DOI: 10.5061/dryad.r4xgxd2m9
title: Data from: Value-related learning in the olfactory bulb occurs through pathway-dependent peri-somatic inhibition of mitral cells

description: Associating values to environmental cues is a critical aspect of learning
 from experiences, allowing animals to predict and maximise future rewards.
 Value-related signals in the brain were once considered a property of
 higher sensory regions, but their wide distribution across many brain
 regions is increasingly recognised. Here, we investigate how
 reward-related signals begin to be incorporated, mechanistically, at the
 earliest stage of olfactory processing, namely, in the olfactory bulb. In
 head-fixed mice performing Go/No-Go discrimination of closely related
 olfactory mixtures, rewarded odours evoke widespread inhibition in one
 class of output neurons, that is, in mitral cells but not tufted cells.
 The temporal characteristics of this reward-related inhibition suggest it
 is odour-driven, but it is also context-dependent since it is absent
 during pseudo-conditioning and pharmacological silencing of the piriform
 cortex. Further, the reward-related modulation is present in the somata
 but not in the apical dendritic tuft of mitral cells, suggesting an
 involvement of circuit component located deep in the olfactory bulb.
 Depth-resolved imaging from granule cell dendritic gemmules suggests that
 granule cells that target mitral cells receive a reward-related extrinsic
 drive. Thus, our study supports the notion that value-related modulation
 of olfactory signals is a characteristic of olfactory processing in the
 primary olfactory area and narrows down the possible underlying mechanisms
 to deeper circuit components that contact mitral cells peri-somatically.
description: # **Value-related learning in the olfactory bulb occurs through
 pathway-dependent peri-somatic inhibition of mitral cells** **Description
 of the data and file structure** Data contained here are the individual
 data points plotted in the main figures of the manuscript currently under
 consideration at PLOS Biology (DOI to be added here upon
 acceptance/publication). Data and file structure: Individual data points
 on scatter are available in double-precision numeric arrays (both .mat and
 .csv formats). Common abbreviations: * MC: Mitral cells * TC: Tufted cells
 * ROI: region of interest * sPlus: Rewarded trials (S+) * sMin: Unrewarded
 trials (S-) * ΔF/F: Normalized calcium fluorescence (see Methods) *
 CaData: Calcium data (reported in ΔF/F) * exc: excitatory * inh:
 inhibitory   **Materials and Methods** *Animals* Tbx21-Cre (doi:
 10.1038/nn.3407), B6J.Cg-Gt(ROSA)26Sortm95.1(CAG- GCaMP6f)Hze/MwarJ, also
 known as Ai95D (), and
 Ai14 (doi: 133-140. 10.1038/nn.2467 ) mice were originally obtained from
 the Jackson Laboratory (stock numbers: 024507, 028865 and 007914,
 respectfully). Lbhd2-CreERT2 mice were generated previously and are also
 available through Jackson Laboratory (stock number 036054; doi:
 10.1523/JNEUROSCI.3076-20.2021 ). Tbx21-Cre::Ai95D and
 Lbhd2-CreERT2::Ai95D mice were generated by crossing parents homozygous
 for each transgene. Lbhd2-CreERT2::Ai14 mice were generated by crossing
 LBHD2-CreERT2 mice with Ai14 mice.   *Surgery* All recovery surgeries were
 conducted in an aseptic manner. For the cranial window and headplate
 implantations, 9-11 week-old male mice were deeply anesthetised with
 isoflurane (3-5% for induction, 1-2% for maintenance; IsoFlo, Zoetis
 Japan). A craniotomy of approximately 1.5 x 1 mm was performed over the
 left olfactory bulb, and a custom cut glass window (thickness No. 1;
 Matsunami, Japan) was implanted. Once the window was sealed with
 cyanoacrylate (Histoacryl, B. Braun, Germany), a custom-made metal
 headplate (26 x 12 mm) was implanted posterior to the cranial window.
