Loss of Ryanodine Receptor 2 impairs neuronal activity-dependent remodeling of dendritic spines and triggers compensatory neuronal hyperexcitability

Dendritic spines are postsynaptic domains that shape structural and functional properties of neurons. Upon neuronal activity, Ca²⁺ transients trigger signaling cascades that determine the plastic remodeling of dendritic spines, which modulate learning and memory. Here, we study in mice the role of the intracellular Ca²⁺ channel Ryanodine Receptor 2 (RyR2) in synaptic plasticity and memory formation. We demonstrate that loss of RyR2 in pyramidal neurons of the hippocampus impairs maintenance and activity-evoked structural plasticity of dendritic spines during memory acquisition. Furthermore, post-developmental deletion of RyR2 causes loss of excitatory synapses, dendritic sparsification, overcompensatory excitability, network hyperactivity and disruption of spatially tuned place cells. Altogether, our data underpin RyR2 as a link between spine remodeling, circuitry dysfunction and memory acquisition, which closely resemble pathological mechanisms observed in neurodegenerative disorders.Subject terms: Neuroscience, Neurological disorders     

Propagation of CaMKII translocation waves in heterogeneous spiny dendrites

CaMKII (Ca²⁺-calmodulin-dependent protein kinase II) is a key regulator of glutamatergic synapses and plays an essential role in many forms of synaptic plasticity. It has recently been observed experimentally that stimulating a local region of dendrite not only induces the local translocation of CaMKII from the dendritic shaft to synaptic targets within spines, but also initiates a wave of CaMKII translocation that spreads distally through the dendrite with an average speed of order 1μm/s. We have previously developed a simple reaction–diffusion model of CaMKII translocation waves that can account for the observed wavespeed and predicts wave propagation failure if the density of spines is too high. A major simplification of our previous model was to treat the distribution of spines as spatially uniform. However, there are at least two sources of heterogeneity in the spine distribution that occur on two different spatial scales. First, spines are discrete entities that are joined to a dendritic branch via a thin spine neck of submicron radius, resulting in spatial variations in spine density at the micron level. The second source of heterogeneity occurs on a much longer length scale and reflects the experimental observation that there is a slow proximal to distal variation in the density of spines. In this paper, we analyze how both sources of heterogeneity modulate the speed of CaMKII translocation waves along a spiny dendrite. We adapt methods from the study of the spread of biological invasions in heterogeneous environments, including homogenization theory of pulsating fronts and Hamilton–Jacobi dynamics of sharp interfaces.     

Expression of phospho‐Ca2+/calmodulin‐dependent protein kinase II in the pre‐Bötzinger complex of rats

The pre‐Bötzinger complex (pre‐BötC) in the ventrolateral medulla oblongata is a presumed kernel of respiratory rhythmogenesis. Ca²⁺‐activated non‐selective cationic current is an essential cellular mechanism for shaping inspiratory drive potentials. Ca²⁺/calmodulin‐dependent protein kinase II (CaMKII), an ideal ‘interpreter’ of diverse Ca²⁺ signals, is highly expressed in neurons in mediating various physiological processes. Yet, less is known about CaMKII activity in the pre‐BötC. Using neurokinin‐1 receptor as a marker of the pre‐BötC, we examined phospho (P)‐CaMKII subcellular distribution, and found that P‐CaMKII was extensively expressed in the region. P‐CaMKII‐ir neurons were usually oval, fusiform, or pyramidal in shape. P‐CaMKII immunoreactivity was distributed within somas and dendrites, and specifically in association with the post‐synaptic density. In dendrites, most synapses (93.1%) examined with P‐CaMKII expression were of asymmetric type, occasionally with symmetric type (6.9%), whereas in somas, 38.1% were of symmetric type. P‐CaMKII asymmetric synaptic identification implicates that CaMKII may sense and monitor Ca²⁺ activity, and phosphorylate post‐synaptic proteins to modulate excitatory synaptic transmission, which may contribute to respiratory modulation and plasticity. In somas, CaMKII acts on both symmetric and asymmetric synapses, mediating excitatory and inhibitory synaptic transmission. P‐CaMKII was also localized to the perisynaptic and extrasynaptic regions in the pre‐BötC.     

