Fig. 4: NRG1 expression in PV neurons is reduced by ketamine.
A–G Testing the effect of ketamine on NRG1 mRNA expression using TRAP. A A schematic illustration of the translating ribosome affinity purification strategy37. Using PV-Cre; fsTRAP mice, translating ribosomes (polysomes) from PV cells (green cells) have EGFP tags from the EGFP-L10 transgene. Lysis of all cells in the PV-Cre; fsTRAP cortex releases both tagged and non-tagged polysomes. The tagged polysomes are selectively captured on an anti-GFP affinity matrix and used for purification of PV-specific mRNAs associated with tagged polysomes. This strategy is also used to purify mRNA from Emx1+ neurons using Emx1-Cre; fsTRAP mice. Mice were treated with saline or ketamine at P56, then sacrificed 24, 48, 72 h or 1 week later for fsTRAP extraction and qPCR analysis. See “Methods” for more information. B Confocal images of genetically labeled PV cells (green), PV immunolabeling (red) and their overlay in layer I-VI of mouse mPFC in P56 PV-Cre; fsTRAP-EGFP mice; Scale bar = 100 µm. The white boxes indicate the region of mPFC imaged at higher magnification (B, right); Scale bar = 50 µm. C Confocal images of genetically labeled excitatory cells (green), GABA immunolabeling (red) and their overlay in layer I-VI of mouse mPFC in P56 Emx1-Cre; fsTRAP-EGFP mice. The white boxes indicate the region of mPFC imaged at higher magnification (C, right). Arrowheads indicate GABA immunopositive cells. D–G All results were analyzed using the ddCt method with gapdh as an endogenous control, along with normalization to Emx1+ P56 nrg1 expression, as indicated by a black mark on the y-axis, so that all panels (D–G) can be compared71. (D) Ketamine treatment (10 mg/kg; s.c.) significantly lowers NRG1 mRNA expression levels from PV neurons (PV-Cre; fsTRAP) in mPFC in P56 mice (n = 5–6 samples; each sample is pooled from 5 mice for PV cell-specific data) (One-way ANOVA: overall p = 0.0006. Two-sample t test (adjusted for multiple comparisons): ketamine vs saline, 24 h (1d) p = 0.0027, 48 h (2d) p = 0.0002; mean ± SEM). E PV cell-specific mRNA expression levels of ErbB4 from mice treated with saline or ketamine (One-way ANOVA: overall p = 0.0018. Two-sample t test (adjusted for multiple comparisons): ketamine vs saline, 1 week p = 4.55 × 10−4); mean ± SEM. F Emx1+ cell-specific (Emx1-Cre; fsTRAP) NRG1 mRNA expression levels from mPFC cortices (n = 4–5 samples; each sample is pooled from two mice for Emx1+ cell-specific data) is not significantly changed with ketamine treatment (10 mg/kg; s.c.). G Emx1+ cell-specific ErbB4 mRNA expression levels are not significantly changed with ketamine treatment (10 mg/kg; s.c.), and are much lower than in PV cells. H–J Testing the effect of ketamine on pCREB expression in excitatory neurons. H Confocal images of genetically labeled PV cells (red), pCREB immunolabeling (green) and their overlay in layer I-VI of mouse mPFC in P56 PV-Cre; Ai9 mice treated with saline; Scale bar = 100 µm. The white box indicates the region of mPFC digitally enlarged (H, right); Scale bar = 50 µm. White arrowheads indicate that PV neurons have very low pCREB immunoreactivity. I Confocal images of genetically labeled PV cells (red), pCREB immunolabeling (green) and their overlay in layer I-VI of mouse mPFC in P56 PV-Cre; Ai9 mice 48 h after ketamine treatment (10 mg/kg; s.c.). The white boxes indicate the region of mPFC digitally enlarged (I, right). White arrowheads indicate that PV neurons have very low pCREB immunoreactivity. J Quantification of the increase in pCREB immunoreactivity in mPFC 24 and 48 h after ketamine treatment. After 72 h to 1 week pCREB levels return to baseline (linear mixed effect model: overall p = 4.3 × 10−32, ketamine vs saline, 24 h p = 2.37 × 10−5, 48 h p = 1.51 × 10−23; mean ± SEM). Please see the “Methods” section for quantification and normalization of immunostaining intensity; the overall normalized values from different mice were compared across treatment groups (n = 6 for saline; n = 3–6 for ketamine groups).
