Graham C. R. Ellis-Davies

A critical time window for dopamine actions on the structural plasticity of dendritic spines

Animal behavior follows rewards Animal behavior is learned and reinforced by rewards. On a molecular level, the reward comes in the form of the neurotransmitter, dopamine, which modulates synapses. The exact timing and mechanism of this process remain unknown. Using optical stimulation, Yagishita et al. found that dopaminergic modulation involved dendritic spine enlargement only during an extremely narrow time window. Known as reinforcement plasticity, this cellular basis for learning could provide insight into psychiatric disorders involving dopaminergic regulation, such as depression, drug addiction, and schizophrenia. Science, this issue p. 1616 Dopamine promotes spine structural plasticity during a narrow time window in mouse neuron distal dendrites. Animal behaviors are reinforced by subsequent rewards following within a narrow time window. Such reward signals are primarily coded by dopamine, which modulates the synaptic connections of medium spiny neurons in the striatum. The mechanisms of the narrow timing detection, however, remain unknown. Here, we optically stimulated dopaminergic and glutamatergic inputs separately and found that dopamine promoted spine enlargement only during a narrow time window (0.3 to 2 seconds) after the glutamatergic inputs. The temporal contingency was detected by rapid regulation of adenosine 3′,5′-cyclic monophosphate in thin distal dendrites, in which protein-kinase A was activated only within the time window because of a high phosphodiesterase activity. Thus, we describe a molecular basis of reinforcement plasticity at the level of single dendritic spines.

Compartmentalization of GABAergic Inhibition by Dendritic Spines

Dendritic Precision Strikes The effects of excitatory synaptic inputs are considered to be highly compartmentalized because of the biophysical properties of dendritic spines. Individual inhibitory synapses, however, are thought to affect dendritic integration in a more extended spatial region. Combining optogenetic stimulation of dendrite-targeting γ-aminobutyric acid—mediated interneurons with two-photon calcium imaging in postsynaptic pyramidal cell dendrites, Chiu et al. (p. 759) challenge this latter view. The findings suggest that the effect of an inhibitory synapse can be as compartmentalized as that of an excitatory synapse, provided that the synapses are localized on spine heads. Inhibitory synapses can control individual dendritic spines independently from their neighbors. γ-aminobutyric acid–mediated (GABAergic) inhibition plays a critical role in shaping neuronal activity in the neocortex. Numerous experimental investigations have examined perisomatic inhibitory synapses, which control action potential output from pyramidal neurons. However, most inhibitory synapses in the neocortex are formed onto pyramidal cell dendrites, where theoretical studies suggest they may focally regulate cellular activity. The precision of GABAergic control over dendritic electrical and biochemical signaling is unknown. By using cell type-specific optical stimulation in combination with two-photon calcium (Ca2+) imaging, we show that somatostatin-expressing interneurons exert compartmentalized control over postsynaptic Ca2+ signals within individual dendritic spines. This highly focal inhibitory action is mediated by a subset of GABAergic synapses that directly target spine heads. GABAergic inhibition thus participates in localized control of dendritic electrical and biochemical signaling.

Disruption of astrocyte–vascular coupling and the blood–brain barrier by invading glioma cells

Astrocytic endfeet cover the entire cerebral vasculature and serve as exchange sites for ions, metabolites, and energy substrates from the blood to the brain. They maintain endothelial tight junctions that form the blood-brain barrier (BBB) and release vasoactive molecules that regulate vascular tone. Malignant gliomas are highly invasive tumors that use the perivascular space for invasion and co-opt existing vessels as satellite tumors form. Here we use a clinically relevant mouse model of glioma and find that glioma cells, as they populate the perivascular space of pre-existing vessels, displace astrocytic endfeet from endothelial or vascular smooth muscle cells. This causes a focal breach in the BBB. Furthermore, astrocyte-mediated gliovascular coupling is lost, and glioma cells seize control over regulation of vascular tone through Ca2+-dependent release of K+. These findings have important clinical implications regarding blood flow in the tumor-associated brain and the ability to locally deliver chemotherapeutic drugs in disease.

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

Activity-dependent competition of synapses plays a key role in neural organization and is often promoted by GABA; however, its cellular bases are poorly understood. Excitatory synapses of cortical pyramidal neurons are formed on small protrusions known as dendritic spines, which exhibit structural plasticity. We used two-color uncaging of glutamate and GABA in rat hippocampal CA1 pyramidal neurons and found that spine shrinkage and elimination were markedly promoted by the activation of GABAA receptors shortly before action potentials. GABAergic inhibition suppressed bulk increases in cytosolic Ca2+ concentrations, whereas it preserved the Ca2+ nanodomains generated by NMDA-type receptors, both of which were necessary for spine shrinkage. Unlike spine enlargement, spine shrinkage spread to neighboring spines (<15 μm) and competed with their enlargement, and this process involved the actin-depolymerizing factor ADF/cofilin. Thus, GABAergic inhibition directly suppresses local dendritic Ca2+ transients and strongly promotes the competitive selection of dendritic spines.

Recombinant probes for visualizing endogenous synaptic proteins in living neurons

The ability to visualize endogenous proteins in living neurons provides a powerful means to interrogate neuronal structure and function. Here we generatexa0recombinant antibody-like proteins, termed Fibronectin intrabodies generated with mRNA display (FingRs), that bind endogenous neuronal proteins PSD-95 and Gephyrin with high affinity and that, when fused to GFP, allow excitatory and inhibitory synapses to be visualized in living neurons. Design of the FingR incorporates a transcriptional regulation system that ties FingR expression to the level of the target and reduces background fluorescence. In dissociated neurons and brain slices, FingRs generated against PSD-95 and Gephyrin did not affect thexa0expression patterns of their endogenous target proteins or the number or strength of synapses. Together, our data indicate that PSD-95 and Gephyrin FingRs can report the localization and amount of endogenous synaptic proteins in living neurons and thus may be used to study changes in synaptic strength inxa0vivo.