Mechanisms for localising calcineurin and CaMKII in dendritic spines

Calcineurin and calmodulin-dependent protein kinase II (CaMKII) are both highly abundant in neurons, and both are activated by calmodulin at similar Ca²⁺ concentrations in the test tube. However, they fulfill opposite functions in dendritic spines, with CaMKII activity driving long-term synaptic potentiation following large influxes of Ca²⁺ through NMDA-type glutamate receptors (NMDARs), and calcineurin responding to smaller influxes of Ca²⁺ through the same receptors to induce long-term depression. In this review, we explore the notion that precise dynamic localisation of the two enzymes at different sites within dendritic spines is fundamental to this behaviour. We describe the structural basis of calcineurin and CaMKII localisation by their interaction with proteins including AKAP79, densin-180, α-actinin, and NMDARs. We then consider how interactions with these proteins likely position calcineurin and CaMKII at different distances from Ca²⁺ microdomains emanating from the mouths of NMDARs in order to drive the divergent responses. We also highlight shortcomings in our current understanding of synaptic localisation of these two important signalling enzymes.     

Regulation of Synaptic Transmission by Presynaptic CaMKII and BK Channels

Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and the BK channel are enriched at the presynaptic nerve terminal, where CaMKII associates with synaptic vesicles whereas the BK channel colocalizes with voltage-sensitive Ca²⁺ channels in the plasma membrane. Mounting evidence suggests that these two proteins play important roles in controlling neurotransmitter release. Presynaptic BK channels primarily serve as a negative regulator of neurotransmitter release. In contrast, presynaptic CaMKII either enhances or inhibits neurotransmitter release and synaptic plasticity depending on experimental or physiological conditions and properties of specific synapses. The different functions of presynaptic CaMKII appear to be mediated by distinct downstream proteins, including the BK channel.     

A diffusion-activation model of CaMKII translocation waves in dendrites

Ca2+-calmodulin-dependent protein kinase II (CaMKII) is a key regulator of glutamatergic synapses and plays an essential role in many forms of synaptic plasticity. It has recently been observed that stimulating dendrites locally with a single glutamate/glycine puff induces a local translocation of CaMKII into spines that subsequently spreads in a wave-like manner towards the distal dendritic arbor. Here we present a mathematical model of the diffusion, activation and translocation of dendritic CaMKII. We show how the nonlinear dynamics of CaMKII diffusion-activation generates a propagating translocation wave, provided that the rate of activation is sufficiently fast. We also derive an explicit formula for the wave speed as a function of physiological parameters such as the diffusivity of CaMKII and the density of spines. Our model provides a quantitative framework for understanding the spread of CaMKII translocation and its possible role in heterosynaptic plasticity.     

Inositol-1,4,5-trisphosphate 3-kinase A regulates dendritic morphology and shapes synaptic Ca2+ transients

Inositol-1,4,5-trisphosphate 3-kinase-A (itpka) accumulates in dendritic spines and seems to be critically involved in synaptic plasticity. The protein possesses two functional activities: it phosphorylates inositol-1,4,5-trisphosphate (Ins(1,4,5)P₃) and regulates actin dynamics by its F-actin bundling activity. To assess the relevance of these activities for neuronal physiology, we examined the effects of altered itpka levels on cell morphology, Ins(1,4,5)P₃ metabolism and dendritic Ca^2 + signaling in hippocampal neurons. Overexpression of itpka increased the number of dendritic protrusions by 71% in immature primary neurons. In mature neurons, however, the effect of itpka overexpression on formation of dendritic spines was weaker and depletion of itpka did not alter spine density and synaptic contacts. In synaptosomes of mature neurons itpka loss resulted in decreased duration of Ins(1,4,5)P₃ signals and shorter Ins(1,4,5)P₃-dependent Ca^2 + transients. At synapses of itpka deficient neurons the levels of Ins(1,4,5)P₃-5-phosphatase (inpp5a) and sarcoplasmic/endoplasmic reticulum calcium ATPase pump-2b (serca2b) were increased, indicating that decreased duration of Ins(1,4,5)P₃ and Ca^2 + signals results from compensatory up-regulation of these proteins. Taken together, our data suggest a dual role for itpka. In developing neurons itpka has a morphogenic effect on dendrites, while the kinase appears to play a key role in shaping Ca^2 + transients at mature synapses.     

