A distinct entorhinal cortex to hippocampal CA1 direct circuit for olfactory associative learning

Journal name:
Nature Neuroscience
Volume:
20,
Pages:
559–570
Year published:
DOI:
doi:10.1038/nn.4517
Received
Accepted
Published online

Abstract

Lateral and medial parts of entorhinal cortex (EC) convey nonspatial 'what' and spatial 'where' information, respectively, into hippocampal CA1, via both the indirect EC layer 2right arrow hippocampal dentate gyrusright arrowCA3right arrowCA1 and the direct EC layer 3right arrowCA1 paths. However, it remains elusive how the direct path transfers distinct information and contributes to hippocampal learning functions. Here we report that lateral EC projection neurons selectively form direct excitatory synapses onto a subpopulation of morphologically complex, calbindin-expressing pyramidal cells (PCs) in the dorsal CA1 (dCA1), while medial EC neurons uniformly innervate all dCA1 PCs. Optogenetically inactivating the distinct lateral EC–dCA1 connections or the postsynaptic dCA1 calbindin-expressing PC activity slows olfactory associative learning. Moreover, optetrode recordings reveal that dCA1 calbindin-expressing PCs develop more selective spiking responses to odor cues during learning. Thus, our results identify a direct lateral ECright arrowdCA1 circuit that is required for olfactory associative learning.

At a glance

Figures

  1. Morphological and molecular characterization of two distinct types of dCA1 PCs.
    Figure 1: Morphological and molecular characterization of two distinct types of dCA1 PCs.

    (a) Representative neuronal morphologies reconstructed from sPCs and cPCs, labeled with values of the LRI and ORI for individual branch-points along apical dendrites. The size and color of circles depict the magnitude of LRI and ORI values. (b) K-means cluster analysis for cataloging sPCs (blue) and cPCs (red) according to their LRImax and ORImax. (c) Sholl analysis of dendritic complexity (60-μm step). Ranges with significant differences between the two subtypes are labeled (P < 0.01, unpaired t-test; exact P-values in the Supplementary Methods Checklist). Error bars show s.e.m. (d) Somata distribution of sPCs and cPCs in the superficial, middle and deep divisions of CA1 stratum pyramidale (SP). SO, stratum oriens. (e) Calb immunostaining in morphologically identified sPCs and cPCs. Arrowhead, principal branch-point. Scale bars, 100 μm (left) and 10 μm (right). (f) Percentages of Calb-immunostained cells in morphologically identified sPCs and cPCs (P = 2.1 × 10−10, Pearson's chi-squared test). (g) Detection of Calb transcripts in morphologically identified sPC and cPC by single-cell RT-PCR. (h) Percentages of Calb-transcription cells among sPCs and cPCs (P = 3.9 × 10−5, Fisher's exact test).

  2. Heterogeneous targeting of direct excitatory inputs from LEC and MEC to dCA1 PCs.
    Figure 2: Heterogeneous targeting of direct excitatory inputs from LEC and MEC to dCA1 PCs.

    (a,b) Histological verification of ChR2(-mCherry) expression (a) restricted within the LEC (left, coronal section) or the MEC (right, horizontal section) and (b) their projection axons in the dorsal hippocampus. Scale bar, 1 mm in a and 100 μm in b. (c) Diagram of CRACM experiments with the 10 × 10 photo-stimulation grids (with 40-μm interspot distances), covering the entire CA1 SLM and DG MO layers, centered on the meeting point of the layer border line and the cell soma. Magnification as in b. (d) Averaged EPSCCRACM traces and their dendritic maps for the LEC TA inputs (activated by 0.5-mW, 473-nm laser) overlaid on the reconstructed morphology of a recorded sPC (left) and cPC (right). Scale bar, 40 μm. (e) Percentages of morphologically identified sPCs and cPCs that were responsive (with EPSCCRACM, black) or unresponsive (white) to laser-grid activation of LEC TA axons at maximum laser intensity (5 mW). CA2 PCs were examined simultaneously in slices from each mouse (P = 9.3 × 10−15, 2.2 × 10−16 and 1.0 × 10−4 for sPC vs. cPC, sPC vs. CA2 and cPC vs. CA2, respectively; Pearson's chi-squared test). (f) Comparison of EPSCCRACM amplitudes from CA1 sPCs, cPCs and CA2 PCs (measured at 2-mW laser intensity). Lines indicate data from the same mouse (P = 0.031 for all two-cell comparisons, Wilcoxon signed-ranked test). Error bars show s.e.m. (gi) As in df but for CRACM assays of MEC TA inputs. Laser power was 0.1 and 2 mW for g and i, respectively. P = 0.031 for CA2 vs. sPC or cPC, Wilcoxon signed-ranked test. Scale bar, 40 μm.

  3. Trans-synaptic virus tracing and EM assay of direct EC-dCA1 PC synapses.
    Figure 3: Trans-synaptic virus tracing and EM assay of direct EC–dCA1 PC synapses.

    (a) Genome structure of HSV-1-G3 recombinant virus: 152 kb with unique long (UL) and unique short (US) segments. HSV-1-G3 contains a GFP driven by an SV40 promoter within the BAC sequence and two GFPs driven by CMV promoter cassettes inserted into the UL and US segments, respectively. (b) Left: Section images of 5.5 and 5 d after HSV-1-G3 was injected in the LEC (top) and MEC (bottom) showing that the anterograde trans-synaptic transport of HSV-1-G3 first infected the ipsilateral hippocampal DG, CA1 and CA2 regions. Scale bar, 1 mm. Right: higher-resolution z-stacked images of the boxed regions in the left panels, showing that HSV-1-G3 injected in the LEC (top) preferentially label the downstream CA1 cPC (arrowheads, principal branch-points of typical bifurcating apical dendrites), while the MEC injection (bottom) trans-synaptically infected most CA1 PCs in the SP layer. Scale bar, 100 μm. (c) Similar anterograde trans-synaptic viral tracing labeled dCA1 cells (GFP, green), 5.5 or 5 d after the HSV-1-G3 injection in the LEC (top) or MEC (bottom) of Calb2-IRES-Cre::Ai9 mice. Scale bar, 100 μm. (d) Colocalization percentage of Calb+tdTomato+ cells labeled by HSV-1-G3 in the dCA1 SP layer across the anterior–posterior axis following the HSV-1-G3 injection in LEC or MEC (n = 6 and 7 for LEC and MEC injections, respectively, P = 5.1 × 10−12; Mann-Whitney U-test). Error bars show s.e.m. (e,f) Electron micrographs showing the double immunogold-labeled direct (e) LEC–cPC or (f) MEC–cPC asymmetric synapses. Presynaptic axonal terminals and postsynaptic dendrites are identified by mCherry immunogold silver-labeled particles (~25 nm) and Calb immunogold silver-labeled particles (~60 nm), respectively. Scale bars, 0.1 μm.

  4. Homogenous targeting of EC direct TA inputs onto most dCA1 inhibitory INs.
    Figure 4: Homogenous targeting of EC direct TA inputs onto most dCA1 inhibitory INs.

    (a) CRACM maps of LEC or MEC TA inputs (stimulated at 1-mW laser power) overlaid on reconstructed morphologies of recorded Pavlb+ axo-axonic cells and basket cells, as well as bistratified Pvalb+Sst+ cells. Black, dendrites; red, axons; scale, 40 μm. (b) Percentages of tested dCA1 INs that were innervated by LEC and MEC axonal projections, measured from the CRACM experiments (under maximum laser intensity; P = 1, 0.57 and 0.81 for comparisons between LEC and MEC innervations on the axo-axonic, basket and bistratified cells, respectively; P = 0.23 and 0.50 for comparisons between different cell types within LEC and MEC, respectively; Fisher's exact test). (c) EPSPCRACM amplitudes (at 2-mW laser) of the direct LEC or MEC synapses on INs. P-values were calculated by Mann-Whitney U-test. Error bars show s.e.m.

  5. Selective optogenetic inactivation of direct LEC TA axons or of dCA1 Calb+ cPCs slows olfactory associative learning.
    Figure 5: Selective optogenetic inactivation of direct LEC TA axons or of dCA1 Calb+ cPCs slows olfactory associative learning.

    (a,b) Diagram of behavioral setup (a) and model (b) of the olfactory cue-based associative Go–No-go task. Odor A, ethyl acetate; odor B, methyl butyrate. (ce) Optogenetic inactivation of the (c) LEC TA–dCA1 path, but not the (d) LEC PP–DG path or the (e) MEC TA–dCA1 path, impaired olfactory associative learning in mice injected with AAV-CaMKIIα-NpHR-EYFP in the LEC or MEC, compared to control mice injected with AAV-CaMKIIα-EYFP. Yellow lines indicate the 589-nm laser illumination (at 10 mW) over trial blocks. Scale bar, 1 mm. Statistical significance was tested by two-way mixed-design ANOVA for the first 2 d and Mann-Whitney U-test for the third day (P = 9.7 × 10−8, 0.44 and 0.98 for the first 2 d; P = 1.68 × 10−4, 0.43 and 0.88 on the third day for LEC–dCA1 (n = 10), LEC–DG (n = 9 or 10) and MEC–dCA1 (n = 10), respectively. (f) Optogenetically suppressing activities of postsynaptic dCA1 Calb+ cPCs impaired associative learning in Calb2-IRES-Cre::Ai35 mice (P = 0.005 for the first 2 d, two-way mixed-design ANOVA; P = 0.04 for the third day, Mann-Whitney U-test; n = 10). (g) Similar inactivation of postsynaptic dCA1 cPCs did not affecting behavioral performance of the well-trained mice on days 4 and 5 (without laser stimuli on the third day). P = 0.011 for the first 2 d, two-way mixed-design ANOVA; P = 0.92 and 0.78, Mann-Whitney U-test for the third day and fourth day, respectively, for comparison between Calb2-IRES-Cre::Ai35 (n = 9) and Ai35 (n = 8) mice; P = 0.48 and 0.36 for the laser ON versus OFF in the two types of mice, respectively, on the fifth day; Mann-Whitney U-test. (h) Optogenetically suppressing activities of Calb+ granule cells in the DG does not affect learning (P = 0.16 and 0.62 for the first 2 d and the third day; n = 10 or 11).

  6. Calb+ cPCs develop more selective odor representations during learning.
    Figure 6: Calb+ cPCs develop more selective odor representations during learning.

    (a) Behavioral performance of Calb2-IRES-Cre::Ai35 mice implanted with optetrodes in dCA1 (insert: scale bar, 1 mm). (b) Top: optogenetically identified units from dCA1 Calb+ PC (red) and Calb PC (blue) in vivo, based on 589-nm, 10-mW laser-induced suppression of baseline spiking activity (raster and peristimulus time histogram (PSTH) plots). Bottom: spike raster, rate plots and corresponding OSI during odor sampling in Go and No-go trials, respectively. Error bars represent s.e.m. (c) OSIs of all recorded dCA1 PCs on individual learning days. Each row represents a single unit, sorted by values of the 1.5-s OSI. Red and blue bars in the right column label Calb+ and Calb units, respectively. (d) PV analysis for odor-specific representations of dCA1 Calb+ and Calb units over 3 d of learning. Black dashed lines, 95% chance level (*P < 0.05 for comparison between original cPC PDI vs. 95% confidence interval of bootstrapped sPC PDIs or vice versa). (e) Histogram distribution of the 1.5-s OSI (top) and cumulative percentage of absolute 1.5-s OSI (bottom) of 46 dCA1 Calb+ and 232 Calb units (P = 0.004 and 4.2 × 10−7, two-sample Kolmogorov-Smirnov test and Mann-Whitney U-test, respectively). (f) Correlation between the behavioral performance and the averaged absolute mean OSI of Calb+ (red) and Calb PCs (blue) between different mice over 3 d of learning. Each dot represents mean values from a mouse on a given day. Lines, linear regression; P = 0.01 and 0.02 for 21 Calb+ PCs and 30 Calb PCs, respectively, measured from 10 mice over 3 d.

  7. Similar place field properties of the dCA1 cPCs and sPCs.
    Figure 7: Similar place field properties of the dCA1 cPCs and sPCs.

    (a) Diagram of the experimental configuration for characterizing place cells using a U-maze with water ports in either end, with a top camera recording the animal's trajectory. (b) Yellow 589-nm laser illumination (2-min duration) to identify Calb+Arch+ cells in vivo, with repeated interleaved illumination ON or OFF (yellow bars indicate ON). Each row of raster spike events represents a single unit from an optetrode recorded Calb2-IRES-Cre::Ai35 mouse. Red blocks indicate Calb+ units. PSTHs: representative Calb (unit 27) and Calb+ (unit 19) dCA1 PCs. (c) Examples of place cells in dCA1 Calb+ and Calb PCs, respectively. Top: place fields of recorded PCs in the U-maze (clockwise and counterclockwise); middle: animal speed and dots representing spikes of the example PC; bottom: animal trajectory and the firing rate of the example PC (yellow bars indicate laser-ON durations). Scale bars, 10 cm. (d) Percentages of place cells in the identified 83 Calb+ cPCs and 235 Calb sPCs in the dCA1. P = 0.33, Pearson's chi-squared test. (e) Comparison of spatial information encoded by place cells in the Calb+ cPC and Calb sPC groups. P = 0.28, Mann-Whitney U-test.

