Interactions of cholecystokinin and mu opioid receptors

在这一年里，我们研究了从幼鼠大脑中急性分离的活神经元中Mu阿片类药物和CCK-B受体之间的相互作用。该方法首先从大鼠大脑的活切片开始，通过用一个小的振动玻璃探头轻轻接近切片来分离这些切片。这种方法的优点在于，非常离散的区域可以被分离。因此，例如，很容易将分离限制在吻侧海马区CA1区的锥体层。这种方法的另一个巨大优点是，由于不需要使用酶，突触前片段仍然附着，它们的存在通过可以在分离的神经元中记录到的自发突触活动来明显。我们发现，CCK和去甲吗啡都减缓了海马锥体神经元这种活动的速度，CCK也降低了幅度。因此，CCK和阿片受体在该制剂中都是活性的。 然而，为了检测这些受体在细胞水平上的相互作用，它们都应该在同一个细胞上表达。海马体之所以令人感兴趣，是因为它众所周知的细胞结构，关于它的大量知识积累，以及它在陈述性记忆和空间映射中的作用。为了表达CCK和MU阿片受体，我们感兴趣的细胞类型将是中间神经元，特别是CCK类型的篮子细胞中间神经元。由于只有约10%的海马神经元是中间神经元，因此候选细胞的产量从一开始就很低。在这个模型中，突触前片段是不必要的，所以用低浓度的酶处理切片，这可以获得更好的健康细胞产量。然而，中间神经元非常稀少，可用的更是稀少。一些数据显示，CCK增加了高电压激活的钙电流，但在对同一细胞进行去甲吗啡测试之前，细胞就解体了。考虑到赔率很低，我改用中脑导水管周围灰质(PAG)细胞。众所周知，PAG参与恐惧和防御反应以及内源性镇痛。与海马区不同，PAG细胞主要是(80%)中间神经元，已知它们同时表达CCK和MU阿片受体。然而，使用酶(蛋白酶XIV或木瓜蛋白酶)解离、研磨或振动探针的变量，使用“穿孔贴片”(β-七叶皂苷或制霉菌素)技术的变化来保存记录细胞的活力，使用丙酮酸或抗坏血酸来延长解离细胞的寿命，数据的产量仍然很低。7月下旬，我开始使用一种新的、更稳定的微操作器，淘汰了已有20年历史的液压操作器。希望增强的机械稳定性将允许进行更长时间和更可靠的记录。

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.