Prediction and validation of a mechanism to control the threshold for inhibitory synaptic plasticity

Synaptic plasticity, neuronal activity‐dependent sustained alteration of the efficacy of synaptic transmission, underlies learning and memory. Activation of positive‐feedback signaling pathways by an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) has been implicated in synaptic plasticity. However, the mechanism that determines the [Ca²⁺]_i threshold for inducing synaptic plasticity is elusive. Here, we developed a kinetic simulation model of inhibitory synaptic plasticity in the cerebellum, and systematically analyzed the behavior of intricate molecular networks composed of protein kinases, phosphatases, etc. The simulation showed that Ca²⁺/calmodulin‐dependent protein kinase II (CaMKII), which is essential for the induction of synaptic plasticity, was persistently activated or suppressed in response to different combinations of stimuli. The sustained CaMKII activation depended on synergistic actions of two positive‐feedback reactions, CaMKII autophosphorylation and CaMKII‐mediated inhibition of a CaM‐dependent phosphodiesterase, PDE1. The simulation predicted that PDE1‐mediated feedforward inhibition of CaMKII predominantly controls the Ca²⁺ threshold, which was confirmed by electrophysiological experiments in primary cerebellar cultures. Thus, combined application of simulation and experiments revealed that the Ca²⁺ threshold for the cerebellar inhibitory synaptic plasticity is primarily determined by PDE1.     

In Vitro Reconstitution of a CaMKII Memory Switch by an NMDA Receptor-Derived Peptide

Ca²⁺/Calmodulin-dependent protein kinase II (CaMKII) has been shown to play a major role in establishing memories through complex molecular interactions including phosphorylation of multiple synaptic targets. However, it is still controversial whether CaMKII itself serves as a molecular memory because of a lack of direct evidence. Here, we show that a single holoenzyme of CaMKII per se serves as an erasable molecular memory switch. We reconstituted Ca²⁺/Calmodulin-dependent CaMKII autophosphorylation in the presence of protein phosphatase 1 in vitro, and found that CaMKII phosphorylation shows a switch-like response with history dependence (hysteresis) only in the presence of an N-methyl-D-aspartate receptor-derived peptide. This hysteresis is Ca²⁺ and protein phosphatase 1 concentration-dependent, indicating that the CaMKII memory switch is not simply caused by an N-methyl-D-aspartate receptor-derived peptide lock of CaMKII in an active conformation. Mutation of a phosphorylation site of the peptide shifted the Ca²⁺ range of hysteresis. These functions may be crucial for induction and maintenance of long-term synaptic plasticity at hippocampal synapses.     

In?Vitro Reconstitution of a CaMKII Memory Switch by an NMDA Receptor-Derived Peptide

Ca²⁺/Calmodulin-dependent protein kinase II (CaMKII) has been shown to play a major role in establishing memories through complex molecular interactions including phosphorylation of multiple synaptic targets. However, it is still controversial whether CaMKII itself serves as a molecular memory because of a lack of direct evidence. Here, we show that a single holoenzyme of CaMKII per se serves as an erasable molecular memory switch. We reconstituted Ca²⁺/Calmodulin-dependent CaMKII autophosphorylation in the presence of protein phosphatase 1 in vitro, and found that CaMKII phosphorylation shows a switch-like response with history dependence (hysteresis) only in the presence of an N-methyl-D-aspartate receptor-derived peptide. This hysteresis is Ca²⁺ and protein phosphatase 1 concentration-dependent, indicating that the CaMKII memory switch is not simply caused by an N-methyl-D-aspartate receptor-derived peptide lock of CaMKII in an active conformation. Mutation of a phosphorylation site of the peptide shifted the Ca²⁺ range of hysteresis. These functions may be crucial for induction and maintenance of long-term synaptic plasticity at hippocampal synapses.     

