Reelin Rescues Behavioral, Electrophysiological, and Molecular Metrics of a Chronic Stress Phenotype in a Similar Manner to Ketamine.

Over the past decade, ketamine, an NMDA receptor antagonist, has demonstrated fast-acting antidepressant effects previously unseen with monoaminergic-based therapeutics. Concerns regarding psychotomimetic effects limit the use of ketamine for certain patient populations. Reelin, an extracellular matrix glycoprotein, has shown promise as a putative fast-acting antidepressant in a model of chronic stress. However, research has not yet demonstrated the changes that occur rapidly after peripheral reelin administration. To address this key gap in knowledge, male Long-Evans rats underwent a chronic corticosterone (CORT; or vehicle) paradigm (40 mg/kg, 21 d). On day 21, rats were then administered an acute dose of ketamine (10 mg/kg, i.p.), reelin (3 µg, i.v.), or vehicle. Twenty-four hours after administration, rats underwent behavioral or in vivo electrophysiological testing before killing. Immunohistochemistry was used to confirm changes in hippocampal reelin immunoreactivity. Lastly, the hippocampus was microdissected from fresh tissue to ascertain whole cell and synaptic-specific changes in protein expression through Western blotting. Chronic corticosterone induced a chronic stress phenotype in the forced swim test and sucrose preference test (SPT). Both reelin and ketamine rescued immobility and swimming, however reelin alone rescued latency to immobility. In vivo electrophysiology revealed decreases in hippocampal long-term potentiation (LTP) after chronic stress which was increased significantly by both ketamine and reelin. Reelin immunoreactivity in the dentate gyrus paralleled the behavioral and electrophysiological findings, but no significant changes were observed in synaptic-level protein expression. This exploratory research supports the putative rapid-acting antidepressant effects of an acute dose of reelin across behavioral, electrophysiological, and molecular measures.

Unlocking the brain: A new method for Western blot protein detection from fixed brain tissue.

BACKGROUND: Aldehyde fixation is a common process used to preserve the complex structure of biological samples ex vivo. This method of fixation relies on the formation of covalent bonds between aldehydes and amines present in the biomolecules of the sample. Aldehyde fixation is routinely performed in histological studies, however fixed tissue samples are rarely used for non-histological purposes as the fixation process is thought to make brain tissue unsuitable for traditional proteomic analyses such as Western blot. Advances in antigen-retrieval procedures have allowed detectable levels of protein to be solubilized from formaldehyde fixed tissue, opening the door for aldehyde-fixed samples to be used in both histological and proteomic approaches. NEW METHOD: Here, we developed a series of antigen-retrieval steps for use on fixed-brain lysates to make them suitable for analysis by Western blot. RESULTS: Prolonged exposure of the tissue homogenate to high temperature (90 °C for 2 h) in the presence of a concentrated formaldehyde scavenger and ionic detergent was sufficient to reveal a variety of synaptic and non-synaptic proteins on membrane blots. CONCLUSION: This protocol has significant utility for future studies using fixed tissue samples in a variety of neuropathological conditions.