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 2 hippocampal dentate gyrusCA3CA1 and the direct EC layer 3CA1 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 ECdCA1 circuit that is required for olfactory associative learning.
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- Supplementary Figure 1: Analyses of dCA1 PC clusters by the Jump method, and principle component analysis (PCA) on different morphological parameters. (259 KB)
(a−c) 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. (f−h) 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.
- 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.
- 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 (a−c) MATLAB TREES toolbox or the (d−g) 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 b−c 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- Supplementary Figure 11: Similar impairments of reversed learning by inactivation of LEC–dCA1 transmissions or of postsynaptic dCA1 Calb+ cPCs. (359 KB)
(a1−3) 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. (a4−5) 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. (b1−5) 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.
- 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 b−c, 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 Calb−Arch− 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 Calb−Arch− 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 Calb−Arch− sPC (blue colors) in the slices of Calb2-IRES-Cre::Ai35 mice.
- 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.
- 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.
- 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.