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Glutamatergic synaptic integration of locomotion speed via septoentorhinal projections

Abstract

The medial septum and diagonal band of Broca (MSDB) send glutamatergic axons to medial entorhinal cortex (MEC). We found that this pathway provides speed-correlated input to several MEC cell-types in layer 2/3. The speed signal is integrated most effectively by pyramidal cells but also excites stellate cells and interneurons. Thus, the MSDB conveys speed information that can be used by MEC neurons for spatial representation of self-location.

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Figure 1: Speed modulation of glutamatergic MEC-projecting neurons in the MSDB.
Figure 2: Glutamatergic monosynaptic projections from MSDB excite MEC pyramidal cells, stellate cells and interneurons.
Figure 3: Speed-dependent septoentorhinal glutamatergic input is most effectively integrated by MEC pyramidal cells.

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Acknowledgements

We thank the light microscopy facility and the image analysis facility of the DZNE for technical support. We thank J. Doerr for providing help with the imaging of cleared tissue and K. Conzelmann for the initial RABV inoculation. We thank M. Harnett and M. Nolan for comments on an early version of this manuscript. We thank the GENIE Project and the Janelia Research Campus, specifically V. Jayaraman, R. Kerr, D. Kim, J. Akerboom, L. Looger and K. Svoboda, for providing GCaMP5 and GCaMP6s. This work was supported by the Deutsche Forschungsgemeinschaft (SFB1089, S.R., S.S., I.S., M.K.S., F.B., L.S.).

Author information

Authors and Affiliations

Authors

Contributions

D.J., D.D., S.B., C.H., H.K., F.F. and L.S. performed and analyzed in vitro and in vivo experiments. F.F., S.B., D.F., I.S., D.A.E., F.B. and M.K.S. performed the tracing and immunohistochemistry. D.J. performed the computational modeling. S.S. and M.K.S. produced and provided viral vectors. D.J. and S.R. wrote the manuscript. S.R. designed and supervised the project.

Corresponding author

Correspondence to Stefan Remy.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Different tuning types of MSDB unit firing

(a) Speed dependence of AP frequency for n = 240 MSDB units. Significantly positive speed modulated units are indicated by black lines, significantly negative speed modulated units are indicated by red lines. (b) Speed dependent output of four representative MSDB units. 30 s of 2500 s total recording time are displayed. Scale bars represent 3 s and 5 cm/s. (c-f) Septal units showing speed and theta modulation (c), anti-speed modulation (d), speed modulation (e) and theta modulation (f) as seen by the speed modulation of AP firing and autocorrelations. The weighted fitting function, the theta modulation index (TMI) and its p-value are given for each unit. (g) Spearman’s rank correlation of the three units displayed in Fig. 1c,e. The linear fitting function, Spearman’s rank correlation coefficient and its p-value are stated. (h) Histogram showing Spearman’s rank correlation coefficient of all units (n = 240). (i) The speed modulation index as used in Fig. 1 and Spearman’s rank correlation coefficient are highly correlated (Spearman’s rank correlation, n = 240 units, significantly speed modulated units are displayed in red).

Supplementary Figure 2 Theta modulation of MSDB unit firing depends on running speed

(a) Autocorrelation of a theta and speed modulated MSDB unit during slow and fast locomotion. (b) Spectra of the autocorrelations displayed in (a). (c-e) Peak modulation frequency during slow and fast running of units showing no velocity modulation (n = 10), of units showing a positive velocity modulation (n = 8, paired Wilcoxon signed rank test, p = 0.008) and of units showing a negative velocity modulation (n = 5).

Supplementary Figure 3 Fluorometric recordings demonstrate the speed modulation of MEC-projecting VGluT2+ neurons in the MSDB

(a) Fluorometric monitoring of MSDB glutamatergic (VGluT2+) population activity in head-fixed mice on a linear treadmill (two representative recordings). Scale bars represent 10 s, 1% ΔF/F and 5 cm/s. (b) Speed dependence of the VGluT2+ population activity (GCaMP5 fluorescence). The upper panel shows the speed modulation of MSDB VGluT2+ activity in representative recordings of three 10-20 minute sessions in one mouse on 3 consecutive days. The lower panel shows linear speed-dependence of GCaMP5 fluorescence (Day 3). (c) Positive slope of linear fit confirms speed-dependence of MSDB VGluT2+ population activity (n = 10 mice, Wilcoxon signed rank test, p = 0.0098, error bars denote s.e.m.). (d) Local expression of GCaMP6s in VGluT2+ neurons in the MSDB. Two magnifications are shown. Scale bars represent 250 μm (overview) and 50 μm (magnifications). (e) GCaMP6s expression in VGluT2+ septo-entorhinal projections. The four different recording depths are indicated. Slight tissue damage above the upper marker identifies the puncture by the optical fiber. Allen Mouse Brain Atlas figure 103 is given as reference. Scale bars represent 500 μm (overview) and 100 μm (magnification). (f) The GCaMP6s fluorescence of VGluT2+ septo-entorhinal projections during resting and locomotion (n = 6 mice, paired Wilcoxon signed rank test) and the linear relationship to running speed (n = 6 mice, one-sample t-test, * p<0.05) in different depth in the MEC.