Ca2+-independent Activation of Ca2+/Calmodulin-dependent Protein Kinase II Bound to the C-terminal Domain of CaV2.1 Calcium Channels

Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) forms a major component of the postsynaptic density where its functions in synaptic plasticity are well established, but its presynaptic actions are poorly defined. Here we show that CaMKII binds directly to the C-terminal domain of Ca_V2.1 channels. Binding is enhanced by autophosphorylation, and the kinase-channel signaling complex persists after dephosphorylation and removal of the Ca²⁺/CaM stimulus. Autophosphorylated CaMKII can bind the Ca_V2.1 channel and synapsin-1 simultaneously. CaMKII binding to Ca_V2.1 channels induces Ca²⁺-independent activity of the kinase, which phosphorylates the enzyme itself as well as the neuronal substrate synapsin-1. Facilitation and inactivation of Ca_V2.1 channels by binding of Ca²⁺/CaM mediates short term synaptic plasticity in transfected superior cervical ganglion neurons, and these regulatory effects are prevented by a competing peptide and the endogenous brain inhibitor CaMKIIN, which blocks binding of CaMKII to Ca_V2.1 channels. These results define the functional properties of a signaling complex of CaMKII and Ca_V2.1 channels in which both binding partners are persistently activated by their association, and they further suggest that this complex is important in presynaptic terminals in regulating protein phosphorylation and short term synaptic plasticity.     

CaMKIIβ is localized in dendritic spines as both drebrin‐dependent and drebrin‐independent pools

Drebrin is a major F‐actin binding protein in dendritic spines that is critically involved in the regulation of dendritic spine morphogenesis, pathology, and plasticity. In this study, we aimed to identify a novel drebrin‐binding protein involved in spine morphogenesis and synaptic plasticity. We confirmed the beta subunit of Ca²⁺/calmodulin‐dependent protein kinase II (CaMKIIβ) as a drebrin‐binding protein using a yeast two‐hybrid system, and investigated the drebrin–CaMKIIβ relationship in dendritic spines using rat hippocampal neurons. Drebrin knockdown resulted in diffuse localization of CaMKIIβ in dendrites during the resting state, suggesting that drebrin is involved in the accumulation of CaMKIIβ in dendritic spines. Fluorescence recovery after photobleaching analysis showed that drebrin knockdown increased the stable fraction of CaMKIIβ, indicating the presence of drebrin‐independent, more stable CaMKIIβ. NMDA receptor activation also increased the stable fraction in parallel with drebrin exodus from dendritic spines. These findings suggest that CaMKIIβ can be classified into distinct pools: CaMKIIβ associated with drebrin, CaMKIIβ associated with post‐synaptic density (PSD), and CaMKIIβ free from PSD and drebrin. CaMKIIβ appears to be anchored to a protein complex composed of drebrin‐binding F‐actin during the resting state. NMDA receptor activation releases CaMKIIβ from drebrin resulting in CaMKIIβ association with PSD.

     

Presynaptic NMDA receptors act as local high‐gain glutamate detector in developing cerebellar molecular layer interneurons

In the classical view, NMDA receptors (NMDARs) are located postsynaptically and play a pivotal role in excitatory transmission and synaptic plasticity. In developing cerebellar molecular layer interneurons (MLIs) however, NMDARs are known to be solely extra‐ or presynaptic and somewhat poorly expressed. Somatodendritic NMDARs are exclusively activated by glutamate spillover from adjacent synapses, but the mode of activation of axonal NMDARs remains unclear. Our data suggest that a volume transmission is likely to stimulate presynaptic NMDARs (preNMDARs) since NMDA puffs directed to the axon led to inward currents and Ca²⁺ transients restricted to axonal varicosities. Using local glutamate photoliberation, we show that pre‐ and post‐synaptic NMDARs share the same voltage dependence indicating their containing NR2A/B subunits. Ca²⁺ transients elicited by NMDA puffs are eventually followed by delayed events reminding of the spontaneous Ca²⁺ transients (ScaTs) described at the basket cell/Purkinje cell terminals. Moreover, the presence of Ca²⁺ transients at varicosities located more than 5 μm away from the uncaging site indicates that the activation of preNMDARs sensitizes the Ca²⁺ stores in adjacent varicosities, a process that is abolished in the presence of a high concentration of ryanodine. Altogether, the data demonstrate that preNMDARs act as high‐gain glutamate detectors.     

