Synthesis of FMRFaNV, a Photoreleasable Caged Transmitter Designed to Study Neuron-Glia Interactions in the Central Nervous System.

Neuroscience studies require technologies able to deliver compounds with both scale and timing compatibility with morphological and physiological synaptic properties. In this light, two-photon flash photolysis has been extensively used to successfully apply glutamate or other neurotransmitters at the synaptic level. However, the set of commercially available caged compounds is restricted and incompatible with studies demanding high cell specificity. The gain in cell specificity is especially relevant and challenging when studying neuron-glia interactions in the central nervous system. Here we develop a system to mimic the metabotropic glutamate receptor-dependent response of astrocytes, a glial cell type, following synaptic glutamate release. For this, we expressed an exogeneous orphan Gq-coupled protein of the Mas-related-gene (Mrg) family in glial cells and generated an MrgR's agonist peptide (FMRFa) that was chemically caged with a nitroveratryl photolabile protecting group (NV). NV has an appropriate quantum yield and a high absorption maximum that makes it very adapted to experiments with very short irradiation time. This novel caged compound allowed the activation of MrgR with both single- and two-photon light sources. Indeed, MrgR activation induced calcium transients and morphological changes in astrocytes as described previously. Thus, FMRFaNV is a very promising tool to study neuron-glia interactions.

Cell-permeant and photocleavable chemical inducer of dimerization.

Chemical inducers of dimerization (CIDs) have been developed to orchestrate protein dimerization and translocation. Here we present a novel photocleavable HaloTag- and SNAP-tag-reactive CID (MeNV-HaXS) with excellent selectivity and intracellular reactivity. Excitation at 360 nm cleaves the methyl-6-nitroveratryl core of MeNV-HaXS. MeNV-HaXS covalently links HaloTag- and SNAP-tag fusion proteins, and enables targeting of selected membranes and intracellular organelles. MeNV-HaXS-mediated translocation has been validated for plasma membrane, late endosomes, lysosomes, Golgi, mitochondria, and the actin cytoskeleton. Photocleavage of MeNV-HaXS liberates target proteins and provides access to optical manipulation of protein relocation with high spatiotemporal and subcellular precision. MeNV-HaXS supports kinetic studies of protein dynamics and the manipulation of subcellular enzyme activities, which is exemplified for Golgi-targeted cargo and the assessment of nuclear import kinetics.

A Simple Reaction for DNA Sensing and Chemical Delivery.

Reactions templated by nucleic acids are currently at the heart of applications in biosensing and drug release. The number of chemical reactions selectively occurring only in the presence of the template, in aqueous solutions, and at room temperature and able to release a chemical moiety is still very limited. Here, we report the use of the p-nitrophenyl carbonate (NPC) as a new reactive moiety for DNA templated reactions releasing a colored reporter by reaction with a simple amine. The easily synthesized p-nitrophenyl carbonate was integrated in an oligonucleotide and showed a very good stability as well as a high reactivity toward amines, without the need for any supplementary reagent, quantitatively releasing the red p-nitrophenolate with a half-life of about 1 h.

Activity-dependent structural plasticity of perisynaptic astrocytic domains promotes excitatory synapse stability.

BACKGROUND: Excitatory synapses in the CNS are highly dynamic structures that can show activity-dependent remodeling and stabilization in response to learning and memory. Synapses are enveloped with intricate processes of astrocytes known as perisynaptic astrocytic processes (PAPs). PAPs are motile structures displaying rapid actin-dependent movements and are characterized by Ca(2+) elevations in response to neuronal activity. Despite a debated implication in synaptic plasticity, the role of both Ca(2+) events in astrocytes and PAP morphological dynamics remain unclear. RESULTS: In the hippocampus, we found that PAPs show extensive structural plasticity that is regulated by synaptic activity through astrocytic metabotropic glutamate receptors and intracellular calcium signaling. Synaptic activation that induces long-term potentiation caused a transient PAP motility increase leading to an enhanced astrocytic coverage of the synapse. Selective activation of calcium signals in individual PAPs using exogenous metabotropic receptor expression and two-photon uncaging reproduced these effects and enhanced spine stability. In vivo imaging in the somatosensory cortex of adult mice revealed that increased neuronal activity through whisker stimulation similarly elevates PAP movement. This in vivo PAP motility correlated with spine coverage and was predictive of spine stability. CONCLUSIONS: This study identifies a novel bidirectional interaction between synapses and astrocytes, in which synaptic activity and synaptic potentiation regulate PAP structural plasticity, which in turn determines the fate of the synapse. This mechanism may represent an important contribution of astrocytes to learning and memory processes.