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. 2016 Jan 6;89(1):194-208.
doi: 10.1016/j.neuron.2015.11.029. Epub 2015 Dec 17.

Local and Distant Input Controlling Excitation in Layer II of the Medial Entorhinal Cortex

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Free PMC article

Local and Distant Input Controlling Excitation in Layer II of the Medial Entorhinal Cortex

Elke C Fuchs et al. Neuron. .
Free PMC article

Abstract

Layer II (LII) of the medial entorhinal cortex (MEC) comprises grid cells that support spatial navigation. The firing pattern of grid cells might be explained by attractor dynamics in a network, which requires either direct excitatory connectivity between phase-specific grid cells or indirect coupling via interneurons. However, knowledge regarding local networks that support in vivo activity is incomplete. Here we identified essential components of LII networks in the MEC. We distinguished four types of excitatory neurons that exhibit cell-type-specific local excitatory and inhibitory connectivity. Furthermore, we found that LII neurons contribute to the excitation of contralateral neurons in the corresponding layer. Finally, we demonstrated that the medial septum controls excitation in the MEC via two subpopulations of long-range GABAergic neurons that target distinct interneurons in LII, thereby disinhibiting local circuits. We thus identified local connections that could support attractor dynamics and external inputs that likely govern excitation in LII.

Figures

Figure 1
Figure 1
Morphological and Electrophysiological Features of Defined Excitatory LII Cell Types in Dorsal MEC (A) Reconstruction of four representative neurons belonging to the indicated cell type (dendrites in black, apical dendrite in blue, axon in red) and their corresponding firing pattern upon somatic current injection (−200 to 600 pA). ISI1/2 plus dAP reveal differences between the four cell types: the stellate cell and intermediate stellate cell exhibit burstiness, the firing pattern of pyramidal cell and intermediate pyramidal cell displays adaptation, and dAP is absent in the pyramidal cell. (B) Distribution of stellate (gray circles) and intermediate stellate cells (green triangles) when using latency to spike firing, ISI1/2, and dAP as distinction criteria. (C) Distribution of pyramidal cells (blue squares) and intermediate pyramidal cells (red triangles) when using dAP, sag potential, and latency as distinction criteria. (D) Principal component analysis based on the same electrophysiological parameters as used in (B) and (C) plus the presence of an apical dendrite. The plot shows the first two principal components, with component 2 representing predominantly latency while component 1 combines information from the four remaining variables. Note the clear separation between stellate (black) and pyramidal cells (blue), whereas both intermediate cells (IM stellate [green] and IM pyramidal [red] cells) display an “intermediate” distribution. (E) Differences in soma size and numbers of primary dendrites of the four excitatory cell types (p < 0.05). (F) Sholl analysis reveals difference between the four cell types when plotting dendritic length as a function of circular distance from the soma in 10-μm steps (two-way ANOVA, F(144,1813) = 4.43, ∗∗∗p < 0.001). Stellate and intermediate pyramidal cells exhibit locally (10–80 μm) a higher density of dendrites compared to pyramidal and intermediate stellate cells. Abbreviations are as follows: ISI, interspike interval; dAP, depolarized afterpotential; and IM, intermediate. See also Figure S1 and Table S1.
Figure 2
Figure 2
Local Excitatory Connectivity in LII of the MEC (A) Firing pattern (left) of indicated excitatory cells in CBCre mice. The PC and IM PC, but not the SC, are excited by ChR2 stimulation (right, stimulation is indicated by red bar above the spike). (B) uEPSCs recorded at −70 mV in indicated cells elicited by a train of 10 action potentials in the presynaptic neuron (40 Hz) (action potential traces in red, upper row). The direction of tested connectivity is indicated by an arrow. uEPSCs are not blocked by Gabazine, but by Gabazine plus CNQX (both at a concentration of 10 μM). (C) Summary graph of investigated connections between indicated cell types. (D) Firing pattern of tested excitatory cells in Uchl1Cre mice. Representative examples showing activation of a SC and IM SC following ChR2 stimulation (stimulation is indicated by red bar above the spike). (E) uEPSCs recorded at −70 mV in indicated cells elicited by a train of 10 action potentials in the presynaptic neuron (40 Hz) (action potential traces in red, upper row). (F) Summary graph of investigated connections between indicated cell types. The numbers above the bars indicate the number of analyzed cell pairs. Abbreviations are as follows: L, layer; IM, intermediate; MEC, medial entorhinal cortex; PC, pyramidal cell; SC, stellate cell; IM PC, intermediate pyramidal cell; and IM SC, intermediate stellate cell. See also Figures S2 and S3.
