Table 1.
Fig 1.
Fezf2 was expressed in layer V/VI neurons of the mouse insular cortex.
Sections were numbered in rostral-caudal order. (a1)–(a8); Nissl staining. (b1)–(b8); adjacent tissue sections of (a1)–(a8). Fezf2 was expressed widely in rostral-caudal axis in layers V/VI. Fezf2 was visualized by immunohistochemistry against RFP in Tg(Fezf2-tdTomato) mice. Dotted lines indicate the borders of GI, DI and AI. Scale bar: 200 μm.
Fig 2.
Fezf2-positive neuron exhibiting fork cell-like shape, having two apical dendrites in layer V neurons of the mouse insular cortex.
A (a), (b), are images taken from the same section Fig 1A4, and (c), (d), (e), (f) are images taken from the same section Fig 1B4. Fezf2 was expressed in layer Ⅴ and Ⅵ with most neurons being densely accumulated in layer V (GI and DI). (a), (b); Nissl staining. (c)—(f); Fezf2. (b), (d) are the higher magnification images of the squares in panels (a), (c), respectively. (e) is the higher magnification image of the square in panel (d). (f) is the enlarged image including the rounded rectangle in panel (e). A Fezf2-positive neuron exhibiting fork cell-like shape, having two apical dendrites, is indicated by an arrow. No VENs-like shaped (bipolar) cells were identified. Scale bars (a), (c): 500 μm, (b), (d): 200 μm, (e): 100 μm, (f): 50 μm.
Fig 3.
A few Fezf2-positive neurons in the mouse insular cortex exhibited fork cell-like shape by immunofluorescence staining.
Immunofluorescence against RFP was performed to visualize Fezf2 signals with frozen brain sections of Tg(Fezf2-tdTomato) mouse. (a)—(c) Fezf2 was expressed in layer Ⅴ and Ⅵ. (b) High magnification image of the red squared box in (a). (d) A fork cell-like neuron indicated by the red arrow in (b), which had two apical dendrites. (e) A fork cell-like neuron indicated by the red arrow in (c). (f) A typical pyramidal neuron in (c). No VEN-like shaped (bipolar) cells were found in the mouse insular cortex. (g) A depicted image of neurons in (d) and (e). Scale bars (a), (c): 200 μm, (b): 100 μm, (d)—(g) 20 μm.
Fig 4.
The ultrastructural analyses of fork cell-like neurons and holding neurons by immunoelectron microscopy.
IHC against RFP was performed to visualize Fezf2 signals with a brain section of Tg(Fezf2-tdTomato) mouse. A. A few Fezf2-positive neurons exhibited fork cell-like neurons that has two apical dendrites and a basal dendrite. (a) Fezf2 was expressed in layer Ⅴ and Ⅵ. (b) High magnification image of the black squared box in (a). (c) High magnification image of the black squared box in (b). A fork cell-like neuron is indicated by the black dotted squared box. (d) Electron microscopy image of the black dotted squared box in (c). The ultrastructure of a fork cell-like neuron, which had two apical dendrites and a basal dendrite were observed in the mouse insular cortex. Scale bars (a): 500 μm, (b): 200 μm, (c): 50 μm, (d) 2 μm. B. A few Fezf2-positive neurons in the mouse insular cortex exhibited enveloping cell-like shape. (a) Fezf2 was expressed in layer Ⅴ and Ⅵ (the black squared box). (b) High magnification image of the black squared box in (a). (c) Electron microscopy image of the black squared box in (b). A holding neuron (see text for further definition), which had two apical dendrites were found in the mouse insular cortex. A cell which was embraced by a holding neuron had weak electron density. (d) High magnification image of (c). The nuclear envelope, having an irregular contour is indicated by arrows. Scale bars (a): 500 μm, (b): 50 μm, (c): 2 μm, (d) 1 μm.
Fig 5.
GRP and NMB were expressed differently in the mouse insular cortex.
A-C. Distribution of GRP or NMB in the mouse insular cortex. Sections were numbered in rostral-caudal order. (a1)—(a10) are Nissl-stained adjacent tissue sections of (b1)—(b10) and (c1)—(c10). Gray arrowheads indicate GRP-expressing neurons. Red brown arrowheads indicate NMB-expressing neurons. Scale bar: 200 μm.
Fig 6.
GRP was expressed widely in layer Ⅱ/Ⅲ and layer Ⅴ, with the highest expression in AI of the mouse insular cortex.
(a), (b). High-magnification image of the squared box in Fig 5Ba4 and 5Bb4. (b). GRP was expressed widely in layer Ⅱ/Ⅲ and layer Ⅴ. (a) and (b) are adjacent sections. Scale bar: 100 μm.
Fig 7.
NMB was expressed in the deep layers, layer Ⅴ and layer Ⅵ (GI, DI, and AI), with the highest expression in layer VI neurons of the mouse insular cortex.
NMB expression was scattered in layer Ⅴ and Ⅵ, with the highest expression in layer VI. (d) and (e) are the higher magnification images of the rounded square boxes in (c) and (d). A square in Nissl stained (a) is shown as (b). (b) and (c) are adjacent sections. Scale bars (a): 500 μm, (b), (c): 200 μm, (d) 100 μm, (e): 50 μm.