The elucidation of the molecular mechanisms of long-Term synaptic plasticity has been hindered by both the compensation that can occur after chronic loss of the core plasticity molecules and by ex vivo conditions that may not reproduce in vivo plasticity. Here we describe a novel method to rapidly suppress gene expression by antisense oligodeoxynucleotides (ODNs) applied to rodent brain slices in an "Oslo-Type" interface chamber. The method has three advantageous features: 1) rapid blockade of new synthesis of the targeted proteins that avoids genetic compensation, 2) efficient oxygenation of the brain slice, which is critical for reproducing in vivo conditions of long-Term synaptic plasticity, and 3) a recirculation system that uses only small volumes of bath solution (5 ml), reducing the amount of reagents required for long-Term experiments lasting many hours. The method employs a custom-made recirculation system involving piezoelectric micropumps and was first used for the acute translational blockade of protein kinase M (PKM) synthesis during long-Term potentiation (LTP) by Tsokas et al., 2016. In that study, applying antisense-ODN rapidly prevents the synthesis of PKM and blocks late-LTP without inducing the compensation by other protein kinase C (PKC) isoforms that occurs in PKC/PKM knockout mice. In addition, we show that in a low-oxygenation submersion-Type chamber, applications of the atypical PKC inhibitor, zeta inhibitory peptide (ZIP), can result in unstable baseline synaptic transmission, but in the high-oxygenation, "Oslo-Type" interface electrophysiology chamber, the drug reverses late-LTP without affecting baseline synaptic transmission. This comparison reveals that the interface chamber, but not the submersion chamber, reproduces the effects of ZIP in vivo. Therefore, the protocol combines the ability to acutely block new synthesis of specific proteins for the study of long-Term synaptic plasticity, while maintaining properties of synaptic transmission that reproduce in vivo conditions relevant for long-Term memory.
- Long-Term Potentiation
- Piezoelectric micropump
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Immunology and Microbiology(all)
- Plant Science