On the Mechanism of Synaptic Depression Induced by CaMKIIN,an Endogenous Inhibitor of CaMKII

Activity-dependent synaptic plasticity underlies, at least in part, learning and memory processes. NMDA receptor (NMDAR)-dependent long-term potentiation (LTP) is a major synaptic plasticity model. During LTP induction, Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is activated, autophosphorylated and persistently translocated to the postsynaptic density, where it binds to the NMDAR. If any of these steps is inhibited, LTP is disrupted. The endogenous CaMKII inhibitor proteins CaMKIINα,β are rapidly upregulated in specific brain regions after learning. We recently showed that transient application of peptides derived from CaMKIINα (CN peptides) persistently depresses synaptic strength and reverses LTP saturation, as it allows further LTP induction in previously saturated pathways. The treatment disrupts basal CaMKII-NMDAR interaction and decreases bound CaMKII fraction in spines. To unravel CaMKIIN function and to further understand CaMKII role in synaptic strength maintenance, here we more deeply investigated the mechanism of synaptic depression induced by CN peptides (CN-depression) in rat hippocampal slices. We showed that CN-depression does not require glutamatergic synaptic activity or Ca²⁺ signaling, thus discarding unspecific triggering of activity-dependent long-term depression (LTD) in slices. Moreover, occlusion experiments revealed that CN-depression and NMDAR-LTD have different expression mechanisms. We showed that CN-depression does not involve complex metabolic pathways including protein synthesis or proteasome-mediated degradation. Remarkably, CN-depression cannot be resolved in neonate rats, for which CaMKII is mostly cytosolic and virtually absent at the postsynaptic densities. Overall, our results support a direct effect of CN peptides on synaptic CaMKII-NMDAR binding and suggest that CaMKIINα,β could be critical plasticity-related proteins that may operate as cell-wide homeostatic regulators preventing saturation of LTP mechanisms or may selectively erase LTP-induced traces in specific groups of synapses.     

Caldendrin and myosin V regulate synaptic spine apparatus localization via ER stabilization in dendritic spines

Excitatory synapses of principal hippocampal neurons are frequently located on dendritic spines. The dynamic strengthening or weakening of individual inputs results in structural and molecular diversity of dendritic spines. Active spines with large calcium ion (Ca²⁺) transients are frequently invaded by a single protrusion from the endoplasmic reticulum (ER), which is dynamically transported into spines via the actin‐based motor myosin V. An increase in synaptic strength correlates with stable anchoring of the ER, followed by the formation of an organelle referred to as the spine apparatus. Here, we show that myosin V binds the Ca²⁺ sensor caldendrin, a brain‐specific homolog of the well‐known myosin V interactor calmodulin. While calmodulin is an essential activator of myosin V motor function, we found that caldendrin acts as an inhibitor of processive myosin V movement. In mouse and rat hippocampal neurons, caldendrin regulates spine apparatus localization to a subset of dendritic spines through a myosin V‐dependent pathway. We propose that caldendrin transforms myosin into a stationary F‐actin tether that enables the localization of ER tubules and formation of the spine apparatus in dendritic spines.     

