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Spatially segregated feedforward and feedback neurons support differential odor processing in the lateral entorhinal cortex

Nature Neuroscience volume 19, pages 935–944 (2016)Cite this article

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Abstract

The lateral entorhinal cortex (LEC) computes and transfers olfactory information from the olfactory bulb to the hippocampus. Here we established LEC connectivity to upstream and downstream brain regions to understand how the LEC processes olfactory information. We report that, in layer II (LII), reelin- and calbindin-positive (RE+ and CB+) neurons constitute two major excitatory cell types that are electrophysiologically distinct and differentially connected. RE+ neurons convey information to the hippocampus, while CB+ neurons project to the olfactory cortex and the olfactory bulb. In vivo calcium imaging revealed that RE+ neurons responded with higher selectivity to specific odors than CB+ neurons and GABAergic neurons. At the population level, odor discrimination was significantly better for RE+ than CB+ neurons, and was lowest for GABAergic neurons. Thus, we identified in LII of the LEC anatomically and functionally distinct neuronal subpopulations that engage differentially in feedforward and feedback signaling during odor processing.

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Figure 1: Differential projection patterns of LEC LII excitatory neurons: RE+ neurons provide feedforward projections to the hippocampus while CB+ neurons project to OB, PIR and contralateral LEC.
Figure 2: Odor-evoked calcium transients in LEC LII neurons are variable.
Figure 3: Odor-responsive regular spiking neurons project to the hippocampus.
Figure 4: CB+ neurons in the LEC provide feedback to the OB and the PIR.
Figure 5: Fast-spiking GABAergic basket cells in the LEC are odor-responsive.
Figure 6: Odor stimulation evokes cell-type specific activity patterns in defined populations of LEC neurons.
Figure 7: Populations of LEC RE+ neurons encode specific odors with high accuracy.

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Acknowledgements

We thank U. Amtmann for immunohistochemical work, E. Fuchs and A. Caputi for providing help at initial stages of the study and for proofreading the manuscript, and G. Giese and the staff from the MPI workshop for their technical assistance. All AAVs were obtained from Penn Vector Core, University of Pennsylvania, Philadelphia, USA. AAVs were provided by the GENIE Project and the Janelia Farm Research Campus of the HHMI and by K. Deisseroth, Stanford University. The research was funded by grants to H.M. from the European Research Council, FP 7 (ERC Adv. grant no. 250047), the German Research Foundation (DFG grants MO432/10 and SFB1134), and the German Ministry of Education and Research (BMBF) (grant 01GQ1003A).

Author information

Author notes

  1. Sarah Melzer & Henry Lütcke

    Present address: Present addresses: Department of Neurobiology, Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts, USA (S.M.). and Scientific IT Services, ETH Zurich, Zurich, Switzerland (H.L.).,

  2. Frauke C Leitner and Sarah Melzer: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Clinical Neurobiology at the Medical Faculty of Heidelberg University, Heidelberg, Germany

    Frauke C Leitner, Sarah Melzer, Roberta Pinna & Hannah Monyer

  2. German Cancer Research Center (DKFZ), Heidelberg, Germany

    Frauke C Leitner, Roberta Pinna & Hannah Monyer

  3. Department of Molecular Neurobiology, Max Planck Institute for Medical Research, Heidelberg, Germany

    Frauke C Leitner & Peter H Seeburg

  4. Laboratory of Neural Circuit Dynamics, Brain Research Institute, University of Zurich, Zurich, Switzerland

    Henry Lütcke & Fritjof Helmchen

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Contributions

F.C.L., S.M. and H.M. designed the experiments and wrote the manuscript with contributions from all authors. F.C.L. performed two-photon calcium imaging experiments, stereotactic injections and immunohistochemistry; S.M. and R.P. performed in vivo and in vitro electrophysiology and cell reconstructions; and H.L. and F.H. analyzed the calcium imaging data. P.H.S. was involved in discussing experiments and provided the infrastructure for the in vivo experiments.

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Integrated supplementary information

Supplementary Figure 1 Relative distribution of RE+, CB+ and GABAergic neurons in LIIa and LIIb.