Supplementary Figure 4 Monosynaptic septoentorhinal glutamatergic projection

(a) Representative medial entorhinal cortex (MEC) injection site referring to Allen Mouse Brain Atlas figure 101. Images available from http://mouse.brain-map.org. The local expression of retrograde tracing virus (RABVΔG-EGFP) and rAAV mRFP is shown in three different magnifications. “Source” neurons, competent for retrograde transsynaptic traversal, are displayed in yellow. Scale bars represent 1 mm (left panel), 500 μm (middle panel) and 100 μm (right panel). (b) Localization of MEC projecting neurons in the medial septum (MS) (left panel), the horizontal part of the vertical limb of the diagonal band (VDBh) (middle panel) and the vertical limb of the diagonal band (VDB) (right panel). Yellow neurons in the magnifications represent the VGluT2+ (tdTomato) MEC projecting (EGFP+) population. Scale bars represent 1 mm (upper panel) and 100 μm (middle panel). Areas were determined using Allen Mouse Brain Atlas figures 43-49 as reference. (c) Scheme of combined MEC retrograde tracing and MSDB cell-type specific labeling. The coronal slicing planes are displayed. (d) Cell count of MSDB neuronal populations; neurons projecting to MEC (green), VGluT2 positive neurons (red) and VGluT2+ neurons projecting to MEC (black) (n = 3 mice with 2 slices each displayed as open circles, filled circles denote mean values ± s.e.m.).

Supplementary Figure 5 MEC retrograde tracing reveals VGluT2+ input from MSDB

(a) Scheme of combined MEC retrograde tracing and MSDB cell-type specific labeling. (b) Light-sheet fluorescence microscope image of entire brain hemisphere, cleared following the CUBIC clearing protocol. Locations in MEC and MSDB for further investigation are marked. Scale bar represents 1 mm. (c) High resolution image of the MEC RABVΔG-EGFP injection site, acquired using two-photon microscopy. Scale bar represents 500 μm. (d) High resolution image and magnification of retrogradely labeled neurons in the MSDB. Yellow neurons represent the VGluT2+ (tdTomato) MEC projecting (EGFP+) population. Scale bars represent 500 μm (left panel) and 50 μm in the magnifications. (e) Image of FluoClerBABB cleared entire MSDB coronal block with magnifications showing the MEC projecting (EGFP+) population and VGluT2+ (tdTomato) neurons. Scale bars represent 500 μm (left panel) and 10 μm in the magnifications.

Supplementary Figure 6 MEC innervation by MSDB VGluT2+ fibers

(a) Scheme of local MSDB cell-type specific ChR2 expression. The coronal cutting plane for MSDB slices and parasagittal cutting plane for MEC slices are displayed. (b) Overview and magnifications of the local expression of floxed ChR2 in a VGluT2-cre mouse. Scale bars represent 1 mm (left panel), 100 μm (middle panel) and 10 μm (right panel). (c) Overview of MEC and its septo-entorhinal VGluT2+ axonal innervation. Magnifications along the dorso-ventral axis confirm a high density of MSDB VGluT2+ fibers in MEC layer 2/3. Scale bars represent 200 μm (overview) and 50 μm (magnifications). (d) Location and layers of the MEC were determined using Allen Mouse Brain atlas sagittal figure 5 as reference (left panel). The distribution of septo-entorhinal VGluT2+ fibers along the dorso-ventral axis is displayed for all MEC layers (right panel, n = 4 mice, error bars denote s.e.m.).

Supplementary Figure 7 Classification of whole-cell recorded neurons according to electrophysiological and immunohistochemical parameters

(a) Representative examples of a whole-cell recorded pyramidal cell, stellate cell and fast-spiking interneuron, biocytin-identification (grey), GAD-67 (red) and WFS-1 (green) immunoreactivity. Voltage responses of different MEC neurons types to 500 ms step current injections at -200 pA, last sub-threshold currents (dark gray), first supra-threshold currents (black), and +500 pA (light gray). Scale bars represent 100 ms, 20 mV and 10 μm. (b) The first 3 principal components separate the different principle cell types. (c) The quantification of monosynaptic response types of all classified pyramidal cells (PY), stellate cells (SC), fast-spiking interneurons (FS) and other interneurons (other) in wild-type animals upon optogenetic stimulation of septo-entorhinal fibers.

Supplementary Figure 8 Morphologies of MEC neuron types

(a-c) Representative morphologies of reconstructed biocytin-filled neurons in the MEC. Scale bars represent 100 μm. (d) Sholl analysis of dendritic length in 10 μm distance intervals from the soma, values are displayed as mean ± s.e.m. (n = 30 PY, n = 13 SC, n = 12 FS). (e) The size of somata differs among MEC cell types. Box plots show medians, 10th, 25th, 75th and 90th percentiles (n = 163 PC, n = 111 SC, n = 27 FS, Kruskal-Wallis with post-hoc Dunn’s test, * p < 0.05, **** p < 0.0001).