Nucleotides and Phosphorylation Bi-directionally Modulate Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII) Binding to the N-Methyl-d-aspartate (NMDA) Receptor Subunit GluN2B

The Ca²⁺/calmodulin (CaM)-dependent protein kinase II (CaMKII) and the NMDA-type glutamate receptor are key regulators of synaptic plasticity underlying learning and memory. Direct binding of CaMKII to the NMDA receptor subunit GluN2B (formerly known as NR2B) (i) is induced by Ca²⁺/CaM but outlasts this initial Ca²⁺-stimulus, (ii) mediates CaMKII translocation to synapses, and (iii) regulates synaptic strength. CaMKII binds to GluN2B around S1303, the major CaMKII phosphorylation site on GluN2B. We show here that a phospho-mimetic S1303D mutation inhibited CaM-induced CaMKII binding to GluN2B in vitro, presenting a conundrum how binding can occur within cells, where high ATP concentration should promote S1303 phosphorylation. Surprisingly, addition of ATP actually enhanced the binding. Mutational analysis revealed that this positive net effect was caused by four modulatory effects of ATP, two positive (direct nucleotide binding and CaMKII T286 autophosphorylation) and two negative (GluN2B S1303 phosphorylation and CaMKII T305/6 autophosphorylation). Imaging showed positive regulation by nucleotide binding also within transfected HEK cells and neurons. In fact, nucleotide binding was a requirement for efficient CaMKII interaction with GluN2B in cells, while T286 autophosphorylation was not. Kinetic considerations support a model in which positive regulation by nucleotide binding and T286 autophosphorylation occurs faster than negative modulation by GluN2B S1303 and CaMKII T305/6 phosphorylation, allowing efficient CaMKII binding to GluN2B despite the inhibitory effects of the two slower reactions.     

Wavelet Transform-Based De-Noising for Two-Photon Imaging of Synaptic Ca2+ Transients

Postsynaptic Ca²⁺ transients triggered by neurotransmission at excitatory synapses are a key signaling step for the induction of synaptic plasticity and are typically recorded in tissue slices using two-photon fluorescence imaging with Ca²⁺-sensitive dyes. The signals generated are small with very low peak signal/noise ratios (pSNRs) that make detailed analysis problematic. Here, we implement a wavelet-based de-noising algorithm (PURE-LET) to enhance signal/noise ratio for Ca²⁺ fluorescence transients evoked by single synaptic events under physiological conditions. Using simulated Ca²⁺ transients with defined noise levels, we analyzed the ability of the PURE-LET algorithm to retrieve the underlying signal. Fitting single Ca²⁺ transients with an exponential rise and decay model revealed a distortion of τ_rise but improved accuracy and reliability of τ_decay and peak amplitude after PURE-LET de-noising compared to raw signals. The PURE-LET de-noising algorithm also provided a ∼30-dB gain in pSNR compared to ∼16-dB pSNR gain after an optimized binomial filter. The higher pSNR provided by PURE-LET de-noising increased discrimination accuracy between successes and failures of synaptic transmission as measured by the occurrence of synaptic Ca²⁺ transients by ∼20% relative to an optimized binomial filter. Furthermore, in comparison to binomial filter, no optimization of PURE-LET de-noising was required for reducing arbitrary bias. In conclusion, the de-noising of fluorescent Ca²⁺ transients using PURE-LET enhances detection and characterization of Ca²⁺ responses at central excitatory synapses.     

Extracellular Ca2+ ions reduce NMDA receptor conductance and gating

Brief intracellular Ca²⁺ transients initiate signaling routines that direct cellular activities. Consequently, activation of Ca²⁺-permeable neurotransmitter-gated channels can both depolarize and initiate remodeling of the postsynaptic cell. In particular, the Ca²⁺ transient produced by NMDA receptors is essential to normal synaptic physiology, drives the development and plasticity of excitatory central synapses, and also mediates glutamate excitotoxicity. The amplitude and time course of the Ca²⁺ signal depends on the receptor’s conductance and gating kinetics; these properties are themselves influenced both directly and indirectly by fluctuations in the extracellular Ca²⁺ concentration. Here, we used electrophysiology and kinetic modeling to delineate the direct effects of extracellular Ca²⁺ on recombinant GluN1/GluN2A receptor conductance and gating. We report that, in addition to decreasing unitary conductance, Ca²⁺ also decreased channel open probability primarily by lengthening closed-channel periods. Using one-channel current recordings, we derive a kinetic model for GluN1/GluN2A receptors in physiological Ca²⁺ concentrations that accurately describes macroscopic channel behaviors. This model represents a practical instrument to probe the mechanisms that control the Ca²⁺ transients produced by NMDA receptors during both normal and aberrant synaptic signaling.     

