|
We perform experiments on brain tissue obtained both from behaviorally characterized rhesus monkeys (as part of an integrated program project) and from rats. While we have experience in recording from many brain areas (hippocampus, brainstem areas, diverse neocortical areas), our research is currently focused on the prefrontal cortex - a brain area essential for higher cognitive function. The prefrontal cortex is removed from the experimental subject's brain as a block and then cut into 400 micron thick living slices which are maintained in oxygenated artificial cerebrospinal fluid for up to 15 hours.
Using infrared differential interference contrast microscopy, we visualize living neurons in the in vitro slices and use whole-cell patch-clamp methodologies to record the electrophysiological and pharmacological response properties of identified neurons. We employ current-clamp techniques to examine action potential firing properties and intrinsic properties (such as resting membrane potential, input resistance and membrane time constant), and voltage-clamp techniques to examine ionic currents, responses to pharmacologic agents (primarily neurotransmitter agonists) and synaptic response properties. In addition, we provide single cells (from which recordings are obtained) to Dr. Carmela Abraham's laboratory for single cell PCR and microarray analyses, and filled neurons to the Peters laboratory for electron microscopy.
At the same time that recordings are obtained, we fill the neurons with intracellular dyes such as biocytin or Lucifer yellow. In addition, we fill neurons in fixed slices obtained from the same subjects. Dye-filled neurons are then manually reconstructed using the 3-D morphometry software Neurolucida. Detailed analyses of dendritic architecture are then undertaken. For quantification of dendritic spines (which is done by our collaborators at the Mount Sinai School of Medicine) cells are visualized by confocal and/or multiphoton laser scanning microscopy.
|