  8. Analyses of dCA1 PC clusters by the Jump method, and principle component analysis (PCA) on different morphological parameters.
    Supplementary Fig. 1: Analyses of dCA1 PC clusters by the Jump method, and principle component analysis (PCA) on different morphological parameters.

    (ac) The jump method (see Methods for details) was used to generate the profiles of (a) distortion, (b) transformed distortion, and (c) jump as a function of the number of cell clusters based on the calculated LRImax and ORImax of apical dendrites from each reconstructed dCA1 PCs. (d,e) 3D scatter plots of dendritic branch points, total length, and branch order of the (d) apical and (e) basal dendrites of all reconstructed dCA1 PCs, respectively. Each red and blue solid dot represents the morphological data measured from individual cPC and sPC, respectively. (fh) Principle component analysis of (f) LRImax and ORImax, (g) other 6 morphological parameters including the total length, total branch points and branch order of the apical and basal dendrite, respectively, and (h) all the above 8 parameters, respectively, showing the resultant first two principal components. Two groups (black and orange) in each panel are determined by the k-means cluster analysis.

  9. Characterization of intrinsic action potential firing patterns and excitability of dCA1 sPCs and cPCs.
    Supplementary Fig. 2: Characterization of intrinsic action potential firing patterns and excitability of dCA1 sPCs and cPCs.

    (a1,b 1) Representative traces showing action potential firing evoked by a train of ten somatic EPSC-like (tau rise = 0.2 ms, tau decay = 6 ms) currents (600−2000 pA) of PCs in the (a1) subiculum and (b1) dCA1. The current trains were injected at 5 Hz to the cells. Asterisks denote the burst spiking. (a2,b 2) Summarized results of experiments shown in a1 and b1, indicating (a2) the subiculum regular spiking (black) PCs and bursting PCs (magenta), respectively, (b2) but only regular spiking pattern for both cPCs (red) and sPCs (blue) in the dCA1. The classification of the regular-spiking and bursting PCs are based on the previous study by Graves et al28. Data were presented as mean±s.e.m. (c1) Example trace of dCA1 sPC (blue) and cPC (red) action potentials evoked by step current injection. (c2) Summarized results of firing rate (upper), inter-spike-interval (left bottom) and ratios of the first ISI and the last ISI (testing spike modulation, right bottom) under increasing step currents. Data were presented as mean±s.e.m. P = 0.11, measured by Mann-Whitney U-test.

  10. Differential propagation of dendritic excitation to the soma in sPCs and cPCs revealed by TREES and NEURON simulations.
    Supplementary Fig. 3: Differential propagation of dendritic excitation to the soma in sPCs and cPCs revealed by TREES and NEURON simulations.

    Simulation was performed under either (ac) MATLAB TREES toolbox or the (dg) NEURON stimulation. (a) A pair of example sPC and cPC from experimental reconstruction showing dendritic sites of current injection (blue and red dots) and the recording at soma. (b) Example of somatic voltage change (ΔV, upper) elicited by injected electronic currents at ~450 μm apical dendrite (bottom) in the sPC (blue) and cPC (red), respectively. (c) Cumulative plot of mean ΔV of 171 sPCs (blue) and 183 cPCs (red). Each mean ΔV for a given PC was averaged from all trials with the injection sites distributed on all distal dendritic sections (> 300 μm). Statistic difference was measured by two-sample Kolmpgorov-Smirnov test, P < 0.001. (d) Morphologies of sPC and cPC NERUON models selected from the ModelDB65,66 depicting distal synaptic inputs, dendritic and somatic recordings. (e,f) Same as bc except using NEURON models. (g) Comparison of mean somatic and dendritic excitation between sPC and cPC. Data were presented as mean±s.e.m. Statistic difference was measured by unpaired t-test, P < 0.001 and P = 0.76 for the somatic and local dendritic injections, respectively.

  11. Histological characterization of viral expression of ChR2 in the LEC, MEC and their axon projections along the anterior-posterior axis of CA1.
    Supplementary Fig. 4: Histological characterization of viral expression of ChR2 in the LEC, MEC and their axon projections along the anterior–posterior axis of CA1.

    (a1,a2) Serial sections of LEC (coronal) and MEC (horizontal), demonstrating the ChR2(-mCherry) expression in LEC and MEC (outlined by dashed lines), respectively. Scale bar, 1 mm. (b1) Representative histology verification of ChR2-expressing axons from the LEC (top) and MEC (bottom) in the hippocampus from anterior (A) to posterior (P) axis, respectively. Scale bar, 1 mm. Note that the ChR2(-mCherry) expressing LEC axons appear an increasing gradient in the CA1 stratum lacunosum-moleculare (SLM) layer along the A−P axis, while that of MEC axons show the opposite pattern. The sections in the dashed line box show the intermingled ChR2-expressing axons from the LEC and MEC projection neurons. (b2) Higher resolution images showing clearer segregation in the DG MO layer and distal−proximal gradients in the CA1 SLM layer of LEC and MEC axons in the boxed regions in b1, Scale bar, 200 μm.

  12. Characterization of optogenetic activation of EC-dCA1 monosynaptic transmission by AOD-based laser stimulation system.
    Supplementary Fig. 5: Characterization of optogenetic activation of EC–dCA1 monosynaptic transmission by AOD-based laser stimulation system.

    (a) Representative traces of EPSCCRACM recorded from the CA1 PC (blue) and the CA2 GABAergic interneuron (red), which, respectively, show consistent mono-synaptic responses and variable poly-synaptic responses, evoked by 473-nm laser pulses with increasing intensities in the CRACM experiment. (b,c) Effects of applying GABAARs blocker picrotoxin (50 μM) on (b) the responsive map, (c) EPSCCRACM amplitudes and the response latency in the dCA1 PC. P = 0.29 and P = 0.004 for the amplitude and latency, respectively, calculated by the paired t-test. (d,e) Effects of application of TTX (0.5 μM) and 4-AP (100 μM) on (d) the discrete dendritic EPSCCRACM map, EPSCCRACM latency (bottom: averaged EPSCCRACM traces from the indicated locations in the map), and (e) the mean maximal amplitude of EPSCCRACM. Data were presented as mean±s.e.m. P = 0.01, measured by paired t-test. (f,g) Effects of HCN channel blocker ZD7288 (20 μM), slimilar as d,e. Note a slight increase of mean amplitude of EPSCCRACM after the ZD compound application. Data were presented as mean±s.e.m. P = 0.16, calculated by the paired t-test. (h) Blocking the GABAARs with picrotoxin (50 μM) does not affect the differential innervations of direct excitatory TA inputs from LEC to dCA1, compared to the data in Fig. 2e. P = 3.8 × 10-8, measured by Person’s Chi-square test. (i) Preserved differential innervations of LEC and MEC direct inputs to sPC and cPC in the distal, medial and proximal part of dCA1.