Supplementary Figure 9 Glutamatergic input characteristics onto different MEC neuron types

(a) Representative examples of whole-cell recorded pyramidal cell, stellate cell and fast-spiking interneuron in MEC brain slices of VGlutT2+ mice with corresponding average postsynaptic responses to 473 nm light stimulations of ChR2-expressing VGlutT2+ septo-entorhinal axons. Scale bars represent 20 mV/100 ms and 2.5 mV/500 ms, respectively. (b) All MEC cell-types are targeted by septo-entorhinal VGluT2+ axons, with more frequent projections onto pyramidal cells (PY, n = 130) and fast-spiking interneurons (FS, n = 21) as compared to stellate cells (SC, n = 63). (c-e) Amplitude and time constants of monosynaptic glutamatergic EPSPs on pyramidal cells (n = 35), stellate cells (n = 7) and fast-spiking interneurons (n = 6) (mean ± s.e.m., Kruskal-Wallis test with post-hoc Dunn’s test, * p < 0.05, *** p < 0.001).

Supplementary Figure 10 Cholinergic inputs to different MEC neuron types

(a-d) Average postsynaptic responses of whole-cell recorded pyramidal cells (a, b), a fast-spiking interneuron (d), and an unidentified interneuron (c) in MEC to 473 nm light stimulations of ChR2-expressing septo-entorhinal axons. Upper three rows: Response to a single light pulse with subsequent bath application of NBQX/D-AP5 and SR-95531/CGP52432. Scale bars represent 100 ms and 5 mV Lower rows: Responses to rhythmic light stimulations with frequencies of 6 to 12 Hz under blockade of glutamatergic and GABAergic transmission using NBQX/D-AP5 and SR-95531/CGP52432 and subsequent additional cholinergic transmission using Mecamylamine hydrochloride/Scopolamine hydrobromide. Scale bars represent 200 ms and 2 mV.

Supplementary Figure 11 Speed- and theta-modulation of simulated neurons

(a,b) Voltage responses of a simulated pyramidal cell (PY), stellate cell (SC) and fast-spiking interneuron (FS) to rhythmic synaptic inputs at 9 Hz. Scale bars represent 400 ms and 2 mV. The passive membrane properties can partly explain a different paired pulse attenuation (a) and different EPSP kinetics (b) of MEC cell-types. Synaptic time constants and weights are kept constant here (experimentally recorded values as in Supplementary Fig. 8, mean ± s.e.m.). (c,d) Speed modulation of simulated MEC neurons in response to realistic input from two MSDB units randomly chosen out of the set of speed modulated units (Friedman test with Dunn’s post hoc test, * p < 0.05, *** p < 0.001). (e,f) In response to speed/theta modulated input (unit 2 from figure 1), the simulated pyramidal cell and stellate cell show stronger speed-modulation. The slopes of the fits are 0.23 (PY, p < 0.05), 0.31 (SC) and 0.07 (FS). Significant theta modulation is exclusively observed in the fast-spiking interneuron. Scale bars represent 2.5 s and 5 cm/s. (g) The pyramidal cell and stellate cell show stronger speed tuning at different proportions of speed tuned input (Friedman test with Dunn’s post hoc test, * p < 0.05). (h,i) The AP output, speed and theta modulation of a modeled pyramidal cell, stellate cell and fast-spiking interneuron in response to speed and theta modulated input (unit 1 from supplementary Fig. 1, scale bars represent 2.5 s and 5 cm/s). Speed modulation was determined as 0.15 cm−1 (PY), 0.19 cm−1 (SC) and 0.05 cm−1 (FS). (j) The speed tuning of simulated neurons at different ratios of the speed tuned and random input (Friedman test with Dunn’s post hoc test, * p < 0.05). (k,l) In response to anti-speed modulated input (unit 2 from supplementary Fig. 1) all three neuron types show mildly anti-speed modulated output. Speed modulation was -0.16 cm−1 (PY), -0.20 cm−1 (SC) and -0.04 cm−1 (FS). Scale bars represent 2.5 s and 5 cm/s. (m) The anti-speed tuning of the simulated pyramidal cell and stellate cell is stronger than the anti-speed tuning of the fast-spiking interneuron at all input compositions (Friedman test with Dunn’s post hoc test, ** p <0.01).

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Supplementary Figures 1–11 and Supplementary Table 1 (PDF 2949 kb)

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MEC retrograde tracing identifies a mono-synaptic septo-entorhinal glutamatergic projection

The video shows a 3D-rendering of the MSDB in a cleared mouse brain. VGluT2-positive neurons are labeled red using flex tdTomato, green labeled neurons are retrogradely traced from MEC using RABVΔG-EGFP. (MOV 18721 kb)

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Justus, D., Dalügge, D., Bothe, S. et al. Glutamatergic synaptic integration of locomotion speed via septoentorhinal projections. Nat Neurosci 20, 16–19 (2017). https://doi.org/10.1038/nn.4447

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