In vivo two‐photon uncaging of glutamate revealing the structure–function relationships of dendritic spines in the neocortex of adult mice

Abstract

Non-technical summary Neurons communicate with each other with synapses using chemical messengers. The major synapses in the cerebral cortex utilize glutamate as a messenger and are made on special submicron structures, called dendritic spines. Dendritic spines are diverse in their size and densely packed in the cortex. Therefore, an optical technique for application of glutamate to single spines (two-photon (TP) uncaging) has been intensively used to clarify their functions in vitro. We have here extended 2P uncaging to living adult brain, and found that spine sizes display tight correlations with their functions, such as rapid glutamate sensing and an increase in cytosolic Ca 2+ concentrations, even in vivo, as they were reported for in vitro preparations. Our data suggest that the structure and motility of dendritic spines play a key role in the adult brain function.Abstract Two-photon (2P) uncaging of caged neurotransmitters can efficiently stimulate individual synapses and is widely used to characterize synaptic functions in brain slice preparations. Here we extended 2P uncaging to neocortical pyramidal neurons in adult mice in vivo where caged glutamate was applied from the pial surface. To validate the methodology, we applied a small fluorescent probe using the same method, and confirmed that its concentrations were approximately homogenous up to 200 μm below the cortical surface, and that the extracellular space of the neocortex was as large as 22%. In fact, in vivo whole-cell recording revealed that 2P glutamate uncaging could elicit transient currents (2pEPSCs) very similar to excitatory postsynaptic currents (EPSCs). A spatial resolution of glutamate uncaging was 0.6-0.8 μm up to the depth of 200 μm, and in vivo 2P uncaging was able to stimulate single identified spines. Automated three-dimensional (3-D) mapping of such 2pEPSCs which covered the surfaces of dendritic branches revealed that functional AMPA receptor expression was stable and proportional to spine volume. Moreover, in vivo 2P Ca 2+ imaging and uncaging suggested that the amplitudes of glutamate-induced Ca 2+ transients were inversely proportional to spine volume. Thus, the key structure-function relationships hold in dendritic spines in adult neocortex in vivo, as in young hippocampal slice preparations. In vivo 2P uncaging will be a powerful tool to investigate properties of synapses in the neocortex.

Optochemical Genetics

“…Caged glutamate, for example, has been very useful for unraveling neural systems, and two-photon cages have enabled the stimulation of single synapses through the spatial precision that can be achieved with two-photon techniques. [34] The photosensitization of P2X 2 receptors and TRPV1 channels with caged ATP and capsaicin, respectively, was the first approach shown to work in living animals (in this case decapitated fruit flies, to ensure that the visual system was inactivated). [35] There are, however, certain functional disadvantages associated with caged ligands.…”

GABA promotes the competitive selection of dendritic spines by controlling local Ca2+ signaling

“…In pyramidal neurons of the cerebral cortices, excitatory synaptic inputs are made on small protrusions of dendrites, called dendritic spines. The sizes of spines are highly variable and correlate with glutamate sensitivity 3, 4 , and the long-term potentiation (LTP) 5, 6 and long-term depression (LTD) 7 of glutamatergic synapses are often associated with the enlargement and shrinkage of dendritic spines 8, 9 . If LTP and LTD are associated with structural changes, they can mediate selection of synapses.…”

In vivo characterization of a bigenic fluorescent mouse model of Alzheimer's disease with neurodegeneration

“…However, the FAD Swedish mutation is located not adjacent to the γ-secretase cleavage site, but to the BACE1 cleavage site. This mutation location results in increased production of all species of Aβ, without a change in the ratio between Aβ42 to Aβ40 (Anderton et al, 1998; Knobloch and Mansuy, 2008; Noguchi et al, 2011). Mutations in PS, a protein involved in the γ-secretase complex, result in an increase in Aβ42 production relative to Aβ40 (Blazquez-Llorca et al, 2011).…”