Verification of AMPK Knockdown in Sprague Dawley Rats using a Vivo-Morpholino Antisense Oligonucleotide

Abstract

Metformin, a drug typically used to treat type II diabetes, can decrease cue-induced cocaine seeking in rats. The proposed mechanism of action for this effect is the activation of adenosine monophosphate-activated protein kinase (AMPK). To confirm this mechanism for the effect of metformin we are validating a technique for knocking down AMPK in the nucleus accumbens core (NAcC) in vivo. This study tests whether microinjections of a vivo-morpholino antisense oligonucleotide (VM-ASO) in the NAcC is an effective method of knocking down AMPK in Sprague Dawley rats. VM-ASO binds target mRNA and prevents translation. This experiment used 8 Sprague Dawley rats (6 male, 2 female). Initially, rats underwent stereotaxic intracranial surgery for the insertion of a cannula into both hemispheres of the NAcC (from bregma: AP +1.5; ML: +/- 1.8; DV: - 5.5mm). Then, 140 pmol of AMPK VM-ASO or scrambled control nucleotide was microinjected in each hemisphere at a rate of 0.2 µl/minute 2mm below the base of the cannula. After a one-minute diffusion period, the microinjectors were removed. This process was repeated for four days. Eight days after the last microinjection, tissue samples were collected and AMPK levels were analyzed via western blot. It was found that microinjections of AMPK VM-ASO did not significantly decrease AMPK levels in the NAcC.