 Dental acrylic (Kulzer, Hanau, Germany) was then added to cover the
 exposed skull and to secure both the headplate and cranial window. For
 experiments involving pharmacological infusion, an additional cannula (10
 mm length; C315GS- 4/SPC, Plastics One) was inserted to target the left
 anterior piriform cortex (coordinate: AP -2.2 mm & ML -2.4 mm from
 bregma; DV -6.1 mm at 45-degree angle from brain surface). *Virus
 injection* To express GCaMP6f in adult-born granule cells, during cranial
 window and headplate implantations, *Lbhd2-CreERT2::Ai14* mice were
 injected with AAV1-syn-GCaMP6f-WPRE-SV40 (Addgene 100837-AAV1; titre was
 1.84 x 1013 GC/mL at the time of synthesis) in the left SVZ (200 μL;
 coordinate: AP 1.0 mm & ML -1.0 mm from bregma; DV -2.2 mm vertically)
 using Nanoject III (Drummond, 3-000-207). *Habituation* Male mice were
 habituated to head fixation on a custom-made running wheel. Thereafter,
 water access was restricted by removing water bottles from their home
 cages. The mice were habituated to receive water from the port at the
 experimental setup on the following two to three days until they learned
 to drink at least 1 ml at the setup. The body weight was recorded daily,
 to ensure that it stayed above 80% of the original weight. Lick responses
 were measured using an IR beam sensor (PM- F25, Panasonic, Osaka, Japan).
 During calcium imaging sessions, respiration of the mice was recorded
 using a flow sensor (AWM3100V, Honeywell, NC) placed close to the right
 nostril. *Discrimination training* After habituation, the mice were
 trained to associate one odour stimulus with a water reward (S+ odour) and
 another odour stimulus with no reward (S- odour). Both S+ and S- odours
 were a binary mix of ethyl butyrate (Sigma-Aldrich; W242705) and methyl
 butyrate (Tokyo Chemical Industry; B0763), but mixed with different ratios
 based on the photoionization detector readings. Odour presentation was
 targeted to the left nostril since the right nostril was used to record
 the nasal airflow with a flow sensor. For the initial, easy,
 discrimination training, an 80/20 vs 20/80 ratio was used. When mice
 reached 80% behavioural accuracy, difficult discrimination training
 started, using 60/40 vs 40/60 odour mixtures. The correct response to S+
 odours was to lick within a 3-second window after stimulus onset
 (anticipatory licks), while the correct response to S- odours was to
 refrain from licking. The water reward consisted of multiple drops, with a
 total of ~18 μL per trial. Olfactory stimuli were presented using a custom
 flow-dilution olfactometer (doi: Elife *8*. 10.7554/eLife.43558). On each
 trial, odour was presented for 1 second and delay to the reward was 3
 seconds from the odour onset. Inter-trial interval was approximately 20
 seconds. * * *Long odour discrimination* The mice proficient at the
 difficult discrimination task were trained to discriminate between the
 same odour mixtures but with4 seconds of odour duration. The response
 window and reward timing on S+ trials was the same between the two
 paradigms.   *Disengagement paradigm* In this paradigm, the same odour
 mixtures as the difficult discrimination were used, but the water reward
 was delivered every trial, approximately 15 seconds before the odour
 onset. The time window used for measuring the anticipatory licks was
 identical to that of the difficult discrimination paradigm. The first
 session was considered a transition session and excluded from imaging
 analysis.   *Random association paradigm* In this paradigm, the water
 reward was presented in 50% of the trials, regardless of the odour
 identity. This decoupled the odour identity and reward, but kept mice
 engaged, as indicated by the anticipatory licks. The first session was
 considered a transition session and excluded from imaging analysis.  
