Dendritic spines are small protrusive structures on dendritic surfaces, and function as postsynaptic compartments for excitatory synapses. Plasticity of spine structure is associated with many forms of long-term neuronal plasticity, learning and memory. Inside these small dendritic compartments, biochemical states and protein-protein interactions are dynamically modulated by synaptic activity, leading to the regulation of protein synthesis and reorganization of cytoskeletal architecture. This in turn causes plasticity of structure and function of the spine. Technical advances in monitoring molecular behaviors in single dendritic spines have revealed that each signaling pathway is differently regulated across multiple spatiotemporal domains. The spatial pattern of signaling activity expands from a single spine to the nearby dendritic area, dendritic branch and the nucleus, regulating different cellular events at each spatial scale. Temporally, biochemical events are typically triggered by short Ca2+ pulses (~10–100 ms). However, these signals can then trigger activation of downstream protein cascades that can last from milliseconds to hours. Recent imaging studies provide many insights into the biochemical processes governing signaling events of molecular assemblies at different spatial localizations. Here, we highlight recent findings of signaling dynamics during synaptic plasticity and discuss their roles in long-term structural plasticity of dendritic spines.

Measurement of Förster resonance energy transfer by fluorescence lifetime imaging microscopy (FLIM-FRET) is a powerful method for visualization of intracellular signaling activities such as protein-protein interactions and conformational changes of proteins. Here, we developed a dark green fluorescent protein (ShadowG) that can serve as an acceptor for FLIM-FRET. ShadowG is spectrally similar to monomeric enhanced green fluorescent protein (mEGFP) and has a 120-fold smaller quantum yield. When FRET from mEGFP to ShadowG was measured using an mEGFP-ShadowG tandem construct with 2-photon FLIM-FRET, we observed a strong FRET signal with low cell-to-cell variability. Furthermore, ShadowG was applied to a single-molecule FRET sensor to monitor a conformational change of CaMKII and of the light oxygen voltage (LOV) domain in HeLa cells. These sensors showed reduced cell-to-cell variability of both the basal fluorescence lifetime and response signal. In contrast to mCherry- or dark-YFP-based sensors, our sensor allowed for precise measurement of individual cell responses. When ShadowG was applied to a separate-type Ras FRET sensor, it showed a greater response signal than did the mCherry-based sensor. Furthermore, Ras activation and translocation of its effector ERK2 into the nucleus could be observed simultaneously. Thus, ShadowG is a promising FLIM-FRET acceptor.

CaMKIIα plays an essential role in decoding Ca 2+ signaling in spines by acting as a leaky Ca 2+ integrator with the time constant of several seconds. However, the mechanism by which CaMKIIα integrates Ca 2+ signals remains elusive. Here, we imaged CaMKIIα-CaM association in single dendritic spines using a new FRET sensor and two-photon fluorescence lifetime imaging. In response to a glutamate uncaging pulse, CaMKIIα-CaM association increases in ~0.1 s and decays over ~3 s. During repetitive glutamate uncaging, which induces spine structural plasticity, CaMKIIα-CaM association did not show further increase but sustained at a constant level. Since CaMKIIα activity integrates Ca 2+ signals over ~10 s under this condition, the integration of Ca 2+ signal by CaMKIIα during spine structural plasticity is largely due to Ca 2+ /CaM-independent, autonomous activity. Based on these results, we propose a simple kinetic model of CaMKIIα activation in dendritic spines.

Activity-dependent structural plasticity at the synapse requires specific changes in the neuronal transcriptome. While much is known about the role of coding elements in this process, the role of the long noncoding transcriptome remains elusive. Here, we report the discovery of an intronic long noncoding RNA (lncRNA)—termed ADEPTR—that is up-regulated and synaptically transported in a cAMP/PKA-dependent manner in hippocampal neurons, independently of its protein-coding host gene. Loss of ADEPTR function suppresses activity-dependent changes in synaptic transmission and structural plasticity of dendritic spines. Mechanistically, dendritic localization of ADEPTR is mediated by molecular motor protein Kif2A. ADEPTR physically binds to actin-scaffolding regulators ankyrin (AnkB) and spectrin (Sptn1) via a conserved sequence and is required for their dendritic localization. Together, this study demonstrates how activity-dependent synaptic targeting of an lncRNA mediates structural plasticity at the synapse.

Key points• Inhibition mediated by GABA and glycine is essential for controlling a balance of excitation and inhibition in the spinal cord.• Although these transmitters are known to be co-released from the same synaptic vesicles, the mechanisms that control the packaging of GABA + glycine into synaptic vesicles have not been fully characterized.• In this study, using paired whole-cell recording, we found that raised extracellular glutamate levels increased the amplitude of GABAergic IPSCs by enhancing glutamate uptake but reduced glycine release.• High-frequency trains of stimulation decreased glycinergic IPSCs more than GABAergic IPSCs at GABA/glycine mixed synapses, and repetitive stimulation occasionally failed to evoke glycinergic but not GABAergic IPSCs.• The present results suggest that the use of GABA as a transmitter at GABA/glycine mixed synapses may afford protection against pathophysiological hyperexcitability associated with increased extracellular glutamate concentration.Abstract Fast inhibitory neurotransmission in the central nervous system is mediated by γ-aminobutyric acid (GABA) and glycine, which are accumulated into synaptic vesicles by a common vesicular inhibitory amino acid transporter (VIAAT) and are then co-released. However, the mechanisms that control the packaging of GABA + glycine into synaptic vesicles are not fully understood. In this study, we demonstrate the dynamic control of the GABA-glycine co-transmission by the neuronal glutamate transporter, using paired whole-cell patch recording from monosynaptically coupled cultured spinal cord neurons derived from VIAAT-Venus transgenic rats. Short step depolarization of presynaptic neurons evoked unitary (cell-to-cell) inhibitory postsynaptic currents (IPSCs). Under normal conditions, the fractional contribution of postsynaptic GABA or glycine receptors to the unitary IPSCs did not change during a 1 h recording. Intracellular loading of GABA or glycine via a patch pipette enhanced the respective components of inhibitory transmission, indicating the importance of the cytoplasmic concentration of inhibitory transmitters. Raised extracellular glutamate levels increased the amplitude of GABAergic IPSCs but reduced glycine release by enhancing glutamate uptake. Similar effects were observed when presynaptic neurons were intracellularly perfused with glutamate. Interestingly, high-frequency trains of stimulation decreased glycinergic IPSCs more than GABAergic IPSCs, and repetitive stimulation occasionally failed to evoke glycinergic but not GABAergic IPSCs. The present results suggest that the enhancement of GABA release by glutamate uptake may be advantageous for rapid vesicular refilling of the inhibitory transmitter at mixed GABA/glycinergic synapses and thus may help prevent hyperexcitability.