  13. Direct long-range GABAergic transmission from LEC and MEC to dCA1 pyramidal neurons.
    Supplementary Fig. 6: Direct long-range GABAergic transmission from LEC and MEC to dCA1 pyramidal neurons.

    (a) Example IPSCCRACM and responsive map of direct GABAergic inputs from the MEC to a dCA1 PC, measured under 0 mV membrane potential and presence of CNQX (10 μM) and AP5 (50 μM). (b) Percentage of cell receiving direct GABAergic projections from LEC or MEC in the dCA1 sPCs and cPCs as well as dCA2 PCs. n, number of tested cells.

  14. Comparison of transmission strength of CA3 SC excitatory synapses on dCA1 sPCs and cPCs.
    Supplementary Fig. 7: Comparison of transmission strength of CA3 SC excitatory synapses on dCA1 sPCs and cPCs.

    (a) Diagram of recording configuration, in which the neighboring sPC and cPC were simultaneously recorded and a filed stimulation electrode (concentric tungsten electrode) was placed in a distance of ~350 μm away in the middle of SR layer. (b) Example EPSC traces recorded from sPC and cPC under increasing stimulus intensities. (c) Summarized EPSC amplitude ratios of the cPC and sPC from the assay shown in a and b. Data were presented as mean±s.e.m.

  15. Characterization of Calb2-IRES-Cre::Ai9 and Calb1-2A-dgCre::Ai9 mice.
    Supplementary Fig. 8: Characterization of Calb2-IRES-Cre::Ai9 and Calb1-2A-dgCre::Ai9 mice.

    (a,b) Example images showing the genetically-tdTomato labeled cells (red) immunostained with monoclonal antibody against calbindin-D28k (green) in the dCA1 of (a) Calb2-IR ES-Cre::Ai9 and (b) Calb1-2A-dgCre::Ai9 (7-d TMP induction, see Methods) mice. Scale bar, 100 μm. (c,d) Characterization of hippocampal labeling from anterior (A) to posterior (P) in (c) Calb2-IR ES-Cre::Ai9 and (d) Calb1-2A-dgCre::Ai9 (7-d induction) mice. Scale bar, 1 mm. All following statistic results are measured in the sections from Bregma -2.0 to -3.5 mm with 0.3 mm step along the A−P axis. (e,f) Comparison of the labeling efficiency (yellow/green) and colocalization rate (yellow/red) in the (e) CA1 SP and (f) DG between the Calb2-IRES-Cre ::Ai9 and Calb1-2A-dgCre ::Ai9 (with 1 or 7 d induction) mice, respectively. (g,h) Fluorescence images showing the (g) neuronal GABA staining in the dCA1 of Calb2-IRES-Cre ::Ai9, and the (h) tdTomato labeling rates in GABAergic cells in different layers of dCA1. Note the co-localization rates are < 10%. Scale bar, 100 μm. (i,j) Similar as g,h except for Cre antibody staining. Note that Calb2-IRES-Cre ::Ai9 mice exhibit high efficiency (> 90%) in labeling neurons in the dCA1 SP layer, but low efficiency (< 15%) in labeling neurons in other layers. Scale bar, 100 μm. Data were presented as mean±s.e.m.

  16. HSV-1-G3 anterograde transportation from dorsal or ventral LEC and MEC to hippocampal CA1.
    Supplementary Fig. 9: HSV-1-G3 anterograde transportation from dorsal or ventral LEC and MEC to hippocampal CA1.

    (a,b) Co-localization of anterograde transportation of HSV-1-G3 (green) from (a) anterior ventral or posterior dorsal LEC; (b) ventral or dorsal MEC to hippocampal CA1 in Calb2-IRES-Cre::Ai9 transgenic mice (tdTomato). Scale bar, 100 μm. (c) Summarized co-localized rates following the HSV-1-G3 injected to different parts of MEC or LEC, respectively. Note that no significant difference was observed between the dorsal and ventral parts within the LEC or MEC. Data were presented as mean±s.e.m. (d,e) Characterization of HSV injected in the (d) anterior ventral or posterior dorsal LEC and (e) ventral or dorsal MEC in Calb2-IRES-Cre::Ai9 transgenic mice. Scales bar, 1 mm. (f) Serial brain sections showing the spreading infection area of HSV-1-G3 3 days after the injection in the anterior-ventral LEC. Scale bar, 1 mm.

  17. Postbehavior histological characterization of NpHR-EYFP expression in LEC or MEC and the locations of implanted optical fibers or optetrodes in dCA1.
    Supplementary Fig. 10: Postbehavior histological characterization of NpHR-EYFP expression in LEC or MEC and the locations of implanted optical fibers or optetrodes in dCA1.

    (a) Post-histology of the mice with AAV-CaMKIIα-NpHR-EYFP or AAV-CaMKIIα-EYFP injected in the bilateral LEC and subsequently behaviorally tested shown in Fig. 5c. The first column: atlas of different coronal sections; 2nd column: example fluorescence images of NpHR-EYFP expression in the bilateral LEC and optical fiber implanted in the dCA1 (scale bar, 1 mm); 3rd column: overlaid image showing the region of NpHR-EYFP expression of all tested mice; 4th column, overlaid image showing the region of EYFP expression of all tested control mice. Grey gradients: viral expression levels in all tested mice, red gradients: optical fiber tracks. (b) Similar as a except for the optical fiber implantation was made in the DG in mice, whose results are shown in Fig. 5d. (c) Similar as a except for the virus was injected in the MEC, whose results are shown in Fig. 5e. (d) Similar as a except for the mice whose results are shown in Fig. 5f−h, respectively. (e) Fluorescence images of hippocampal sections show the expression of Arch-GFP (green) and the optetrode recording sites (arrows) in the dCA1. Uppers 4 mice were recorded in the right hemisphere, while the bottoms 6 were done in the left hemisphere. Scale bar, 1 mm.