 *Pharmacological inactivation of anterior piriform cortex* For
 pharmacological inactivation of the anterior piriform cortex, muscimol
 (M1523, Sigma- Aldrich, MO) was infused (2 mM in Ringer; 500 nL at 100
 nL/min) through the previously implanted cannula, using a Hamilton syringe
 (1 mL Model 7001KH PST-3 80100, Hamilton Company, Nevada USA),
 approximately 10 minutes before the start of the imaging sessions. Two
 days prior to the first muscimol infusion, Ringer solution (500 nL at 100
 nl/min) was infused.   *In vivo calcium imaging* All the calcium data
 presented in this manuscript were obtained from awake mice. Two-photon
 fluorescence of GCaMP6f and tdTomato were measured simultaneously with a
 custom-made microscope (INSS, UK) fitted with a 25x objective (Nikon
 N25X-APO-MP1300, 1.1 N.A.) or a 16x objective (Nikon N16XLWD-PF, 0.8 NA),
 and high-power laser (980 nm; Insight DeepSee, MaiTai HP, Spectra-Physics,
 USA) at depths 50–400 μm below the surface of the olfactory bulb. Images
 from a single plane were obtained at ~30 Hz with a resonant scanner. In
 each trial, 400 image frames were acquired, with 100 frames before odour
 stimulus to obtain a baseline. Each day, the stage coordinates were chosen
 relative to a reference location, which was determined by the surface
 blood vessel pattern. Fields of view were 512 μm x 512 μm for apical
 dendrites, 256 μm x 256 μm for tufted and mitral cell somata, and 128 μm x
 128 μm and for adult-born granule cell gemmules. Calcium data during
 difficult discrimination and disengaged experiments were obtained from
 Tbx21-Cre::Ai95D mice (Figs. 1 and 4). 6 male mice were used for somata
 imaging. All 6 were used to image mitral cell somata, while a subset (3
 mice) were used to image tufted cell somata. To obtain calcium data from
 different subcelluar compartments of mitral cells, we used
 Lbhd2-CreERT2::Ai95D mice. For long odour discrimination, random
 association, and muscimol infusion experiments, calcium data was obtained
 from both Tbx21-Cre::Ai95D and Lbhd2-CreERT2::Ai95D mice. Finally,
 Lbhd2-CreERT2::Ai14 mice were used in to record red (tdTomato, mitral
 cells) and green (calcium indicator, gemmules) fluorescent signals during
 adult-born granule cell imaging experiments . The “superficial” and “deep”
 levels were approximately 40-50 mm above and below from the transition in
 the red fluorescence density, respectively.   *Data analysis* All data was
 analyzed offline using custom MATLAB (MathWorks, USA) routines. To
 calculate the behavioural accuracy, the number of licks during a 3 second
 window from final valve opening until reward presentation was counted for
 each trial (anticipatory licks). Correct response to the rewarded odour
 was a minimum of 2 anticipatory licks, and correct response to the
 unrewarded odour was less then 2 anticipatory licks. Behavioural accuracy
 was calculated as the percentage of correct trials from the total number
 of trials.   To calculate the sniffing frequency and speed of inhalation,
 the sniffing signal was first filtered (1 Hz high-pass and 30 Hz low-pass)
 and normalised (z-score). Inhalation peaks were detected using the
 *findpeaks* MATLAB function. Sniff onsets were determined by searching
 back in time from each detected inhalation peak to the point where the
 signal crossed a threshold value. The detected onsets and peaks were then
 used to calculate the frequency (as 1/inter-onset time) and speed of
 inhalation (as onset-to-peak time).   *Image analysis* For each field of
 view, the imaging data was manually curated based on motion artifacts and
 drift over time. Data with motion artifacts and/or drift were motion
 corrected using the NoRMCorre toolbox
 ( [https://doi.org/10.1016/j.jneumeth.2017.07.031)
 ](https://doi.org/10.1016/j.jneumeth.2017.07.031)and, when unsuccessfully
 corrected, excluded from analysis. Regions of interest (ROIs) were
 manually drawn using ImageJ (NIH, Bethesda, USA) based on the average
 field of view from each imaging session and exported for usage in MATLAB.
