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. 2021 Mar 26;10:e67262. doi: 10.7554/eLife.67262

Figure 1. Lateral entorhinal cortex (LEC) and medial entorhinal cortex (MEC) layer Vb (LVb) neurons show distinct morphological features.

(A–C) Expression of tetracycline-controlled transactivator (tTA) in the enhancer-driven gene expression (EDGE) mouse line (MEC-13-53D), which is visualized with mCherry (green) by crossing to a tTA-dependent mCherry line. A horizontal section was immunostained with an anti-Purkinje cell protein 4 (PCP4) antibody (magenta) to label entorhinal LVb neurons. Images of LEC (B) and MEC (C) correspond with the boxed areas in (A) and show from left to right PCP4 expression, mCherry expression, and a merged image. (D) Percentage of tTA-expressing neurons among layers in LEC and MEC. (E) Percentage of tTA-expressing neurons among the total PCP4-positive neurons in LEC and MEC. (F) Percentage of PCP4-positive neurons among the total tTA-expressing neurons in LEC and MEC. Error bars: mean ± standard errors. The tTA-expressing neurons mainly distributed in LVb of EC and colocalized with PCP4. (G–I) Morphology of LVb neurons targeted in MEC-13-53D in MEC (G, H) and LEC (I). tTA-expressing LVb neurons were first labeled with green fluorescent protein (GFP) (green) by injecting AAV2/1-TRE-Tight-EGFP in MEC-13-53D, and then intracellularly filled with biocytin (magenta, G) Images of MEC (H) and LEC (I) show biocytin labeling, which correspond with the boxed area in each inset. The four neurons shown in (G) correspond to the neurons in (H). Double arrowheads show the cell bodies, the single arrowheads show their dendrites, and different neurons are marked in different colors (green, blue, red, and yellow). The distribution of apical dendrites largely differs between MEC-LVb and LEC-LVb neurons. (J) Proportion of morphologically identified cell types of LVb neurons in LEC and MEC. These data were obtained in 10 animals and 22 slices. Scale bars represent 500 μm for (A) and inset of (H) and (I), 100 μm for (H) and (I), 50 μm for (B) and (C), and 20 μm for (G). Figure 1—source data 1. See also Figure 1—figure supplement 1, Figure 1—figure supplement 2, Figure 1—figure supplement 3, and Figure 1—figure supplement 4. 

Figure 1—source data 1. Specificity of tetracycline-controlled transactivator expression in MEC-13-53D.

Figure 1.

Figure 1—figure supplement 1. Laminar organization of lateral entorhinal cortex (LEC) and medial entorhinal cortex (MEC).

Figure 1—figure supplement 1.

(A) Horizontal sections showing Purkinje cell protein 4 (PCP4) immunolabeling (magenta) and retrograde labeling with tdTomato (green) at different dorsoventral levels, following injection of Cre-expressing retrograde adeno-associated virus (AAV) (AAVrg-pmSyn1-EBFP-cre) into nucleus accumbens (NAc) of Gt(ROSA)26Sortm9(CAG-tdTomato)Hze mice. The injection site is shown by a double arrowhead in the inset. Note that PCP4-positive neurons are localized in layer Vb (LVb), whereas NAc-projecting neurons distribute in layer Va (LVa) of both MEC and LEC. The thickness of EC LVa/LVb varies along the dorsoventral and mediolateral axis, and between LEC and MEC. LVa is especially thin in the medial and dorsal portion of MEC, whereas LVb is relatively thick. LVa gets thicker in lateral and ventral portions, and in the LEC the thickness of LVa and LVb becomes similar. (B, C) Distribution of NeuN-, tdTomato-, and PCP4-positive neurons in MEC (B) and LEC (C) corresponding with the boxed area in (A). White arrowheads show the gaps between the tdTomato-positive retrogradely labeled MEC-LVa neurons, which are also shown in (A) and (D). Note that in MEC these gaps do contain PCP4-positive neurons, as well as PCP4-positive neuropil likely representing apical dendrites of LVb neurons. This patchy pattern of alternating somata of neurons and neuropil was never observed in LVa of LEC. (D, E) Distribution of PCP4-positive neurons and retrogradely labeled neurons in MEC following AAVrg-pmSyn1-EBFP-cre injection into retrosplenial cortex (RSC) of Gt(ROSA)26Sortm9(CAG-tdTomato)Hze mice. Double arrowhead in the inset indicates the injection site. Scale bars represent 1000 μm for insets in (A) and (D), 500 μm for EC images in (A) and (D), and 100 μm for (B), (C), and (E).
Figure 1—figure supplement 2. Purkinje cell protein 4 (PCP4) but not chicken ovalbumin upstream promoter transcription factor interacting protein 2 (Ctip2) is expressed mainly in excitatory layer Vb (LVb) neurons in both lateral entorhinal cortex (LEC) and medial entorhinal cortex (MEC).