Figure 3
Figure 3
Local Inhibitory Connectivity in LII of the MEC (A) WFS1+ neurons in an island (left) receive innervation from PV+ axons visualized by mCherry expression following AAV DIO ChR2-mCherry injection into the MEC of a PVCre mouse (middle). The merged picture shows immunostaining for VGAT, a marker of GABAergic terminals (right). Scale bar, 100 μm. (B) Confocal image of PV+ and VGAT+ axon terminals surrounding the soma of a WFS1+ neuron in LII. (C) Firing pattern (left) and representative traces (middle) of unitary IPSCs (uIPSCs, black) and unitary EPSCs (uEPSCs, red) recorded in FS interneurons and excitatory cells. uIPSCs were recorded at −50 mV, and uEPSCs at −70 mV in the respective postsynaptic neuron, and were elicited by a train of 10 action potentials (40 Hz) (train of 10 action potentials in red). The summary graph (right) shows the investigated connections between LII FS interneurons and excitatory cells. (D) MCherry expression in SOM+ interneurons in LII following AAV DIO ChR2-mCherry injection into the MEC of a SOMCre mouse (left). Quantification of mCherry+ interneurons that expressed SOM or PV in MEC LII of SOMCre mice (right). The numbers indicate analyzed mCherry+ neurons from two mice. (E) PV+ and SOM+ (labeled by mCherry following AAV DIO ChR2-mCherry injection into the MEC of a SOMCre mouse) interneurons in LII are localized preferentially around islands (left). mCherry expression reveals that fluorescently labeled axons of SOM+ interneurons are localized between islands and in LI (right). Scale bar, 100 μm. (F) Firing pattern (left) and representative traces (middle) of uIPSCs (black) and uEPSCs (red) recorded in SOM+ interneurons and excitatory cells. Connectivity was tested by eliciting a train of 10 action potentials in the presynaptic neuron (train of action potentials in red). The summary graph (right) shows the quantitative evaluation of connectivity between SOM+ interneurons and indicated excitatory cells. (G) PV (left), calretinin (middle), and SOM (right) immunostaining in MEC LII of a 5-HT3AEGFP mouse. The boxed double-labeled CR+/EGFP+ interneuron is shown at a higher magnification in the upper left corner. Scale bar, 100 μm. (H) Quantification of EGFP+ interneurons expressing PV, CR, or SOM in LII of the MEC in 5-HT3AEGFP mice. The numbers indicate analyzed EGFP+ neurons from two or three mice. (I) Firing pattern (left) and representative traces (middle) of uIPSCs (black) and uEPSCs (red) detected in 5-HT3AEGFP+ interneurons and excitatory cells. The summary graph (right) shows quantitative evaluation of investigated connections between indicated cell types. Data are represented as percentage of analyzed connections. The total number for the different cell pairs is indicated above the bars. Abbreviations are as follows: PC, pyramidal cell; SC, stellate cell; IM PC, intermediate pyramidal cell; IM SC, intermediate stellate cell; CR, calretinin; 5-HT3A, 5-HT3A receptor; 5HT3, 5-HT3A+ interneuron; L, layer; PV, parvalbumin; SOM, somatostatin; and VGAT, vesicular glutamate transporter. See also Figures S4 and S5.
Figure 4
Figure 4
LII CB+ Neurons Target Multiple Cell Types in LII of the Contralateral MEC (A) Retrogradely labeled LII and LIII neurons following CTB injection into the contralateral MEC (sagittal section). (B) Most retrogradely labeled neurons (blue) in LII were CB+ (red) and localized in CB+ islands (the confines of this island are indicated by a dashed line). The Inset is a higher magnification of the indicated area (white square) and shows a CTB+/CB+ neuron. Scale bar, 100 μm. (C) Confocal image of injection site in LII following AAV DIO-ChR2-mCherry injection into the MEC of CBCre mice. Scale bar, 100 μm. (D) Sagittal MEC section showing innervation of LII following AAV DIO-ChR2-mCherry injection into the contralateral MEC of CBCre mice. Scale bar, 100 μm. (E) Synaptic responses and firing pattern of a targeted SC and a FS interneuron. ChR2-mCherry-expressing axons were stimulated by 5-ms laser pulses (blue bar) and EPSCs were recorded. Excitatory and monosynaptic inputs were identified in the presence of the indicated antagonists. Abbreviations are as follows: L, layer; CTB, cholera toxin subunit B; MEC, medial entorhinal cortex; FS, fast-spiking interneuron; and SC, stellate cell. See also Figure S6 and Table S2.
