Calcium ion (Ca2+) and Calmodulin (CaM) are important signaling molecules that have been shown to play a significant role in a wide variety of neuronal functions, including neurite outgrowth of axon and dendrites. The growth cone is located on the tip of the growing neurite. The morphology and motility of the growth cone depend on Ca2+ and the stimulation of Ca2+ dependent protein kinases (CaMK) in developing neurons. CaM-kinase II (CaMKII) is a member of the CaMK family, and it is highly expressed in the cytosol of developing neurons, including the growth cone. We hypothesized that CaMKII activity could be necessary for growth cones morphology and motility. To test this possibility, the identified Pedal A (PeA) neurons from the central ring ganglia of the freshwater pond snail Lymnaea stagnalis were cultured for 24 hrs -48 hrs in a 2 ml brain conditioned medium. Following neurite outgrowth, we have examined the effect of CaMKII inhibitor on growth cone structure and motility rate by using a CaMKII specific inhibitor KN-93 and its inactive analog KN-92; both drugs were pressure applied directly onto individual growth cones. Here we demonstrate that inhibition of CaMKII dramatically changes the morphology and motility of growth cone in cultured neurons. Growth cones of neurons treated with KN-93 exhibit collapsed morphology. In contrast, growth cone exposure to KN-92 did not affect growth cone morphology and motility rate. Taken together, the data presented here provide the first direct evidence for the involvement of CaMKII activity to maintain growth cone morphology and motility in the freshwater pond snail Lymnaea stagnalis.
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