During this year, we have examined the interactions between the mu opioid and CCK- B receptors in acutely isolated living neurons obtained from brains of rat pups. The method begins with living slices of rat brain, which are dissociated by gently approaching the slice with a small vibrating glass probe. The beauty of this method is that very discrete regions can be dissociated. Thus for example, it is easy to confine the dissociation to say the pyramidal layer of the CA1 region in the rostral hippocampus. Another great advantage to the method is that, as no enzymes need be used, presynaptic fragments remain attached, and their presence is evident by spontaneous synaptic activity that can be recorded in the isolated neuron. We found that both CCK and normorphine slow the rate of this activity in hippocampal pyramidal neurons, and that CCK also diminishes the amplitudes. Therefore, both CCK and opioid receptors are active in this preparation. To detect a cell-level interaction of these receptors, however, they should both be expressed on the same cell. The hippocampus is of interest because of it's well known cellular architecture, the vast accumulation of knowledge about it, and its role in declarative memory and spacial mapping. The cell type of interest for our purpose of expressing both CCK and mu opioid receptors would then be an interneuron, and a CCK-type basket cell interneuron in particular. As only about 10% of hippocampal neurons are interneurons, the yield of candidate cells is low to begin with. Presynaptic fragments are unnecessary in this model, so the slices were treated with low concentrations of enzymes, which gave better yields of healthy cells. However, interneurons were very scarce, and usable ones even more scarce. Some data showed that CCK increased high voltage activated calcium currents, but the cells disintegrated before a test of normorphine could be made on the same one. Given the bad odds, I switched to periaqueductal gray (PAG) cells. The PAG is known to be involved in, among others, fear and defense reactions and in endogenous analgesia. In contrast to the hippocampus, PAG cells are mostly (80%) interneurons, and they are known to express both CCK and mu opioid receptors. However, using variables in enzyme (protease XIV or papain) dissociation, trituration vs. vibrating probe, employing variations in """"""""perforated patch"""""""" (beta escin or nystatin) technique to preserve the viability of recorded cells, using pyruvate or ascorbate to enhance the lifetime of dissociated cells, the yield of data was still very poor. In late July I began using a new, more stable micromanipulator, retiring the 20 year old hydraulic one. It is hoped that the enhanced mechanical stability will permit longer and more reliable recordings.