(a) WFS1 is co-expressed in CB+ neurons as shown already previously for the MEC25,52. Immunohistochemical analysis of WFS1- and CB-expression in the LEC revealed high overlap in excitatory neurons: 93.8 ± 0.5% of all WFS1+ neurons in LIIb were CB+. Note that no WFS1+/GAD+ neurons were detected. Thus, immunostaining against WFS1 allows the analysis of excitatory CB+ neurons. (b) There is a clear segregation of RE- and WFS1- positivity in LIIa and LIIb, akin to what is observed when employing RE and CB immunohistochemistry. (c) We quantified the number of RE+, NeuN+ and GAD+ neurons in brain slices of GAD-EGFP mice immunostained against RE and NeuN. Excitatory RE+ neurons represent almost all excitatory neurons in LIIa, but only a small fraction of excitatory neurons in LIIb. The six panels on the right show higher magnifications of the boxed area. (d) Same as in c, but slices were immunostained against WFS1 and NeuN. WFS1+ neurons represent the vast majority of all excitatory neurons in LIIb, but only a very small population of excitatory neurons in LIIa. The six panels on the right show higher magnifications of the boxed area. (e) Table summarizing the relative distribution of RE+, WFS1+ and GAD+ neurons in LEC LII. For each data point, the quantification was performed in 4 slices from 3 hemispheres. The ‘neuron total’ for the data point regarding the quantification of GABAergic neurons includes all NeuN+ neurons plus a minority of GABAergic neurons that were EGFP+ but NeuN- (it is known that some GABAergic neurons do not express NeuN). Scale bars in a to d, 50 μm and 10 μm (magnified images). Abbreviations: EGFP, enhanced green fluorescent protein; exc., excitatory; GAD+ neuron, GABAergic neuron; L, layer; NeuN, neuronal nuclei; WFS1, Wolfram syndrome 1.

Supplementary Figure 2 RE+ and CB+ excitatory neurons in LEC LII exhibit differential projection patterns.

(a) Confocal image showing the injection site of the retrograde tracer FG in the DG of a GAD67EGFP mouse. The two panels below depict CB-immunostained and FG+ neurons in the LEC. The merged image reveals a clear segregation of the two populations, with FG+ neurons in LIIa and CB+ neurons in LIIb, and indicates that CB+ neurons do not project to the DG. (b) Confocal image showing the injection site of the retrograde tracer CTB in the OB of a GAD67EGFP mouse. Only few CTB+ neurons in LIIa co-localize with RE+ neurons. (c) Confocal image of the PIR injected with the retrograde tracer CTB (RE staining was used here with the purpose to visualize the brain structure). CTB labeling can be detected in LEC LIIb and deeper layers, but not in RE+ neurons in LIIa. (d) Same as in c, except that the tracer CTB was injected in the contralateral LEC. CTB positivity can be detected in LEC LIIb, but not in RE+ neurons in LIIa. Scale bars, 200 μm (injection sites) and 50 μm. Abbreviations: cLEC, contralateral LEC; CTB, cholera toxin subunit B; DG, dentate gyrus; EGFP, enhanced green fluorescent protein; FG, fluorogold; L, layer; OB, olfactory bulb; PIR, piriform cortex.

Supplementary Figure 3 Interneuron diversity in the LEC.

Confocal images of sections from a GAD67EGFP mouse brain immunostained with antibodies against (a) PV, (b) SOM, (c) CR, (d) CB, (e) RE and (f) 5-HT3AR with corresponding graphs showing the distribution in LIIa and LIIb. Counting was performed in 3-4 hemispheres from 2 mice. For experiments as illustrated in f, 5-HT3-EGFP mice were used. Furthermore, RE and CB stainings were employed with the purpose to visualize the border between LIIa and LIIb. Scale bar, 100 μm. Abbreviations: CR, Calretinin; EGFP, enhanced green fluorescent protein; L, layer; PV, Parvalbumin; SOM, Somatostatin; 5-HT3AR, ionotropic serotonin receptor 5-HT3A.

Supplementary Figure 4 Dependence of calcium responses on odor concentration.