  18. Similar impairments of reversed learning by inactivation of LEC-dCA1 transmissions or of postsynaptic dCA1 Calb+ cPCs.
    Supplementary Fig. 11: Similar impairments of reversed learning by inactivation of LEC–dCA1 transmissions or of postsynaptic dCA1 Calb+ cPCs.

    (a13) The inactivation of (a1) LEC−dCA1, but not (a2) LEC−DG or (a3) MEC−dCA1 transmissions impairs the reverse learning, in which the odor−licking/no n -licking contingencies were reversed, in the days 4−6 with the same mice which underwent the initial learning shown in Fig. 5c−e, respectively. Data were presented as mean±s.e.m. (a45) Suppressing Calb+ PCs activities in the (a4) dCA1, but not in the (a5) DG impairs the reversal learning in the days 4−6 in the same mice which underwent the initial learning shown in Fig. 5f and g, respectively. Data were presented as mean±s.e.m. (b15) Detailed changes of the hit rate (upper) and the correct rejection rate (bottom) for behavioral performance data shown in the Fig. 5c−g and Supplementary Fig. 12a1−5 during the initial and reversal learning, respectively. Statistic differences were presented in the Supplementary Table 4−15. Data were presented as mean±s.e.m.

  19. Optogenetic tagging of dCA1 Calb+ cPC units with Arch in vivo in comparison with that of tagging with ChR2.
    Supplementary Fig. 12: Optogenetic tagging of dCA1 Calb+ cPC units with Arch in vivo in comparison with that of tagging with ChR2.

    (a) Comparisons of waveforms of the spontaneous spikes (top traces) and the optical ChR2-excitation induced spikes (by 5 ms blue laser pulses, 0.5 mW, bottom) recorded from dCA1 PV+IN and PCs with the optetrode in the Pavlb-Cre::Ai27 and Calb2-IRES-Cre::Ai27 mice, respectively. Note that the optical ChR2-excitation significantly alters the waveform of PCs’ spikes, but not that of PV+INs, indicated by cross correlation (cc) values (r). (b,c) Example recording of the LFPs and spike units from (b) PCs and (c) PV+INs with 5 ms blue-laser pulse stimulation (0.5 mW) in the dCA1 area of the Calb2-IRES-Cre::Ai27 and Pavlb-Cre::Ai27 mice, respectively. Waveforms: averaged spontaneous spikes (left), individual ChR2-induced spikes (blue) or spontaneous spikes (black, corresponding to the *-labeled spikes in the middle). r: cc between the averaged spike and the individual spike. (d) Similar to bc, but showing the example of two tagged dCA1 Calb+ PCs with Arch in Calb2-IRES-Cre::Ai35 mice. Note that the yellow-laser illumination (2 min, 10 mW, indicated by yellow bars) significant suppresses the rate of spontaneous spikes, but exhibiting identical waveforms recorded during the ON and OFF of laser stimulation. (e) Example of simultaneous recordings from the Calb+Arch+ cPC (red) and CalbArch sPC (blue) in hippocampal slices of the Calb2-IRES-Cre::Ai35 mouse, showing differential effects of 2-min yellow-light illumination (10 mW, indicated by the yellow bar) on evoked rhythmic spikes (by intracellular injection of 5 ms current pulses at 0.5 Hz, black traces) between these two subtypes of dCA1 PCs. (f) Statistic results of the changes of spike rate and resting membrane potential during the ON and OFF of the yellow-light illumination in the Calb+Arch+ cPC (red) and CalbArch sPC (blue) in the experiments shown in d. P = 0.008, measured by the Wilcoxon signed-ranked test. (g) Example recordings and (h) statistical results showing changes of the frequency and amplitude of spontaneous IPSCs (Vclamp = 0 mV) during the ON and OFF of 2-min laser illumination in the dCA1 Calb+Arch+ cPC (red colors) and CalbArch sPC (blue colors) in the slices of Calb2-IRES-Cre::Ai35 mice.

  20. Varied spiking responses and selectivities in response to odors A and B during odor sampling in trials.
    Supplementary Fig. 13: Varied spiking responses and selectivities in response to odors A and B during odor sampling in trials.

    (a) Raster plots of spikes (upper two panels) and peri-stimuli time plots of firing rate changes (in 100 ms bins) for single-units recorded in the Go (red, odor A) and No-go (blue, odor B) trials in different mice (see the unit identity). The odor delivery duration is indicated by the green bar. The numbers for individual types are presented in the Supplementary Table 16.

  21. Comparison of neuronal spiking activities in Correct and Error trials in the behavioral task.
    Supplementary Fig. 14: Comparison of neuronal spiking activities in Correct and Error trials in the behavioral task.

    (a) Comparison of the mean firing rates of all PC units (left), sPC (middle), or cPC (right) in the Correct and Error trials in Go (upper) and No-go trials (bottom), respectively. The upper dots indicate the significant differences between Correct and Error trials (Paired t-test, P < 0.05). Data were presented as mean±s.e.m. (b) D’-prime analysis of differential firing of individual units in Correct and Error trials in Go (left) and No-go trials (right), respectively. Red and blue columns in the upper right indicate the cPC and sPC, respectively and sorted by the mean d’-prime of 1.5-s after odor delivery. The lower panels are the mean population d’-prime of sPC (blue) and cPC (red) during Correct and Error trials in Go (left) and No-go trials (right), respectively. Black dashed line, 95% chance level. All units were recorded in the first learning day.

  22. Comparison of spontaneous firing rates during different brain states between the identified Calb+ and Calb- dCA1 PCs.
    Supplementary Fig. 15: Comparison of spontaneous firing rates during different brain states between the identified Calb+ and Calb dCA1 PCs.

    (a) Examples of neuronal spikes of units and local field potentials in the running, rapid-eye movement (REM) sleep and slow-wave sleep (SWS) states, respectively. Asterisks label the units of light-responsive Calb+ cells. Scale bar, 1 sec (middle panel) or 100 ms (enlarged, bottom panel), 0.5 mV. (b) Distributions of Calb+ (red) and Calb (blue) dCA1 PCs showing different spontaneous firing rates during the RUN, REM, SWS, respectively. P = 0.0127, 0.0282 and 0.0025 for RUN, SWS and REM state, respectively, calculated by the Mann-Whitney U-test. Note that this result is consistent with a previous study by Mizuseki et al29.

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Author information

  1. These authors contributed equally to this work.

    • Yiding Li &
    • Jiamin Xu

Affiliations

  1. Institute of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.

    • Yiding Li,
    • Jia Zhu &
    • Chengyu Li
  2. State Key Laboratory of Cognitive Neuroscience & Learning and IDG/McGovern Institute for Brain Research, Beijing Normal University, Beijing, China.

