GENE DELIVERY OF IMMEDIATE EARLY GENE, ARC, VIA RECOMBINANT ADENOASSOCIATED VIRAL VECTORS IMPROVES IMPAIRED EXPLORATORY BEHAVIOR IN RATS REARED IN ISOLATION AFTER EARLY-LIFE SEIZURES

Objectives: We have previously found that complex social and sensory-motor stimulations, provided by exposing young animals (postnatal day (P) 21) to an enriched environment for 7–14 days after kainic acid (KA)-induced seizures effectively reverse decrease in exploratory behavior. Correlated with the behavioral change, genes involved in synaptic plasticity, memory consolidation and cell proliferation such as Arc, Homer1a, Egr1, Egr2, Egr4, and JunB are significantly increased by environmental enrichment. Here we treat P22 rats 24h after KA or saline with rAAV-Arc to determine whether behavioral deficits can be reversed by delivery of Arc to the hippocampus.

Methods: The entire coding sequence of Arc (2.76Kb) was amplified from a rat brain cDNA pool using PCR, followed by cloning into the mammalian expression vector, pcDNA3.1 (+). Western blot confirmed intact Arc protein expression. The rAAV vector construct containing cloned cDNA was stereotactically injected into hilus of the hippocampus of anesthetized rats. rAAV-GFP injected rats served as sham controls. Four groups (n=4–5): saline-GFP, saline-Arc, KA-GFP and KAArc, received intrahippocampal injection on P22, one day after KA or saline injection. Two groups, isolated and enriched enrichment were added as littermate controls. Exploratory behavior in open field was quantified at 7, 14 and 21 days.

Results: Three weeks after gene therapy, but not at one or two weeks, significant group difference in exploratory behavior was noted (p<0.007, ANOVA). KA-Arc explored significantly more than KA-GFP (95.2±11.3 vs. 65.0±3.1, p<0.04) while trends toward improved behavior was observed in Controls (Cont Arc, 78±13.2 vs. combined Cont GFP+isolated, 47.5±7.6, p<0.07).

Conclusion: Intrahippocampal injection of rAAV-Arc increased exploratory behavior in young rats after KA-induced seizures in three weeks, likely at the peak of transgene protein expression. Our results support the hypothesis that Arc plays a causative role in behavioral improvement.