 Average pixel value from each ROI was offset with a value from the darkest
 region in the frame (e.g. a blood vessel). To account for bleaching over
 the course of the imaging session, the mean pixel values for all trials
 were concatenated and detrended using the MATLAB function *detrend*, then
 reshaped back into an array (individual trials x frames) before relative
 fluorescence change was obtained. For each ROI, the change in fluorescence
 (ΔF/F) was calculated by subtracting the mean pixel value from the
 baseline period (1 second before odour stimulus onset) and dividing by the
 baseline value. Odour evoked responses were calculated as the mean
 fluorescence change during the odour stimulus presentation, and post-odour
 evoked responses as the mean fluorescence change between odour stimulus
 offset and reward presentation. For the ‘long odour’ and ‘random
 association’ experiments, the time windows to calculate the evoked
 responses were based on the difficult odour discrimination experiments.
 All visible ROIs from each field of view are included in the plots unless
 otherwise stated. *Lick-aligned average* Rewarded trials were analysed for
 each imaging session. Onsets of anticipatory licks were defined as the
 average time of the first two licks observed after the start of odour
 presentation. Rewarded trials were grouped into early vs late lick trials
 if the anticipatory lick onsets occurred before or after the median onset
 time, respectively. Within each group, calcium transients were aligned to
 the anticipatory lick onset time for each rewarded trial and averaged.
 Note that the reaction time considers only the timing of lick onsets and
 is distinct from the discrimination time which considers the time at which
 S+ vs. S- responses diverge.   *Normalised S+ vs. S- difference* For each
 trial, the average value of relative fluorescence change was calculated
 for the odour period (first 1 second after the odour onset) and the
 post-odour period (1-3 s after the odour onset). The normalized difference
 between S+ and S- response amplitudes for the odour period, as well as the
 post-odour period, is calculated as follows: ![A mathematical equation
 with black text Description automatically
 generated](file:////Users/izumifukunaga/Library/Group%20Containers/UBF8T346G9.Office/TemporaryItems/msohtmlclip/clip_image002.jpg "A mathematical equation with black text Description automatically generated") where *i* denotes the trial index, *n* is the number of trials, x is the evoked fluorescence change in response to the rewarded odour, and y is the evoked fluorescence change in response to the unrewarded odour.   Figure 1: * HeatplotData_MCTbx (for 2D colormap; aligned to start of odour; Mitral cells)\ \- Trial average ΔF/F values over time for 428 ROIs; frame rate = 30 Hz (see Methods). * HeatplotData_TC (for 2D colormap; aligned to start of odour; Tufted cells)\ \- Trial average ΔF/F values over time for 150 ROIs * MeanEvokedAmp_MCTbx (mean GCaMP6f transients; Mitral cells)\ \- Mean odour and post-odour evoked ΔF/F values for 428 ROIs\ \- Includes vector of significant divergence per ROI * MeanEvokedAmp_TC (mean GCaMP6f transients; Tufted cells)\ \- Mean odour and post-odour evoked ΔF/F values for 150 ROIs\ \- Includes vector of significant divergence per ROI * ScatterData_discrimination_MCtbx (Evoked calcium amplitudes for scatter plots; Mitral cells; odour = during 1 second odour presentation; post = post-odour period)\ \- Mean odour and post-odour evoked ΔF/F values for 428 ROIs\ \- Includes vectors with all values (all), only significant divergent values (sign), and only non-significant values (nonsign) vectors * ScatterData_discrimination_TC (Evoked calcium amplitudes for scatter plots; Tufted cells)\ \- Mean odour and post-odour evoked ΔF/F values for 150 ROIs\ \- Includes vectors with all values (all), only significant divergent values (sign), and only non-significant values (nonsign) vectors Figure 2: * HeatplotData_MCTbx_LickAligned (for 2D colormap; aligned to lick onsets; Mitral cells)\ \- Trial average ΔF/F