Figure 1.
GAD65 and GAD 67 expression in the E14.5 neocortical meninges.
(A) The images for the section stained with DAPI (blue), anti-GAD65 antibody (red) and anti-GAD67 antibody (green). The stable signals for both isoforms of GAD were found in the meningeal region just above the upper edge of the neocortical primordium (arrows). The developing neurons in the neocortex, in the marginal zone (MZ) and the subplate (SP) also exhibited the faint signals for GAD67 (arrowheads). (B) The images for the negative control section stained without anti-GAD65 (NC for GAD65) and anti-GAD67 (NC for GAD67). Me, meninge, MZ, marginal zone; CP, cortical plate; SP, subplate. Bar, 50 µm.
Figure 2.
GAD65/67 expression in the meninges and the presence of GABA beneath the meninges.
(A) Double immunolabeling for GAD65/67 (Green) and Laminin (Red) in the E14.5 neocortical primordium. The signals for GAD65/67 were present along the basal lamina which is laminin-positive (arrow). (B) Double immunostaining for GAD65/67 (green) and pan-Zic proteins (Red) in the E14.5 neocortex. Most of the cells expressing GAD65/67 also express Zic proteins, which are the transcriptional factors working as the markers of the meningeal fibroblasts (arrowheads), although, to be noted, a few GAD65/67-positve cells do not express Zic proteins (arrow). A few Cajal-Retzius cells in the marginal zone (MZ) express Zic proteins (asterisks). (C) Immunostaining for GABA in the upper layers of developing neocortex. GABA signals could be observed at the upper edge of the E14.5 developing neocortex just beneath the meninge (arrows). GABA-containing neurons in the marginal zone and in the subplate are also visualized (several representatives are indicated by arrowheads). The counterstaining by 7-amino-actinomycin D (7-AAD) helps to visualize the contours and the density of the cells in the developing cortical layers. (D) The E14.5 sections stained for GAD65/67 and Rdh10, an RA-synthesizing enzyme. RA is known to be released from the meningeal fibroblasts [11]. The signals for both proteins were present in the meninges (arrows). Me, meninge; MZ, marginal zone; CP, cortical plate; SP, subplate. Bars, 50 µm.
Figure 3.
GAD65/67-positive cells in the meninges cover the entire developing neocortex by E14.5.
(Upper panels) E12.5 developing neocortex was stained for GAD65/67 and developed with DAB. By E12.5, in the rostral region of the developing neocortex, GAD65/67-positive cells were present in the dorsal meninges, although the GAD65/67-positive cells did not cover the entire neocortex. In the middle region of the E12.5 neocortex, GAD65/67-expressing cells were present in the lateral region but not the dorsal region of the neocortex. In the mid-caudal region, the GAD65/67-expressing cells did not reach even the lateral end of the neocortex. Arrows in the upper panels indicate the dorsal ends of the distribution of GAD65/67-expressing cells in the meninges at each rostro-caudal position. (lower panels) In E14.5 sections stained for GAD65/67, the GAD65/67-expressing cells cover the entire neocortex at any position along rostro-caudal axis. Because boiling the sections in sodium citrate buffer is needed before staining with anti-GAD65/67 antibody, the sections were injured during the staining process. Bar, 5 mm.
Figure 4.
GAD 65 expression is apparent from the start of the development of the choroid plexus.
(A–C) The distribution of GAD65 and GAD67 in the choroid plexus on E12.5 (A), E13.5 (B) and E14.5 (C). The signals for GAD65 were observed in the epithelial cells of the choroid plexus throughout the stages examined, whilst few signals for GAD67 were detected in the developing choroid plexus. The signals for GAD65 were stable on E12.5, and the level of expression of GAD65 appeared to become higher as the development proceeds. (D) GABA distribution in the E14.5 choroid plexus. The numbers and the intensity of signals for GABA vary among the epithelial cells of choroid plexus. Some cells in the choroid plexus showed bright signals in its cytoplasm (arrows), while many cells in the epithelium of choroid plexus exhibited faint signals for GABA. CPe, choroid plexus epithelium: CPm, choroid plexus mesenchyme: Bars, 50 µm.
Figure 5.
Bestropin-1 (Best1), a GABA-permeable channel, is expressed in the meninges and the choroid plexus.
(A) The faint signals for Best1 were ubiquitously detected in the cells in the E14.5 neocortical structures including GAD65/67-positive meninges. (B) The cells in the choroid plexus epithelium exhibited rather strong signals for Best1 on E14.5. Me, meninge; MZ, marginal zone; CP, cortical plate; SP, subplate, CPe, choroid plexus epithelium: CPm, choroid plexus mesenchyme: Bars, 50 µm.