Studying Signal Transduction in Single Dendritic Spines

Many forms of synaptic plasticity are triggered by biochemical signaling that occurs in small postsynaptic compartments called dendritic spines, each of which typically houses the postsynaptic terminal associated with a single glutamatergic synapse. Recent advances in optical techniques allow investigators to monitor biochemical signaling in single dendritic spines and thus reveal the signaling mechanisms that link synaptic activity and the induction of synaptic plasticity. This is mostly in the study of Ca²⁺-dependent forms of synaptic plasticity for which many of the steps between Ca²⁺ influx and changes to the synapse are now known. This article introduces the new techniques used to investigate signaling in single dendritic spines and the neurobiological insights that they have produced.Each neuron typically receives 1000–10,000 synaptic inputs and sends information to an axon, which branches to produce a similar number of synaptic outputs. Most excitatory postsynaptic terminals are associated with dendritic spines, small protrusions emanating from the dendritic surface (Nimchinsky et al. 2002; Alvarez and Sabatini 2007). Each spine has a volume of ∼0.1 femtoliter, and connects to the parent dendrite through a narrow neck, which acts as a diffusion barrier and compartmentalizes biochemical reactions. Ca²⁺ influx into spines initiates a cascade of biochemical signals leading to various forms of synaptic plasticity including long-term potentiation (LTP).Because LTP in hippocampal CA1 pyramidal neurons is a cellular mechanism that may underlie long-term memory formation, the signal transduction underlying LTP has been extensively studied by pharmacological and genetic methods (Bliss and Collingridge 1993; Derkach et al. 2007). It is now well established that LTP is induced by Ca²⁺ influx into dendritic spines through NMDA-type glutamate receptors (NMDARs), which induces the insertion of AMPA-type glutamate receptors (AMPARs) into the synapse, thereby increasing the sensitivity of the postsynaptic terminal to glutamate (Derkach et al. 2007; Kessels and Malinow 2009). An increase of release probability during LTP has also been reported (Enoki et al. 2009), and thus both pre- and postsynaptic mechanisms may contribute to LTP (Lisman and Raghavachari 2006).Manipulations of signal transduction using specific pharmacological inhibitors or genetic perturbations have identified many signaling pathways that connect Ca²⁺ to LTP induction. For example, LTP requires the activation of many signaling proteins, including Ca²⁺/calmodulin-dependent kinase II (CaMKII), extracellular signal-related kinase (ERK), Phoshoinositide 3 kinase (PI3K), protein kinase A and C, and GTPases such as Ras, Rab, and Rho (Kennedy et al. 2005). The list is continually growing, and the hundreds of implicated proteins form a complex signaling network whose contribution to LTP is still unclear (Bromberg et al. 2008).Signaling dynamics in neurons have traditionally been measured using biochemical analyses (Bromberg et al. 2008). However, the spatiotemporal resolution of conventional biochemistry is limited, restricting analysis to the time scale of many minutes and requiring the homogenization of tissue containing millions of synapses and other cellular elements. Furthermore, resolving synaptically induced changes in signaling by biochemical analysis typically requires stimulating many synapses at the same time, which may produce unintended effects, for instance, excitotoxicity or homeostatic plasticity.The size of dendritic spines is similar to the resolution of an optical microscope, permitting the optical analysis of biochemical signaling in each dendritic spine (Svoboda and Yasuda 2006). In particular, the advent of two-photon-based FRET techniques and the development of appropriate fluorescent reporters of specific biochemical reactions (see below) have provided readouts for signal transduction with high spatiotemporal resolution in live brain tissue (Svoboda and Yasuda 2006; Yasuda 2006). This has provided detailed information about the dynamics of signal transduction in spines and dendrites, and insights into the molecular mechanisms of synaptic plasticity.