Figure 3

The Mossy cell identity is corroborated by reactivity to GluR2/3 antibodies. (A) Pattern of expression of GluR2 mRNA as revealed by in situ hybridization (http://mouse.brain-map.org)²¹, which resemble the expression of Shh-nlacZ and CR⁺ cells (bar 100 m) (mouse P57) (Bar = 100 µm). (B) Double fluorescent in situ hybridization for GAD1 and CR (http://mouse.brain-map.org)²¹ showing colocalization of the two mRNAs (CR/GAD-1 = 71.8 ± 5.2%; GAD-1/CR = 50.2 ± 2.4%,estimated from 2 mice, P56) (Bar = 45 µm). (C) IHC staining for GluR2/3 and Shh-nlacZ showing an orthogonal view to demonstrate colocalization (see Table 1, columm-7). (100X, Bar = 20 µm). For the cell numbers marked by GluR 2/3 and LacZ see Table 1. (D) Staining for GluR2/3 and GABA confirm the colocalization, revealing that mossy cells are immunoreactive to GABA. (20X, bar = 50 µm). (E) Zoom into D (100X) showing hilar neurons different channels confirming colocalization of GluR2/3 and GABA at high magnification (100X, bar = 20μm). (F) An additional double fluorescent IHC section that shows GABA and GluR2/3 colocalization, and high (orange arrow) and low (white arrow) levels of GABA staining in cell positive for both GluR2/3 and GABA or cells that are only immunoreactive to GABA (40X, bar = 50μm). (G) Analysis of the GABA and GluR2/3 co-expression show that a 76.5% of GABA⁺ cells colocalized with GluR2/3; while an 88.3% of GlutR2/3⁺ cells colocalized with GABA. (n = 3 mice × 10 slices). Abbr: GCL, granular cell layer; SGZ subgranular zone; and h, hilus.