Calcium/calmodulin-dependent protein kinase II contributes to activity-dependent filopodia growth and spine formation

Abstract

Remodeling of synaptic networks through an activity-dependent formation or elimination of synaptic connections is believed to contribute to information processing and long-term memory. Recent work showed that enhanced synaptic activation, including induction of long-term potentiation and sensory stimulation, promote a rapid growth of dendritic filopodia and the formation of new spines or new types of synapses. Here, we investigated whether calcium/calmodulin-dependent protein kinase II (CaMKII), an enzyme implicated in the control of synaptic efficacy, also participated in these mechanisms. We show that the intracellular application of autophosphorylated CaMKII reproduced these morphological changes and triggered filopodia growth and spine formation. In addition, we find that activation of endogenous kinase through the inhibition of phosphatases or the application of calmodulin in the cell produced similar effects. Conversely, blockade of CaMKII activity prevented the synaptic enhancement, the growth of filopodia and formation of new spines triggered by LTP induction, and a short anoxia/hypoglycemia. Together, these results support the interpretation that CaMKII contributes to the control of activity-dependent structural plasticity.

Figure 1.

Growth of filopodia and formation of new spines induced by intracellular injection of activated CaMKII. A, Confocal microscopic images of dendritic spines taken 15 and 60 min after intracellular injection of the fluorescent dye sulforhodamine in a control experiment. B, Growth of two filopodia (arrows) and the formation of three new spines (asterisks) in a cell injected with activated CaMKII. C, Absence of changes in a cell injected with heat-inactivated CaMKII. Scale bars, 2 μm. D, Changes in EPSC slope measured as a function of time in seven cells injected with activated CaMKII (black circles) and five cells injected with the heat-inactivated enzyme (open circles). Data are means ± SEM. E, Quantitative analysis of the growth of filopodia (black columns) and formation of new spines (open columns) expressed as events per 100 μm of dendritic segment and per 60 min under control condition (n = 16), after the injection of activated (n = 14) or heat-inactivated (n = 9) CaMKII (^*p < 0.01; t test).

Figure 2.

Structural plasticity is induced by the activation of endogenous CaMKII. A, Formation of three new spines (arrows) in a cell injected with the phosphatase inhibitor calyculin A (150 nm). B, Growth of two filopodia in a cell injected with calmodulin (150 nm; bars: 2 μm). C, Growth of filopodia (black columns) and formation of new spines (open columns), expressed as events per 100 μm of dendritic segment and per 60 min, under control conditions (n = 16), after the intracellular injection of activated CaMKII (n = 14), or the phosphatase inhibitor calyculin A (150 nm; n = 13) or calmodulin (150 and 450 nm; n = 15). Data are means ± SEM. ^*p < 0.01; t test.

Figure 3.

Blockade of LTP-induced filopodia growth and spine formation by inhibition of CaMKII. A, Formation of two new spines (asterisks) induced by theta burst stimulation. B, Application of high-frequency stimulation in the presence of KN93 (10 μm) did not induce the growth of filopodia or the formations of new spines. Scale bars, 2 μm. C, Changes in EPSC amplitude triggered by theta burst stimulation under control conditions (black circles) or in the presence of KN93 (10 μm, open circles). Data are means ± SEM (n = 5, 6). D, Quantitative assessment of filopodia growth (black columns) and spine formation (open columns) observed under control conditions (n = 16), after the application of theta burst stimulation in the absence of drugs (LTP; n = 10), or after stimulation in the presence of MK801 (40 μm; n = 8) or KN93 (10 μm; n = 10). Data are means ± SEM. ^*p < 0.05; t test.

Figure 4.

Blockade of anoxia/hypoglycemia-induced structural plasticity by inhibitors of CaMKII. A, Growth of two filopodia (arrows, top) and formation of two new spines (asterisks, bottom) observed after the application of a brief (5 min) period of anoxia/hypoglycemia. B, Anoxia/hypoglycemia applied in the presence of AIP (200 nm) failed to induce structural plasticity. Scale bars, 2 μm. C, Growth of filopodia (black columns) and formation of new spines (open columns) expressed as events per 100 μm of dendritic segments and per 60 min under control conditions (n = 16), after a short (5 min) period of anoxia/hypoglycemia, (n = 12), or after anoxia/hypoglycemia, but in the presence of KN93 (40 μm; n = 7) or AIP (200 nm; n = 11). Data are means ± SEM. ^*p < 0.01; t test.