Volume 14, Issue 1, Pages 63-68 (July 2006) Adeno-associated Virus-Mediated Expression and Constitutive Secretion of Galanin Suppresses Limbic Seizure Activity in Vivo  Thomas J. McCown  Molecular Therapy  Volume 14, Issue 1, Pages 63-68 (July 2006) DOI: 10.1016/j.ymthe.2006.04.004 Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 1 The effects of AAV-FIB-GFP and AAV-FIB-GAL vectors on the expression of limbic seizure behaviors. Seven days after infusion of AAV-FIB-GFP (N = 8) into the piriform cortex, kainic acid administration (10 mg/kg, ip) elicited wet dog shakes, class III and class IV seizure activity, with the same latency as in untreated controls (N = 8) (t test, P > 0.1). Seven days after infusion of AAV-FIB-GAL (N = 12) into the piriform cortex, kainic acid administration elicited wet dog shake behaviors with the same latency as in untreated control animals. In contrast, a single, brief class III seizure was exhibited by only 1 of the 12 rats over the 240-min post-kainic acid observation period, while the other 11 rats did not exhibit any limbic seizure behaviors. None of the animals exhibited any class IV seizure behaviors over the entire 240-min post-kainic acid observation period (*t test, P < 0.01) (mean ± SEM). Molecular Therapy 2006 14, 63-68DOI: (10.1016/j.ymthe.2006.04.004) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 2 The in vivo presence of FIB-GAL mRNA 1 week after AAV-FIB-GAL infusion into the piriform cortex. AAV-FIB-GAL vectors were infused into the rat piriform cortex and 1 week later the mRNA was extracted. Subsequent RT-PCR shows that the appropriate 155-bp FIB-GAL product is present in the injected piriform cortex (lane A), while no product was found in the contralateral, uninjected cortex (lane B). Omission of the RT step (lane C) indicated the absence of contaminating viral DNA. The two outside lanes contain a 50 bp DNA step ladder with the base pair size indicated on the right. Molecular Therapy 2006 14, 63-68DOI: (10.1016/j.ymthe.2006.04.004) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 3 Illustrations of AAV vector infusion sites and expression of GFP in the piriform cortex. (A) The range of AAV-FIB-GFP and AAV-FIB-GAL infusion sites in the piriform cortex is shown (diagrams adapted from Paxinos and Watson [19]). (B) Typical GFP expression in the piriform cortex prior to treatment with kainic acid. This level of GFP expression coursed approximately 250 μm in a rostral–caudal direction. (C) The level of GFP expression 2 weeks after kainic acid administration in the only animal of eight total in which GFP expression was evident. The other seven animals exhibited no discernable GFP expression. Horizontal bars, 40 μm. Molecular Therapy 2006 14, 63-68DOI: (10.1016/j.ymthe.2006.04.004) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 4 EEG recordings from the piriform cortex or the hippocampus following kainic acid administration. (A) Afterdischarge activity in the piriform cortex of an untreated control animal during a class IV seizure. (B) Afterdischarge activity in the piriform cortex of an AAV-FIG-GFP-treated animal during a class IV seizure, further reinforcing the lack of effect exerted by GFP expression and constitutive secretion. (C) The total lack of any afterdischarge activity in the piriform cortex of an animal that received an AAV-FIB-GAL infusion 7 days prior to the kainic acid treatment (10 mg/kg, ip.. At no time during the 240-min observation period was any behavioral or electrographic seizure activity noted. (D) EEG recording from the hippocampus of an animal that received an AAV-FIB-GAL infusion 7 days prior to the kainic acid treatment. During this recording the animal was motionless, yet the recording shows clear afterdischarge activity. Periods of this electrographic seizure activity occurred over the entire 240-min observation period, but at no time were any limbic seizure behaviors noted. Molecular Therapy 2006 14, 63-68DOI: (10.1016/j.ymthe.2006.04.004) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 5 The effects of AAV-FIB-GAL (N = 9) or AAV-FIB-GFP (N = 5) infusion on the threshold for seizure genesis in animals that had been electrically kindled to class V seizure activity. As seen on the left, AAV-FIB-GFP infusion did not alter the stimulation threshold for seizure genesis, but AAV-FIB-GAL infusion significantly elevated the amount of current necessary to elicit seizure activity (paired t test, P < 0.02) (mean ± SEM). The right shows GFP expression around the electrode tip, demarcated by *. Horizontal bar, 40 μm. Molecular Therapy 2006 14, 63-68DOI: (10.1016/j.ymthe.2006.04.004) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions