Select expression of H1aV in adult dorsal hippocampus impairs spatial working memory
Selective lesions of the hippocampus (e.g., Reisel et al., 2002) and dorsomedial prefrontal cortex (Kellendonk et al., 2006) impair spatial working memory in the mouse. Expression of H1aV in TgH1aV.Fb mice occurs in both structures. To question the role of hippocampal H1aV expression for acquisition of spatial working memory in isolation from H1aV expression in prefrontal cortex, we stereotaxically delivered rAAV expressing H1aV under the human synapsin core promoter bilaterally in the dorsal hippocampus of 16 week-old male wild-type mice. Recombinant pseudo-typed rAAV1/2 virus with preferred neuronal tropism (Burger et al., 2004; Kaplitt et al., 1994; Klugmann et al., 2005; Peel et al., 1997; Xu et al., 2001) was used for the injections. Gene expression in the hippocampus mediated by these rAAV subtypes requires 3 weeks to reach peak and persists stably for ∼18 months without overt inflammation or immunogenicity (Mastakov et al., 2002; Richichi et al., 2004; Xu et al., 2001). Efficacy and spread of viral gene transfer was confirmed by immunocytochemistry on serial coronal brain sections upon completion of the behavioral experiments (Figure 6A–D). Histological analysis of the rAAV-injected mice 6 weeks after infusion revealed no gross abnormalities in the brain (data not shown). No GFP signal was detectable in control hippocampi having received empty rAAV (rAAV-empty, Figure 6D). We detected robust H1aV expression from at least 1 mm on either side of the injection point and covering the entire dorsal aspect of the hippocampus (Figure 6A–C). GFP fluorescence, as revealed by confocal microscopy, was found in neuronal cell bodies and dendrites in both hippocampi in the CA1-3 fields and the dentate gyrus of injected but not control mice (Figure 6A–D, left and right panels). Counterstaining with NeuN and counting double positive cells from representative regions showed transgene expression in ∼80% of principal hippocampal CA1 neurons (N = 600, ∼480 double positive cells), as shown for representative sections from rAAV-H1aV-injected mice (Figure 6A–C, right panels). The subcellular distribution of H1aV in the hippocampus was reminiscent of that in TgH1aV.Fb mice (see Figure 1C, inset).
Figure 6  rAAV1/2-mediated H1aV expression in the hippocampus. (A–D, left and middle panels) Alexa Fluor 488 or DAB enhanced anti-GFP immunohistochemistry for the H1aV fusion-protein on representative coronal sections approximately −1 and +1 mm from the injection site, 8 weeks after bilateral injection of rAAV1/2-H1aV/-empty. (A–D, left panels) Z-projections of the GFP-enhanced H1aV expressing green cells/neurites (oriens layer, stratum radiatum, and molecular layer) on NeuN positive Cy-3 fluorescent neurons reveal transgene expression almost exclusively in the hippocampus. rAAV-empty injected brains show no GFP fluorescence antibody enhancement. (A–C, middle panels) DAB stainings of adjacent sections demonstrating robust labeling of pyramidal cell bodies in all subregions of the dorsal hippocampus. (D, middle panel) No DAB positive cells were detected in hippocampi of rAAV-empty injected mice. (A–C, right panels) High-magnification confocal dual fluorescent images (H1aV in green, NeuN in red) of representative regions from the CA1 pyramidal cell layer (marked by white boxes in A–D left panels) showing ∼80% double labeling over the total extent of the dorsal hippocampus. Note that the extent of transgene expression in the hippocampus of rAAV-infected cells is comparable with the distribution of H1aV in TgH1aV.Fb mice (Figure 1C inset). (D, right panel) No GFP fluorescence was detected in rAAV-empty infected neurons. αNeuN, anti-NeuN staining; αGFP, anti-GFP staining. Selective expression of H1aV in dorsal hippocampus impaired acquisition of working memory to a similar extent as that seen in TgH1aV.Fb mice. At the start of training (6 weeks postinfection), control (rAAV-empty, N = 6) and H1aV expressing (rAAV-H1aV, N = 7) mice were comparable in their success rates (block #1; rAAV-empty, 67 ± 6.1% vs. rAAV-H1aV, 70 ± 10.5%; unpaired T-test, P > 0.8; Figure 4E). Control mice learned the task and reached a success rate of 92 ± 6.2% on the fifth day of training (paired T-test, P < 0.05, block#1 vs. block#5). rAAV-H1aV injected mice, however, failed to improve their performance significantly (Block#1 vs. Block#5, 70 ± 10.5% vs. 80 ± 5.4%; paired T-test; P > 0.35), indicating that H1aV overexpression in the hippocampus could mediate the working memory deficit in TgH1aV.Fb mice.