Figure 5
Figure 5
LII Neurons Project to the MS (A) CTB+ neurons in MEC LII (red arrows) following tracer injection into the MS. Scale bar, 100 μm. (B) CTB+/CB+ neurons located in CB islands (left two panels) and CTB+/RE+ cells (right two panels) following CTB injection into the MS. Scale bars, 20 μm. (C) CB+ neurons (red) located in the same CB island can project to either the MS (green CTB labeling) or the contralateral MEC (blue CTB labeling). Boxed double-labeled neurons are shown below as a merged image and the single channel for green and blue CTB. Scale bar, 50 μm. (D) GAD67EGFP+ neuron in LII co-labeled with CTB following tracer injection into the MS (upper panel). The boxed double-labeled neuron is shown below as a merged image and the single channel for EGFP and blue CTB. Scale bar, 50 μm. Abbreviations are as follows: CB, calbindin; c, contralateral; CTB, cholera toxin subunit B; L, layer; MEC, medial entorhinal cortex; MS, medial septum; and RE, reelin.
Figure 6
Figure 6
Septal GABAergic Neurons Projecting to the MEC (A) FG-labeled neurons in the MS (left, upper panel) following tracer injection into the MEC. PV staining of the same section (left, lower panel). The overlay is shown at higher magnification (right panel). Scale bar, 100 μm. Higher magnification images below show the double-labeled neuron indicated by the arrow in the right panel. Scale bar, 20 μm. (B) FG-labeled neurons in the MS (left, upper panel) following tracer injection into the MEC. CB staining of the same section (left, lower panel). The overlay is shown at higher magnification (right panel). Scale bar, 100 μm. Higher magnification images below show the double-labeled neuron indicated by the arrow in the right panel. Scale bar, 20 μm. (C) Image of a FG+/CTB+/PV+ neuron in the MS following FG (blue) injection into the hippocampus and CTB (red) injection into the MEC. Scale bar, 20 μm. (D) Image of a FG+/CTB+/CB+ neuron in the MS following FG injection into the hippocampus and CTB injection into the MEC. Scale bar, 20 μm. Abbreviations are as follows: CB, calbindin; CTB, cholera toxin subunit B; FG, fluorogold; HC, hippocampus; MEC, medial entorhinal cortex; MS, medial septum; and PV, parvalbumin.
Figure 7
Figure 7
Septal PV+ Neurons Inhibit Preferentially FS Interneurons in LII of the MEC (A) Schematic drawing indicating the site of virus injection into the MS (top). MCherry expression following AAV DIO ChR2-mCherry injection into the MS of a PVCre mouse (coronal section; bottom). (B) ChR2-mCherry+ axons in LII of the dorsal (left) and intermediate (right) MEC (sagittal sections). Scale bar, 50 μm. (C) Responses of a targeted FS cell in MEC LII at the indicated potentials and in the presence of indicated antagonists. Blue bars show the duration of laser pulses. (D) Histogram indicating percentage of responding neurons (red) in LII. The numbers above the bars indicate the number of analyzed cells. (E) Representative firing pattern and reconstruction of a targeted FS (left) and a targeted non-FS GABAergic neuron (right). Dendrites are indicated in black and axons in red. Scale bar, 100 μm. (F) Stimulation of septal PV+ long-range projections reduced spiking in LII FS neurons. Responding cells were depolarized to suprathreshold potentials, and long-range axons were stimulated with 60-ms pulses at 8 Hz. Superimposed traces of a representative cell (left) and histogram (right) showing significant reduction of the firing rate during 60-ms pulses (p < 0.05 and ∗∗p < 0.01). Data represent mean ± SEM. Abbreviations are as follows: L, layer; MEC, medial entorhinal cortex; MS, medial septum; FS, fast-spiking interneuron; non-FS, non-fast-spiking interneuron; and PV, parvalbumin. See also Figures S7 and S8.

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