011184)

  • Nathan N Urban

National Institute on Deafness and Other Communication Disorders (F31-DC013490)

  • Shawn D Burton

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

Thanks to Brent Doiron, Claire Cheetham and members of the Urban laboratory for helpful comments and discussion, and Greg LaRocca for excellent technical assistance. This work was supported by the National Institute of Deafness and Other Communication Disorders Grants R01-DC011184 (NNU) and F31-DC013490 (SDB).

Ethics

Animal experimentation: All experiments were completed in compliance with the guidelines established by the Institutional Animal Care and Use Committee of Carnegie Mellon University.(IACUC # AS15-010) and University of Pittsburgh (IACUC # 15116582).

Reviewing Editor

  1. Naoshige Uchida, Reviewing Editor, Harvard University, United States

Publication history

  1. Received: March 15, 2016
  2. Accepted: June 27, 2016
  3. Accepted Manuscript published: June 28, 2016 (version 1)
  4. Version of Record published: August 3, 2016 (version 2)

Copyright

© 2016, Geramita et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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