The synapse is a highly organized cellular specialization whose structure and composition are reorganized, both positively and negatively, depending on the strength of input signals. The mechanisms orchestrating these changes are not well understood. A plausible locus for the reorganization of synapse components and structure is actin, because it serves as both cytoskeleton and scaffold for synapses and exists in a dynamic equilibrium between F-actin and G-actin that is modulated bidirectionally by cellular signaling. Using a new FRET-based imaging technique to monitor F-actin/G-actin equilibrium, we show here that tetanic stimulation causes a rapid, persistent shift of actin equilibrium toward F-actin in the dendritic spines of rat hippocampal neurons. This enlarges the spines and increases postsynaptic binding capacity. In contrast, prolonged low-frequency stimulation shifts the equilibrium toward G-actin, resulting in a loss of postsynaptic actin and of structure. This bidirectional regulation of actin is actively involved in protein assembly and disassembly and provides a substrate for bidirectional synaptic plasticity.

Single-particle electron microscopy (EM) combined with biochemical measurements revealed the molecular shape of SAP97 and a monomer-dimer transition that depended on the N-terminal L27 domain. Overexpression of SAP97 drove GluR1 to synapses, potentiated AMPA receptor (AMPAR) excitatory postsynaptic currents (EPSCs), and occluded LTP. Synaptic potentiation and GluR1 delivery were dissociable by L27 domain mutants that inhibit multimerization of SAP97. Loss of potentiation was correlated with faster turnover of monomeric SAP97 mutants in dendritic spines. We propose that L27-mediated interactions of SAP97 with itself or other proteins regulate the synaptic delivery of AMPARs. RNAi knockdown of endogenous PSD-95 depleted surface GluR1 and impaired AMPA EPSCs. In contrast, RNAi knockdown of endogenous SAP97 reduced surface expression of both GluR1 and GluR2 and inhibited both AMPA and NMDA EPSCs. Thus SAP97 has a broader role than its close relative, PSD-95, in the maintenance of synaptic function.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) and actin are two crucial molecules involved in long-term potentiation (LTP). In addition to its signaling function, CaMKII plays a structural role via direct interaction with actin filaments, thus coupling functional and structural plasticity in dendritic spines. The status of F-actin, regulated by CaMKII, determines the postsynaptic protein binding capacity and thus may act as a synaptic tag that consolidates LTP.

QZ1, 2-[2-chloro-6-hydroxy-3-oxo-5-(quinolin-8-ylaminomethyl)-3H-xanthen-9-yl]benzoic acid, and QZ2, 2-[6-hydroxy-3-oxo-4,5-bis-(quinolin-8-ylaminomethyl)-3H-xanthen-9-yl]benzoic acid, two fluorescein-based dyes derivatized with 8-aminoquinoline, have been prepared and their photophysical, thermodynamic, and zinc-binding kinetic properties determined. Because of their low background fluorescence and highly emissive Zn(II) complexes, QZ1 and QZ2 have a large dynamic range, with 42-and 150-fold fluorescence enhancements upon Zn(II) coordination, respectively. These dyes have micromolar K d values for Zn(II) and are selective for Zn(II) over biologically relevant concentrations of the alkali and alkaline earth metals. The Zn(II) complexes also fluoresce brightly in the presence of excess Mn(II), Fe(II), Co(II), Cd(II), and Hg(II), offering improved specificity for Zn(II) over di(2-picolyl)amine-based Zn(II) sensors. Stopped-flow kinetic investigations indicate that QZ1 and QZ2 bind Zn(II) with k on values of (3-4) × 10 6 M -1 s -1 , compared to (6-8) × 10 5 M -1 s -1 for select ZP (Zinpyr) dyes, at 4.3 °C. Dissociation of Zn(II) from QZ1 and QZ2 occurs with k off values of 150 and 160 s -1 , over 5 orders of magnitude larger than those for ZP probes, achieving reversibility on the biological (millisecond) time scale. Laser scanning confocal and two-photon microscopy studies reveal that QZ2 is cell-permeable and Zn(II)-responsive in vivo. Because of its weaker affinity for Zn(II), QZ2 responds to higher concentrations of intracellular Zn (II) than members of the ZP family, illustrating that binding affinity is an important parameter for Zn(II) detection in vivo. (S5-S9); plots of k obs versus [Zn(II)] (S10-S14); Eyring plots (S15-S19); relative fluorescence emission of the Zn(II)-bound sensors (S20); and both confocal and two-photon microscopy images of biological samples (S21-S23); and experimental details for the representative kinetic analyses. This material is available free of charge via the Internet at