    • Yiding Li,
    • Nan Liu,
    • Malte J Rasch,
    • Xiang Gu,
    • Xiang Li &
    • Xiaohui Zhang
  3. University of Chinese Academy of Sciences, Shanghai, China.

    • Yiding Li &
    • Jia Zhu
  4. Key Laboratory of Brain Functional Genomics-Ministry of Education, School of Life Science, East China Normal University, Shanghai, China.

    • Jiamin Xu &
    • Longnian Lin
  5. Britton Chance Center for Biomedical Photonics and Department of Biomedical Engineering, Wuhan National Laboratory for Optoelectronics–Huazhong University of Science and Technology, Wuhan, China.

    • Yafeng Liu &
    • Shaoqun Zeng
  6. State Key Laboratory of Virology, CAS Center for Excellence in Brain Science and Intelligence Technology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.

    • Wenbo Zeng,
    • Haifei Jiang &
    • Minhua Luo
  7. Key Laboratory of Cell Proliferation and Differentiation of the Ministry of Education and State Key Laboratory of Membrane Biology, College of Life Sciences, Peking University, Beijing, China.

    • Ning Huang,
    • Junlin Teng &
    • Jianguo Chen

Contributions

X.Z. conceived and supervised the project, and X.Z. and Y. Li designed the experiments. Y. Li performed the virus injection, in vitro electrophysiology, immunostaining, single-cell RT-PCR, cellular imaging, morphological reconstruction and behavioral tests. J.X. performed the in vivo optetrode recording, spike sorting and place field analysis. N.L. and M.J.R. performed the TREE analysis, cluster analysis and computational simulation. W.Z., H.J. and M.L. designed and prepared the HSV virus. N.H., J.T. and J.C. designed and conducted the EM experiment. Y. Liu and S.Z. designed and built the AOD-based rapid laser stimulation system. J.Z. and C.L. participated in the behavioral experiment. X.G. and X.L. prepared and genotyped all transgenic mice. Y. Li, L.L. and X.Z. analyzed the data. X.Z. and Y. Li wrote the manuscript.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: Analyses of dCA1 PC clusters by the Jump method, and principle component analysis (PCA) on different morphological parameters. (259 KB)

    (ac) The jump method (see Methods for details) was used to generate the profiles of (a) distortion, (b) transformed distortion, and (c) jump as a function of the number of cell clusters based on the calculated LRImax and ORImax of apical dendrites from each reconstructed dCA1 PCs. (d,e) 3D scatter plots of dendritic branch points, total length, and branch order of the (d) apical and (e) basal dendrites of all reconstructed dCA1 PCs, respectively. Each red and blue solid dot represents the morphological data measured from individual cPC and sPC, respectively. (fh) Principle component analysis of (f) LRImax and ORImax, (g) other 6 morphological parameters including the total length, total branch points and branch order of the apical and basal dendrite, respectively, and (h) all the above 8 parameters, respectively, showing the resultant first two principal components. Two groups (black and orange) in each panel are determined by the k-means cluster analysis.

  2. Supplementary Figure 2: Characterization of intrinsic action potential firing patterns and excitability of dCA1 sPCs and cPCs. (202 KB)

    (a1,b 1) Representative traces showing action potential firing evoked by a train of ten somatic EPSC-like (tau rise = 0.2 ms, tau decay = 6 ms) currents (600−2000 pA) of PCs in the (a1) subiculum and (b1) dCA1. The current trains were injected at 5 Hz to the cells. Asterisks denote the burst spiking. (a2,b 2) Summarized results of experiments shown in a1 and b1, indicating (a2) the subiculum regular spiking (black) PCs and bursting PCs (magenta), respectively, (b2) but only regular spiking pattern for both cPCs (red) and sPCs (blue) in the dCA1. The classification of the regular-spiking and bursting PCs are based on the previous study by Graves et al28. Data were presented as mean±s.e.m. (c1) Example trace of dCA1 sPC (blue) and cPC (red) action potentials evoked by step current injection. (c2) Summarized results of firing rate (upper), inter-spike-interval (left bottom) and ratios of the first ISI and the last ISI (testing spike modulation, right bottom) under increasing step currents. Data were presented as mean±s.e.m. P = 0.11, measured by Mann-Whitney U-test.

  3. Supplementary Figure 3: Differential propagation of dendritic excitation to the soma in sPCs and cPCs revealed by TREES and NEURON simulations. (212 KB)

    Simulation was performed under either (ac) MATLAB TREES toolbox or the (dg) NEURON stimulation. (a) A pair of example sPC and cPC from experimental reconstruction showing dendritic sites of current injection (blue and red dots) and the recording at soma. (b) Example of somatic voltage change (ΔV, upper) elicited by injected electronic currents at ~450 μm apical dendrite (bottom) in the sPC (blue) and cPC (red), respectively. (c) Cumulative plot of mean ΔV of 171 sPCs (blue) and 183 cPCs (red). Each mean ΔV for a given PC was averaged from all trials with the injection sites distributed on all distal dendritic sections (> 300 μm). Statistic difference was measured by two-sample Kolmpgorov-Smirnov test, P < 0.001. (d) Morphologies of sPC and cPC NERUON models selected from the ModelDB65,66 depicting distal synaptic inputs, dendritic and somatic recordings. (e,f) Same as bc except using NEURON models. (g) Comparison of mean somatic and dendritic excitation between sPC and cPC. Data were presented as mean±s.e.m. Statistic difference was measured by unpaired t-test, P < 0.001 and P = 0.76 for the somatic and local dendritic injections, respectively.

  4. Supplementary Figure 4: Histological characterization of viral expression of ChR2 in the LEC, MEC and their axon projections along the anterior–posterior axis of CA1. (365 KB)

    (a1,a2) Serial sections of LEC (coronal) and MEC (horizontal), demonstrating the ChR2(-mCherry) expression in LEC and MEC (outlined by dashed lines), respectively. Scale bar, 1 mm. (b1) Representative histology verification of ChR2-expressing axons from the LEC (top) and MEC (bottom) in the hippocampus from anterior (A) to posterior (P) axis, respectively. Scale bar, 1 mm. Note that the ChR2(-mCherry) expressing LEC axons appear an increasing gradient in the CA1 stratum lacunosum-moleculare (SLM) layer along the A−P axis, while that of MEC axons show the opposite pattern. The sections in the dashed line box show the intermingled ChR2-expressing axons from the LEC and MEC projection neurons. (b2) Higher resolution images showing clearer segregation in the DG MO layer and distal−proximal gradients in the CA1 SLM layer of LEC and MEC axons in the boxed regions in b1, Scale bar, 200 μm.