(a) Average calcium responses evoked by air only and by odors (benzaldehyde and amyl acetate) at the indicated concentration in air. Data shown are grand averages for 159 RE+ neurons and 36 UNC. (b) Maximum calcium response amplitude evoked by air only and by the two odors presented at the indicated concentration. (c) Calcium event probability for air only and the two odors presented at the indicated concentrations. Abbreviation: UNC, unclassified neurons.

Supplementary Figure 5 Odor-responsive excitatory RE+ neurons in LIIa of the LEC are regular-spiking or bursty-spiking projecting neurons.

Four reconstructed odor-responsive neurons with corresponding firing pattern (left panels) and higher magnification of the reconstruction (cell bodies and dendrites in black, axons in red). 2D images on the right show a ventral (grey) and dorsal (black) outline of the hippocampus and the LEC. Cell bodies and dendrites are localized more ventrally, whereas axons extend to dorsal areas. Scale bars, 20 mV, 200 ms (firing pattern), 400 μm (reconstructions, if not indicated otherwise).

Supplementary Figure 6 Additional excitatory RE+ neurons in LEC LIIa that were not tested for odor responsiveness.

The neurons are largely regular spiking, have spiny dendrites and project towards alveus/presubiculum/hippocampus. Six reconstructed excitatory neurons with corresponding firing pattern (left panels) and higher magnification of the reconstruction (cell bodies and dendrites in black, axons in red). 2D images on the right show a ventral (grey) and dorsal (black) outline of the hippocampus and LEC. Cell bodies and dendrites are localized more ventrally, whereas axons extend to dorsal areas. Scale bars, 20 mV, 200 ms (firing pattern), 400 μm (reconstructions, if not indicated otherwise).

Supplementary Figure 7 RE+ and CB+ neurons segregate into two distinct cell populations based on their intrinsic electrophysiological and morphological properties tested in vitro.

(a) Selected electrophysiological properties were compared. Means ± s.e.m. and t-test based p-values are shown for normally distributed data. Medians ± interquartile range and Mann-Whitney-U test based p-values are shown for non-normally distributed data (indicated by black asterisks). All p-values were corrected by the Holm-Bonferroni method. Please note differences in physiological properties when comparing in vitro (this table) and in vivo (Supplementary Table 1) data: RE+ neurons measured in vivo had a significantly more depolarized resting membrane potential (t(33) = 5.67, P < 0.0001) and action potential threshold (t(22) = 6.59, P < 0.0001), a smaller AP amplitude (t(18) = -5.75, P = 0.0001) and maximal firing frequency (t(49) = -10.66, P < 0.0001) and saturate at lower injected currents (U = 6, P < 0.0001). Similarly, GABAergic LII neurons were more depolarized (t(61) = -10.78, P < 0.0001), had a lower maximal firing frequency (t(59.6) = 3.7, P = 0.003) and saturated earlier (t(54.2) = 10.2, P < 0.0001) in vivo (Supplementary Table 1) than in vitro (n = 49 GAD-EGFP+ neurons patched in vitro in LII in 7 mice). (b,c) Representative reconstructions with corresponding in vitro firing patterns of RE+ (b) and CB+ (c) neurons. Scale bars, 20 mV, 200 ms (firing pattern), 200 μm (reconstruction). (d) k-means clustering suggests that RE+ and CB+ neurons form two mostly non-overlapping cell clusters. Four selected parameters are plotted. Blue and red dots indicate cell cluster 1 and 2 respectively that correspond largely to CB+ und RE+ neurons respectively. Calculated centroids are indicated as black crosses. Green arrows indicate cells that were assigned to a cluster that would not have been predicted based on CB/RE expression: 5 CB+ neurons and 1 RE+ neuron were unexpectedly assigned to cluster 2 (5 red dots with arrow) and cluster 1 (1 blue dot with arrow) respectively. Abbreviations: AP, action potential; AHP, after hyperpolarization.

Supplementary Figure 8 Examples of in vivo patched and filled GABAergic odor-responsive neurons in LIIa of the LEC.

Three additional reconstructed odor-responsive neurons with their corresponding firing pattern, higher magnification and overview of the hippocampus and LEC. Axons are shown in red, cell bodies and dendrites in black. Scale bars, 20 mV, 200 ms (firing pattern), 200 μm (higher magnification reconstruction), 400 μm (overview).