values over time for 428 ROIs Figure 3: * HeatplotData_MC_LongOdour (for 2D colormap; aligned to odour onset; Mitral cells)\ \- Trial average ΔF/F values over time for 210 ROIs * ScatterData_longOdour (Evoked calcium amplitudes for scatter plots for the long odour experiments; Mitral cells)\ \- Mean evoked ΔF/F values for 210 ROIs during the early (odour) and late (post) phase of stimulus presentation\ \- Includes vectors with all values (all), only significant divergent values (sign; dark datapoints in figure), and only non-significant values (nonsign; light datapoints in figure) vectors Figure 4: * HeatplotData_MC_Disengaged (for 2D colormap; Mitral cells during disengagement paradigm)\ \- Trial average ΔF/F values over time for 147 ROIs * HeatplotData_MC_RandomAssoc (for 2D colormap; Mitral cells during random association paradigm)\ \- Trial average ΔF/F values over time for 301 ROIs * MeanEvokedAmp_disengaged_MC (mean GCaMP6f transients for Mitral cells during disengagement paradigm)\ \- Mean odour and post-odour evoked ΔF/F values for 147 ROIs\ \- Includes vector of significant divergence per ROI * MeanEvokedAmp_randomassoc_MC (mean GCaMP6f transients for Mitral cells during random association paradigm)\ \- Mean odour and post-odour evoked ΔF/F values for 301 ROIs\ \- Includes vector of significant divergence per ROI * ScatterData_disengaged (Evoked calcium amplitudes for scatter plots; disengagement paradigm; Mitral cells)\ \- Mean evoked ΔF/F values for 147 ROIs during the early (odour) and late (post) phase of stimulus presentation\ \- Includes vectors with all values (all), only significant divergent values (sign; dark datapoints in figure), and only non-significant values (nonsign; light datapoints in figure) vectors * ScatterData_randomassoc (Evoked calcium amplitudes for scatter plots; random association paradigm; Mitral cells)\ \- Mean evoked ΔF/F values for 301 ROIs during the early (odour) and late (post) phase of stimulus presentation\ \- Includes vectors with all values (all), only significant divergent values (sign; dark datapoints in figure), and only non-significant values (nonsign; light datapoints in figure) vectors * AnticipatoryLicks (Anticipatory licks for individual sessions of Discrimination, Random Association, and Disengagement paradigms).\ \- Mean number of anticipatory licks (see Methods) per session\ \- Discr: Discrimination paradigm; Diseng: Disengagement paradigm; Rand: Random association paradigm Figure 5: * HeatplotData_ROImatched_MC_Control (for 2D colormap; Mitral cells during control sessions; ROIs matched with corresponding muscimol sessions)\ \- Trial average ΔF/F values over time for 123 ROIs * HeatplotData_ROImatched_MC_Muscimol (for 2D colormap; Mitral cells during muscimol sessions)\ \- Trial average ΔF/F values over time for 123 ROIs * MeanEvokedAmp_ROImatched_MC_Control (Mean GCaMP6f transients; control sessions)\ \- Mean odour and post-odour evoked ΔF/F values for 123 ROIs\ \- Includes vector of significant divergence per ROI * MeanEvokedAmp_ROImatched_MC_Muscimol (Mean GCaMP6f transients; muscimol sessions)\ \- Mean odour and post-odour evoked ΔF/F values for 123 ROIs\ \- Includes vector of significant divergence per ROI * NormalizedDiff_ROImatched_MC_Control (Normalised difference in rewarded vs. unrewarded odours during control sessions; see Methods)\ \- Difference between odour and post-odour evoked ΔF/F values in response to rewarded and unrewarded odours for 123 ROIs * NormalizedDiff_ROImatched_MC_Muscimol (Normalised difference in S+ vs. S- odours during muscimol sessions; see Methods)\ \- Difference between odour and post-odour evoked ΔF/F values in response to rewarded and unrewarded odours for 123 ROIs * BehaviouralAccuracy (Behavioural accuracy during control vs. muscimol sessions).