  5. Supplementary Figure 5: Characterization of optogenetic activation of EC–dCA1 monosynaptic transmission by AOD-based laser stimulation system. (212 KB)

    (a) Representative traces of EPSCCRACM recorded from the CA1 PC (blue) and the CA2 GABAergic interneuron (red), which, respectively, show consistent mono-synaptic responses and variable poly-synaptic responses, evoked by 473-nm laser pulses with increasing intensities in the CRACM experiment. (b,c) Effects of applying GABAARs blocker picrotoxin (50 μM) on (b) the responsive map, (c) EPSCCRACM amplitudes and the response latency in the dCA1 PC. P = 0.29 and P = 0.004 for the amplitude and latency, respectively, calculated by the paired t-test. (d,e) Effects of application of TTX (0.5 μM) and 4-AP (100 μM) on (d) the discrete dendritic EPSCCRACM map, EPSCCRACM latency (bottom: averaged EPSCCRACM traces from the indicated locations in the map), and (e) the mean maximal amplitude of EPSCCRACM. Data were presented as mean±s.e.m. P = 0.01, measured by paired t-test. (f,g) Effects of HCN channel blocker ZD7288 (20 μM), slimilar as d,e. Note a slight increase of mean amplitude of EPSCCRACM after the ZD compound application. Data were presented as mean±s.e.m. P = 0.16, calculated by the paired t-test. (h) Blocking the GABAARs with picrotoxin (50 μM) does not affect the differential innervations of direct excitatory TA inputs from LEC to dCA1, compared to the data in Fig. 2e. P = 3.8 × 10-8, measured by Person’s Chi-square test. (i) Preserved differential innervations of LEC and MEC direct inputs to sPC and cPC in the distal, medial and proximal part of dCA1.

  6. Supplementary Figure 6: Direct long-range GABAergic transmission from LEC and MEC to dCA1 pyramidal neurons. (161 KB)

    (a) Example IPSCCRACM and responsive map of direct GABAergic inputs from the MEC to a dCA1 PC, measured under 0 mV membrane potential and presence of CNQX (10 μM) and AP5 (50 μM). (b) Percentage of cell receiving direct GABAergic projections from LEC or MEC in the dCA1 sPCs and cPCs as well as dCA2 PCs. n, number of tested cells.

  7. Supplementary Figure 7: Comparison of transmission strength of CA3 SC excitatory synapses on dCA1 sPCs and cPCs. (72 KB)

    (a) Diagram of recording configuration, in which the neighboring sPC and cPC were simultaneously recorded and a filed stimulation electrode (concentric tungsten electrode) was placed in a distance of ~350 μm away in the middle of SR layer. (b) Example EPSC traces recorded from sPC and cPC under increasing stimulus intensities. (c) Summarized EPSC amplitude ratios of the cPC and sPC from the assay shown in a and b. Data were presented as mean±s.e.m.

  8. Supplementary Figure 8: Characterization of Calb2-IRES-Cre::Ai9 and Calb1-2A-dgCre::Ai9 mice. (403 KB)

    (a,b) Example images showing the genetically-tdTomato labeled cells (red) immunostained with monoclonal antibody against calbindin-D28k (green) in the dCA1 of (a) Calb2-IR ES-Cre::Ai9 and (b) Calb1-2A-dgCre::Ai9 (7-d TMP induction, see Methods) mice. Scale bar, 100 μm. (c,d) Characterization of hippocampal labeling from anterior (A) to posterior (P) in (c) Calb2-IR ES-Cre::Ai9 and (d) Calb1-2A-dgCre::Ai9 (7-d induction) mice. Scale bar, 1 mm. All following statistic results are measured in the sections from Bregma -2.0 to -3.5 mm with 0.3 mm step along the A−P axis. (e,f) Comparison of the labeling efficiency (yellow/green) and colocalization rate (yellow/red) in the (e) CA1 SP and (f) DG between the Calb2-IRES-Cre ::Ai9 and Calb1-2A-dgCre ::Ai9 (with 1 or 7 d induction) mice, respectively. (g,h) Fluorescence images showing the (g) neuronal GABA staining in the dCA1 of Calb2-IRES-Cre ::Ai9, and the (h) tdTomato labeling rates in GABAergic cells in different layers of dCA1. Note the co-localization rates are < 10%. Scale bar, 100 μm. (i,j) Similar as g,h except for Cre antibody staining. Note that Calb2-IRES-Cre ::Ai9 mice exhibit high efficiency (> 90%) in labeling neurons in the dCA1 SP layer, but low efficiency (< 15%) in labeling neurons in other layers. Scale bar, 100 μm. Data were presented as mean±s.e.m.

  9. Supplementary Figure 9: HSV-1-G3 anterograde transportation from dorsal or ventral LEC and MEC to hippocampal CA1. (477 KB)

    (a,b) Co-localization of anterograde transportation of HSV-1-G3 (green) from (a) anterior ventral or posterior dorsal LEC; (b) ventral or dorsal MEC to hippocampal CA1 in Calb2-IRES-Cre::Ai9 transgenic mice (tdTomato). Scale bar, 100 μm. (c) Summarized co-localized rates following the HSV-1-G3 injected to different parts of MEC or LEC, respectively. Note that no significant difference was observed between the dorsal and ventral parts within the LEC or MEC. Data were presented as mean±s.e.m. (d,e) Characterization of HSV injected in the (d) anterior ventral or posterior dorsal LEC and (e) ventral or dorsal MEC in Calb2-IRES-Cre::Ai9 transgenic mice. Scales bar, 1 mm. (f) Serial brain sections showing the spreading infection area of HSV-1-G3 3 days after the injection in the anterior-ventral LEC. Scale bar, 1 mm.

  10. Supplementary Figure 10: Postbehavior histological characterization of NpHR-EYFP expression in LEC or MEC and the locations of implanted optical fibers or optetrodes in dCA1. (340 KB)

    (a) Post-histology of the mice with AAV-CaMKIIα-NpHR-EYFP or AAV-CaMKIIα-EYFP injected in the bilateral LEC and subsequently behaviorally tested shown in Fig. 5c. The first column: atlas of different coronal sections; 2nd column: example fluorescence images of NpHR-EYFP expression in the bilateral LEC and optical fiber implanted in the dCA1 (scale bar, 1 mm); 3rd column: overlaid image showing the region of NpHR-EYFP expression of all tested mice; 4th column, overlaid image showing the region of EYFP expression of all tested control mice. Grey gradients: viral expression levels in all tested mice, red gradients: optical fiber tracks. (b) Similar as a except for the optical fiber implantation was made in the DG in mice, whose results are shown in Fig. 5d. (c) Similar as a except for the virus was injected in the MEC, whose results are shown in Fig. 5e. (d) Similar as a except for the mice whose results are shown in Fig. 5f−h, respectively. (e) Fluorescence images of hippocampal sections show the expression of Arch-GFP (green) and the optetrode recording sites (arrows) in the dCA1. Uppers 4 mice were recorded in the right hemisphere, while the bottoms 6 were done in the left hemisphere. Scale bar, 1 mm.