Supplementary Figure 9 Additional examples of in vivo patched and filled GABAergic neurons with different firing patterns and extensive axon arborization in the superficial layers of the LEC.

Neurons are shown with corresponding firing pattern (left panels), higher magnification, and overview of the hippocampus and LEC. Axons are shown in red, cell bodies and dendrites in black. Scale bars, 20 mV, 200 ms (firing pattern), 200 μm (higher magnification reconstruction), 400 μm (overview).

Supplementary Figure 10 Properties of odor-evoked calcium responses of RE+, CB+ and GABAergic neurons (top to bottom row).

(a) Overlay of all trial-averaged GCaMP6 fluorescence traces during odor stimulation, sorted into significant positive responses ('Pos'), non-significant traces ('NS'), and significant negative responses ('Neg'). Significance was evaluated within the 4-s odor stimulation period. Note that for RE+ and CB+ neurons some NS and Neg traces display late fluorescence changes (number of responses for RE+: 723 Pos, 2021 NS, 340 Neg; CB+: 871 Pos, 1413 NS, 158 Neg; GAD: 378 Pos, 720 NS, 66 Neg). (b) Group-wise average of the respective Pos, NS, and Neg traces shown in a. (c) Histograms of fluorescence peaks during odor stimulation for significant positive and negative responses for the different cell types. Note that the tail of responses larger than 400% is truncated (cut events: 11 RE+, 6 CB+, 2 GAD). (d) Histograms of estimated onset latencies for significant fluorescence responses for the different cell types (peak position/half-width: RE+ 0.4 s/0.7 s, CB+ 0.4 s/0.6 s, GAD 0.3 s/0.5 s). (e) Histograms of fluorescence response durations (full width at half-maximum) for the different cell types (evaluated in a 19-s time window following stimulus onset). Note the bimodality of the distributions, indicating the fraction of neurons displaying a prolonged, late response component.

Supplementary Figure 11 Selectivity of LEC neuronal subtypes quantified by lifetime sparseness.

(a) Cumulative distributions of lifetime sparseness for RE+, CB+ and GAD+ neurons. Lifetime sparseness was quantified as kurtosis of the maximum ∆F/F response for each odor. A kurtosis of 0 indicates normally distributed responses while kurtosis < 0 indicates more uniformly distributed responses. All three distributions are significantly different from each other (Kolmogorov-Smirnov test, all p < 0.01). (b) Average (± s.e.m.) values of lifetime sparseness measured as kurtosis for RE+, CB+ and GAD+ neurons. Abbreviations: GAD+ neuron, GABAergic neuron. *** P < 0.001; ** P < 0.01; * P < 0.05, unpaired t-test.

Supplementary Figure 12 Odor-specific information content of neuronal responses quantified by mutual information.

(a) Cumulative distributions of mutual information for odor responsive RE+, CB+ and GAD+ neurons. RE+ neurons contain significantly more stimulus-related information than CB+ and GAD+ neurons (Kolmogorov-Smirnov test, p < 0.001). (b) Average mutual information (± s.e.m.) for RE+, CB+ and GAD+ neurons. Abbreviations: GAD+ neuron, GABAergic neuron. *** P < 0.001, unpaired t-test.

Supplementary Figure 13 Interdependence between odor selectivity and odor-specific information content of neuronal responses.

(a-b) Correlations between neuronal selectivity (measured as lifetime sparseness) and odor-specific information content (measured as mutual information in a or classification accuracy in b). Selectivity and information content are significantly correlated for RE+ neurons (r > 0.15, P < 0.001), but not for CB+ and GAD+ neurons (r < 0.1, P > 0.05). (c) By contrast, information-based metrics are significantly correlated for all three cell types (r > 0.2, P < 0.01 for all cell types).

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Leitner, F., Melzer, S., Lütcke, H. et al. Spatially segregated feedforward and feedback neurons support differential odor processing in the lateral entorhinal cortex. Nat Neurosci 19, 935–944 (2016). https://doi.org/10.1038/nn.4303

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