Fig. 6: Intranasal adeno-associated virus delivery of AAV9-MeCP2-GFP-mouse HTR2A-shRNA improves memory in rats.

The target sequence used to synthesize the shRNA is 100% conserved between mice and rats. To test whether shRNA-knockdown of the rat 5HT-2A receptor improves memory, Wistar rats (12 animals per group) were randomly assigned to two different groups consisting of vehicle- or AAV9-MeCP2-GFP-mHTR2A-shRNA. Following treatment on day 1, animals were assessed behaviorally 3- (A–D) and 5-weeks later (E–H). Details of the novel object recognition test can be found in the methods. At 3 weeks, there was no significance difference in the time spent or the number of physical contacts with the novel object during the acquisition (training) period (p > 0.05) (A, B). Rats were tested 24-h later, and AAV9-treated rats (blue bars) showed a significant increase in both the contact-recognition index (p < 0.000003) (C) and the time recognition-index (p < 0.0003) (D). The same groups of rats were retested at 5-weeks and again no significant difference was noted in the time spent or the number of physical contacts with the novel object during the acquisition (training) period (p > 0.05) (E, F). Twenty-four hours later there was a significant increase in the contact-recognition index (p < 0.01) (G), however, there was no significant difference in the contact-recognition index (p = 0.114) (H). I Data show the results of qPCR real-time assays to analyze mRNA levels of Htr2a following extraction of rat brain RNA. Results display relative mRNA levels after 5-weeks post-treatment with AAV9-HTR2A-shRNA. 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 = 0.038. J Representative, merged immunofluorescence image of vehicle-control animals depicting the presence of 5HT-2A receptor protein labeling (red fluorescence) within the olfactory bulb. K Representative, merged immunofluorescence image of AAV9-HTR2A-shRNA-treated animals. The blue staining reflects nuclear staining with DAPI. As expected, there was no expression of GFP in vehicle controls (J, top panel) while strong GFP labeling was observed in cell bodies of neurons of shRNA-treated rats (K, bottom panel). Images are representative of 3 separate mice for each group.