Acute dissociation for analyses of NMDA receptor function in cortical neurons during aging

The present study was designed to determine if functional age differences in the NMDA epsilon2 (NR2B) subunit were detectable at the level of individual cortical neurons. Neurons were acutely dissociated from the frontal and prefrontal cortices of young adult, middle-aged, or old mice, using a combination of proteinase K and trypsin followed by manual trituration. After overnight culture, patch-clamp electrophysiology and rapid perfusion were used to obtain whole-cell responses to 300 μM NMDA, with or without the potent NR2B antagonist ifenprodil. Healthy, phase-bright cortical neurons were isolated from animals of all ages. Cell diameter and capacitance was consistent between ages. We were able to perform kinetic analyses of the NMDA-evoked response, and demonstrated a significant increase in the rate of deactivation with increasing age. In addition, we observed a significant effect of high-concentration ifenprodil on the NMDA-evoked response in old animals. Thus, this method is ideal for the dissociation of neurons from the brain of both young and old animals, and offers a powerful tool for functional analysis at the level of the individual cell.