\ \- Mean behavioural accuracy in percentage for all sessions Figure 6 * HeatplotData_ApicalDendriteLBHD2 (for 2D colormap; Mitral cell apical dendrites imaged in the glomerular layer using Lbhd2-CreERT2::Ai95D mice)\ \- Trial average ΔF/F values over time for 140 ROIs * HeatplotData_MC_LBHD2 (for 2D colormap; Mitral cell somata from the Lbhd2-CreERT2::Ai95D mice)\ \- Trial average ΔF/F values over time for 321 ROIs Figure 8: * HeatplotData_abGC_deep (for 2D colormap; deep adult-born granule cell gemmules)\ \- Trial average ΔF/F values over time for 43 ROIs * HeatplotData_abGC_superficial (for 2D colormap; superficial adult-born granule cell gemmules)\ \- Trial average ΔF/F values over time for 28 ROIs * meanEvokedAmp_abGC_deep (mean GCaMP6f transients from deep adult-born granule cell gemmules)\ \- Mean odour and post-odour evoked ΔF/F values for 43 ROIs\ \- Includes vector of significant divergence per ROI * meanEvokedAmp_abGC_superficial (mean GCaMP6f transients from superficial adult-born granule cell gemmules)\ \- Mean odour and post-odour evoked ΔF/F values for 28 ROIs\ \- Includes vector of significant divergence per ROI * NormlizedDiff_abGC_deep (Normalised difference in rewarded vs. unrewarded Evoked calcium amplitudes for deep adult-born granule cell gemmules; see Methods)\ \- Difference between odour and post-odour evoked ΔF/F values in response to rewarded and unrewarded odours * NormlizedDiff_abGC_superficial (Normalised difference in rewarded vs. unrewarded Evoked calcium amplitudes for superficial adult-born granule cell gemmules; see Methods)\ \- Difference between odour and post-odour evoked ΔF/F values in response to rewarded and unrewarded odours Supplementary Figure 1(figureSupp1) * DiscriminationTime (Time in ms at which cumulative rewarded vs. unrewarded lick counts diverge significantly during discrimination paradigm)\ \- Histogram data for discrimination time in ms Supplementary Figure 2(figureSupp2) * SniffingFrequency (Reciprocal of sniff intervals in Hz around the time of odour presentation)\ \- Session average sniffing frequency in Hz. * SniffingFrequencyCumHist (Cumulative histogram of all instantaneous sniff frequency during odour/post-odour period from individual sessions)\ \- Cumulative histogram data for sniffing frequency in Hz * SniffingVelocity (Time to peak in ms from inhalation onset to peak)\ \- Session average sniffing velocity in ms. * SniffingVelocityCumHist (Cumulative histogram of sniffing velocity for individual sessions)\ \- Cumulative histogram data for sniffing velocity in ms Supplementary Figure 3 (figureSupp3) * SignificantDivergence_perFOV_MC (Frequencies of observing significant excitatory and inhibitory responses for Mitral cells)\ \- Percentage of significantly divergent ROIs per field of view (FOV) during odour and post-odour periods; 17 FOVs * SignificantDivergence_perFOV_TC (Frequencies of observing significant excitatory and inhibitory responses for Tufted cells)\ \- Percentage of significantly divergent ROIs per field of view (FOV) during odour and post-odour periods; 6 FOVs Supplementary Figure 5 (figureSupp5) * NormalizedDiff_longOdour (Normalised difference in rewarded vs. unrewarded responses during discrimination paradigm with long odour; see Methods)\ \- Cumulative histogram data for normalized difference data in ΔF/F * NormalizedDiff_shortOdour (Normalised difference in S+ vs. S- responses during standard discrimination paradigm; see Methods)\ \- Cumulative histogram data for normalized difference data in ΔF/F Supplementary Figure 6 (figureSupp6) * ScatterData_easyDiscrimination (Evoked calcium amplitudes for scatter plots; easy discrimination paradigm)\ \- Mean odour and post-odour evoked ΔF/F values for 141 ROIs\ \- Includes vectors with all values (all), only significant divergent values (sign; dark datapoints in figure), and only non-significant values (nonsign; light datapoints in figure) vectors Supplementary Figure 7 (figureSupp7) * ScatterData_MCdendrites_Discrimination (Evoked calcium amplitudes for scatter plots for individual apical dendrite ROIs during standard discrimination paradigm)\ \- Mean odour and post-odour evoked ΔF/F values for 48 ROIs\ \- Includes vectors with all values (all), only significant divergent values (sign; dark datapoints in figure), and only non-significant values (nonsign; light datapoints in figure) vectors