  11. Supplementary Figure 11: Similar impairments of reversed learning by inactivation of LEC–dCA1 transmissions or of postsynaptic dCA1 Calb+ cPCs. (359 KB)

    (a13) The inactivation of (a1) LEC−dCA1, but not (a2) LEC−DG or (a3) MEC−dCA1 transmissions impairs the reverse learning, in which the odor−licking/no n -licking contingencies were reversed, in the days 4−6 with the same mice which underwent the initial learning shown in Fig. 5c−e, respectively. Data were presented as mean±s.e.m. (a45) Suppressing Calb+ PCs activities in the (a4) dCA1, but not in the (a5) DG impairs the reversal learning in the days 4−6 in the same mice which underwent the initial learning shown in Fig. 5f and g, respectively. Data were presented as mean±s.e.m. (b15) Detailed changes of the hit rate (upper) and the correct rejection rate (bottom) for behavioral performance data shown in the Fig. 5c−g and Supplementary Fig. 12a1−5 during the initial and reversal learning, respectively. Statistic differences were presented in the Supplementary Table 4−15. Data were presented as mean±s.e.m.

  12. Supplementary Figure 12: Optogenetic tagging of dCA1 Calb+ cPC units with Arch in vivo in comparison with that of tagging with ChR2. (229 KB)

    (a) Comparisons of waveforms of the spontaneous spikes (top traces) and the optical ChR2-excitation induced spikes (by 5 ms blue laser pulses, 0.5 mW, bottom) recorded from dCA1 PV+IN and PCs with the optetrode in the Pavlb-Cre::Ai27 and Calb2-IRES-Cre::Ai27 mice, respectively. Note that the optical ChR2-excitation significantly alters the waveform of PCs’ spikes, but not that of PV+INs, indicated by cross correlation (cc) values (r). (b,c) Example recording of the LFPs and spike units from (b) PCs and (c) PV+INs with 5 ms blue-laser pulse stimulation (0.5 mW) in the dCA1 area of the Calb2-IRES-Cre::Ai27 and Pavlb-Cre::Ai27 mice, respectively. Waveforms: averaged spontaneous spikes (left), individual ChR2-induced spikes (blue) or spontaneous spikes (black, corresponding to the *-labeled spikes in the middle). r: cc between the averaged spike and the individual spike. (d) Similar to bc, but showing the example of two tagged dCA1 Calb+ PCs with Arch in Calb2-IRES-Cre::Ai35 mice. Note that the yellow-laser illumination (2 min, 10 mW, indicated by yellow bars) significant suppresses the rate of spontaneous spikes, but exhibiting identical waveforms recorded during the ON and OFF of laser stimulation. (e) Example of simultaneous recordings from the Calb+Arch+ cPC (red) and CalbArch sPC (blue) in hippocampal slices of the Calb2-IRES-Cre::Ai35 mouse, showing differential effects of 2-min yellow-light illumination (10 mW, indicated by the yellow bar) on evoked rhythmic spikes (by intracellular injection of 5 ms current pulses at 0.5 Hz, black traces) between these two subtypes of dCA1 PCs. (f) Statistic results of the changes of spike rate and resting membrane potential during the ON and OFF of the yellow-light illumination in the Calb+Arch+ cPC (red) and CalbArch sPC (blue) in the experiments shown in d. P = 0.008, measured by the Wilcoxon signed-ranked test. (g) Example recordings and (h) statistical results showing changes of the frequency and amplitude of spontaneous IPSCs (Vclamp = 0 mV) during the ON and OFF of 2-min laser illumination in the dCA1 Calb+Arch+ cPC (red colors) and CalbArch sPC (blue colors) in the slices of Calb2-IRES-Cre::Ai35 mice.

  13. Supplementary Figure 13: Varied spiking responses and selectivities in response to odors A and B during odor sampling in trials. (742 KB)

    (a) Raster plots of spikes (upper two panels) and peri-stimuli time plots of firing rate changes (in 100 ms bins) for single-units recorded in the Go (red, odor A) and No-go (blue, odor B) trials in different mice (see the unit identity). The odor delivery duration is indicated by the green bar. The numbers for individual types are presented in the Supplementary Table 16.

  14. Supplementary Figure 14: Comparison of neuronal spiking activities in Correct and Error trials in the behavioral task. (410 KB)

    (a) Comparison of the mean firing rates of all PC units (left), sPC (middle), or cPC (right) in the Correct and Error trials in Go (upper) and No-go trials (bottom), respectively. The upper dots indicate the significant differences between Correct and Error trials (Paired t-test, P < 0.05). Data were presented as mean±s.e.m. (b) D’-prime analysis of differential firing of individual units in Correct and Error trials in Go (left) and No-go trials (right), respectively. Red and blue columns in the upper right indicate the cPC and sPC, respectively and sorted by the mean d’-prime of 1.5-s after odor delivery. The lower panels are the mean population d’-prime of sPC (blue) and cPC (red) during Correct and Error trials in Go (left) and No-go trials (right), respectively. Black dashed line, 95% chance level. All units were recorded in the first learning day.

  15. Supplementary Figure 15: Comparison of spontaneous firing rates during different brain states between the identified Calb+ and Calb dCA1 PCs. (177 KB)

    (a) Examples of neuronal spikes of units and local field potentials in the running, rapid-eye movement (REM) sleep and slow-wave sleep (SWS) states, respectively. Asterisks label the units of light-responsive Calb+ cells. Scale bar, 1 sec (middle panel) or 100 ms (enlarged, bottom panel), 0.5 mV. (b) Distributions of Calb+ (red) and Calb (blue) dCA1 PCs showing different spontaneous firing rates during the RUN, REM, SWS, respectively. P = 0.0127, 0.0282 and 0.0025 for RUN, SWS and REM state, respectively, calculated by the Mann-Whitney U-test. Note that this result is consistent with a previous study by Mizuseki et al29.

PDF files

  1. Supplementary Text and Figures (4,697 KB)

    Supplementary Figures 1–15 and Supplementary Tables 1–16

  2. Supplementary Methods Checklist (481 KB)

Additional data