Fig. 4: Intranasal adeno-associated virus delivery of AAV9-MeCP2-GFP-mouse HTR2A-shRNA leads to a decrease in 5HT-2A receptor protein expression.

Following behavioral analysis, a subset of brains from these mice were fixed, and 4 µm paraffin-embedded sagittal tissue sections were stained with a specific antibody against GFP (green fluorescence, 1:1000), anti-5HT2A receptor (red fluorescence, 1:500), or Hoechst nuclear labeling (blue fluorescence). Whole slide imaging was performed using a Pannoramic Midi II scanner (see “Materials and methods” for details). A Representative, low-field immunofluorescence sagittal image following treatment with MeCP2-GFP-mouse HTR2A-shRNA identified neuronal labeling in most brain regions including the olfactory bulb (OB), cortex, cerebellum, and numerous sub-cortical areas including the interpeduncular nucleus (IPN), a major connectome for stress-mediated pathways. Three different brain regions were examined at higher magnification including the cerebellum (B–G), IPN (H–M), and olfactory bulb (N–S). Data are representative images from either vehicle controls in each brain region (B–D, H–J, or N–P) and AAV-treated mice (E–G, K–M, or Q–S). Treatment with MeCP2-GFP-mouse HTR2A-shRNA led to a general pattern of less robust staining profile of the 5HT2A receptor in cell body regions and apical dendrites (merged image Panel G as an example). All scale bars represent 50 µm. T–V Quantitative analysis using ImageJ software indicated a significant decrease in 5HT-2A receptor fluorescence intensity of AAV-treated mice (red bar) versus vehicle-controls (green bar) in both total brain sections (T) and olfactory bulb (U). Although there was a trend for a decrease in 5HT-2A receptor fluorescence intensity in the cerebellum, data did not reach significance. Data represent the mean gray value ± SD in three different cases. *Denotes significant difference, p-value < 0.05. W, X Data show the results of qPCR real-time assays to analyze mRNA levels of Htr2a following extraction of total brain RNA from frozen brain (W) or olfactory tissue (X) in either vehicle-controls (black bars) or shRNA treated (red bars). Results display relative mRNA levels after 8-weeks of treatment with AAV9-MeCP2-GFP-mouse HTR2A as compared to vehicle-controls. Real-time PCR results represent a total of N = 5 animals for each group performed in triplicate ±SEM. *Denotes significant difference between the two groups, p = 0.006 in olfactory mRNA.