Figure 1—figure supplement 2.

(A, B) Horizontal section showing Ctip2 (blue) and PCP4 immunolabeling (magenta). (C, D) Distribution of Ctip2- (blue), PCP4- (magenta), and GAD67-GFP-positive neurons (green) in LEC (C) and MEC (D) corresponding with the boxed areas in (B). White arrowheads point to Ctip2+/PCP4-/GAD67- neurons, while yellow arrowheads point to Ctip2+/PCP4-/GAD67+ neurons. (E) In both LEC and MEC, the percentage of GAD67-GFP-positive neurons double labeled for Ctip2 (Ctip2+/GAD67+; blue) was significantly higher than the percentage double labeled for PCP4 (PCP4+/GAD67+; magenta) (error bars: mean ± standard errors, N = 5, two-tailed paired t-test for LEC Ctip2 vs. PCP4: t3 = 15.34, ***p=0.0006, MEC Ctip2 vs. PCP4: t3 = 13.74, ***p=0.0008). Scale bars represent 1000 μm for insets in (A), 500 μm for (B), and 50 μm for (C) and (D). Figure 1—figure supplement 2—source data 1.
Figure 1—figure supplement 2—source data 1. Specificity of Purkinje cell protein 4 and chicken ovalbumin upstream promoter transcription factor interacting protein 2 expression in entorhinal layer Vb neurons.
Figure 1—figure supplement 3. Dendrites of lateral entorhinal cortex-layer Vb (LEC-LVb) neurons do not reach layer IIa and I.

Figure 1—figure supplement 3.

Horizontal sections showing the distribution of green fluorescent protein (GFP)-labeled LEC-LVb neurons at different dorsoventral levels, following injection of AAV2/1-TRE-Tight-GFP into the deep layers of LEC. Neurites of labeled LEC-LVb neurons are visualized with anti-GFP staining shown in magenta. Cell bodies of these infected cells can be seen in white resulting from the overlap of GFP (green) and anti-GFP labeling (magenta). Scale bars represent 500 μm for (A) (also apply to B and C) and 200 μm for (A’) (also apply to B’ and C’).
Figure 1—figure supplement 4. Medial entorhinal cortex (MEC) and lateral entorhinal cortex-layer Va (LEC-LVa) neurons share similar morphological features.

Figure 1—figure supplement 4.

(A) LVa neurons were labeled in C57BL/6N mice by injecting Cre-expressing retrograde adeno-associated virus (AAV) (AAVrg-pmSyn1-EBFP-cre) in nucleus accumbens (NAc) while injecting AAV-CMV-FLEX-mCherry in entorhinal cortex (EC). (B–E) Horizontal sections showing labeled LVa neurons in MEC (C) and LEC (E), corresponding with the boxed area in (B) and (D), respectively. In both regions, the apical dendrites of LVa neurons reach layer I. Labeled neurons in the hippocampus (B, D) are NAc-projecting subicular neurons that were also infected due to the spread of AAV-CMV-FLEX-mCherry from the injection site. Scale bars represent 500 μm for (B) (also apply to D) and 100 μm for (C) (also apply to E).