Ca2ϩ /calmodulin-dependent protein kinase II (CaMKII) is highly enriched in excitatory synapses in the CNS and critically involved in synaptic plasticity, learning, and memory. However, the precise temporal and spatial regulation of CaMKII activity in living cells has not been well described, because of a lack of specific methods. We tried to address this by optically detecting the conformational change in CaMKII during activation using fluorescence resonance energy transfer (FRET). The engineered FRET probe Camui␣ detects calmodulin binding and autophosphorylation at threonine 286 that renders the enzyme constitutively active. In combination with two-photon microscopy, we demonstrate that Camui␣ can be used to observe temporal and spatial regulation of CaMKII activity in living neurons.

BackgroundGlycogen synthase kinase-3 (GSK-3) is a widely expressed and highly conserved serine/threonine protein kinase encoded by two genes that generate two related proteins: GSK-3α and GSK-3β. Mice lacking a functional GSK-3α gene were engineered in our laboratory; they are viable and display insulin sensitivity. In this study, we have characterized brain functions of GSK-3α KO mice by using a well-established battery of behavioral tests together with neurochemical and neuroanatomical analysis.ResultsSimilar to the previously described behaviours of GSK-3β+/-mice, GSK-3α mutants display decreased exploratory activity, decreased immobility time and reduced aggressive behavior. However, genetic inactivation of the GSK-3α gene was associated with: decreased locomotion and impaired motor coordination, increased grooming activity, loss of social motivation and novelty; enhanced sensorimotor gating and impaired associated memory and coordination. GSK-3α KO mice exhibited a deficit in fear conditioning, however memory formation as assessed by a passive avoidance test was normal, suggesting that the animals are sensitized for active avoidance of a highly aversive stimulus in the fear-conditioning paradigm. Changes in cerebellar structure and function were observed in mutant mice along with a significant decrease of the number and size of Purkinje cells.ConclusionTaken together, these data support a role for the GSK-3α gene in CNS functioning and possible involvement in the development of psychiatric disorders.

Activity-dependent plasticity of synaptic structure and function plays an essential role in neuronal development and in cognitive functions including learning and memory. The formation, maintenance and modulation of dendritic spines are mainly controlled by the dynamics of actin filaments (F-actin) through interaction with various actin-binding proteins (ABPs) and postsynaptic signaling messengers. Induction of long-term potentiation (LTP) triggers a cascade of events involving Ca signaling, intracellular pathways such as cAMP and cGMP, and regulation of ABPs such as CaMKII, Cofilin, Aip1, Arp2/3, α-actinin, Profilin and Drebrin. We review here how these ABPs modulate the rate of assembly, disassembly, stabilization and bundling of F-actin during LTP induction. We highlight the crucial role that CaMKII exerts in both functional and structural plasticity by directly coupling Ca signaling with F-actin dynamics through the β subunit. Moreover, we show how cAMP and cGMP second messengers regulate postsynaptic structural potentiation. Brain disorders such as Alzheimer's disease, schizophrenia or autism, are associated with alterations in the regulation of F-actin dynamics by these ABPs and signaling messengers. Thus, a better understanding of the molecular mechanisms controlling actin cytoskeleton can provide cues for the treatment of these disorders.

SUMMARY The structural modification of dendritic spines plays a critical role in synaptic plasticity. CaMKII is a pivotal molecule involved in this process through both kinase-dependent and independent structural functions, but the respective contributions of these two functions to the synaptic plasticity remain unclear. We demonstrate that the transient interplay between the kinase and structural functions of CaMKII during the induction of synaptic plasticity temporally gates the activity-dependent modification of the actin cytoskeleton. Inactive CaMKII binds F-actin, thereby limiting access of actin regulating proteins to F-actin and stabilizing spine structure. CaMKII-activating stimuli trigger dissociation of CaMKII from F-actin through specific autophosphorylation reactions within the F-actin binding region and permits F-actin remodeling by regulatory proteins followed by reassociation and restabilization. Blocking the autophosphorylation impairs both functional and structural plasticity without affecting kinase activity. These results underpin the importance of the interplay between the kinase and structural functions of CaMKII in defining a time window permissive for synaptic plasticity.