Optically Selective Two-Photon Uncaging of Glutamate at 900 nm

We have synthesized a 7-diethylaminocoumarin (DEAC) derivative that allows wavelength-selective two-photon uncaging at 900 nm versus 720 nm. This new caging chromophore, called DEAC450, has an extended π-electron moiety at the 3-position that shifts the absorption spectrum maximum of DEAC from 375 to 450 nm. Two-photon excitation at 900 nm was more than 60-fold greater than at 720 nm. Two-photon uncaging of DEAC450-Glu at 900 nm at spine heads on pyramidal neurons in acutely isolated brain slices generated postsynaptic responses that were similar to spontaneous postsynaptic excitatory miniature currents, whereas significantly higher energies at 720 nm evoked no currents. Since many nitroaromatic caged compounds are two-photon active at 720 nm, optically selective uncaging of DEAC450-caged biomolecules at 900 nm may allow facile two-color optical interrogation of bimodal signaling pathways in living tissue with high resolution for the first time.

Calcium Sensitivity of Neurotransmitter Release Differs at Phasic and Tonic Synapses

The efficacy of synaptic transmission varies greatly among synaptic contacts. We have explored the origins of differences between phasic and tonic crustacean neuromuscular junctions. Synaptic boutons of a phasic motor neuron release three orders of magnitude more quanta to a single action potential and show strong depression to a train, whereas tonic synapses are nearly unresponsive to single action potentials and display an immense facilitation. Phasic and tonic synapses display a similar nonlinear dependence on extracellular [Ca2+]. We imposed similar spatially uniform intracellular [Ca2+] ([Ca2+]i) steps in phasic and tonic synapses by photolysis of presynaptic caged calcium. [Ca2+]i was measured fluorometrically while transmitter release was monitored electrophysiologically from single boutons in which the [Ca2+]i was elevated. Phasic synapses released the readily releasable pool (RRP) of vesicles at a much higher rate and with a shorter delay than did tonic synapses. Comparison of several kinetic models of molecular events showed that a difference in Ca2+-sensitive priming of vesicles in the RRP combined with a revision of the kinetic Ca2+-binding sequence to the secretory trigger produced the best fit to the markedly different responses to Ca2+ steps and action potentials and of the characteristic features of synaptic plasticity in phasic and tonic synapses. The results reveal processes underlying one aspect of synaptic diversity that may also regulate changes in synaptic strength during development and learning and memory formation.

Three-Dimensional Mapping of Unitary Synaptic Connections by Two-Photon Macro Photolysis of Caged Glutamate

To understand the precise microarchitecture of the cortical circuitry, it is crucial to know the distribution of synaptic connections and their synaptic strengths at the level of a single cell, rather than a group of cells. Here, we describe a new application of two-photon photolysis of caged glutamate that enabled us to induce an action potential in only a small number (about five) of pyramidal neurons by increasing the volume of two-photon excitation by reducing the effective numerical aperture of the objective. We performed whole cell patch-clamp recordings from layer 2/3 pyramidal neurons in the rat visual cortex and stimulated many neurons in a large three-dimensional space (approximately 600 x 600 x 100 microm) including neurons in layers 2/3 and 4 using this new technique. We mapped the density and amplitude of unitary excitatory postsynaptic currents and found that the basic microarchitecture of excitatory synaptic connections consists of two regions: a columnar, dense core region with a radius of 150 microm and an outer, sparse region. The dense core region includes the majority of strong synaptic connections in layer 2/3. Our results reveal the columnar organization of synaptic connectivity in the rat visual cortex, where functional columns have not been clearly demonstrated. Thus this technique will be a uniquely powerful tool for quantifying synaptic connectivity and manipulating neural activity at the single-cell level.

Spectral Evolution of a Photochemical Protecting Group for Orthogonal Two-Color Uncaging with Visible Light

Caged compounds are molecules rendered functionally inert by derivatization with a photochemical protecting group. We describe the design logic behind the development of a diethylaminocoumarin (DEAC) caging chromophore, DEAC450, that absorbs blue light strongly (ε450 = 43,000 M(-1) cm(-1)) and violet light 11-fold more weakly. The absorption minimum is in the wavelength range (340-360 nm) that is traditionally used for photolysis of many widely used nitroaromatic caged compounds (e.g., 4-carboxymethoxy-5,7-dinitroindolinyl(CDNI)-GABA). We used this chromophore to synthesize DEAC450-caged cAMP and found this probe was very stable toward aqueous hydrolysis in the electronic ground state but was photolyzed with a quantum efficiency of 0.78. When DEAC450-cAMP and CDNI-GABA where co-applied to striatal cholinergic interneurons, the caged compounds were photolyzed in an chromatically orthogonal manner using blue and violet light so as to modulate the neuronal firing rate in a bidirectional way.

Caged compounds for multichromic optical interrogation of neural systems

Caged compounds are widely used by neurophysiologists to study many aspects of cellular signaling in glia and neurons. Biologically inert before irradiation, they can be loaded into cells via patch pipette or topically applied in situ to a defined concentration; photolysis releases the caged compound in a very rapid and spatially defined way. As caged compounds are exogenous optical probes, they include not only natural products such neurotransmitters, calcium and IP3 but non‐natural products such as fluorophores, drugs and antibodies. In this Technical Spotlight we provide a short introduction to the uncaging technique by discussing the nitroaromatic caging chromophores most widely used in such experiments [e.g. α‐carboxy‐ortho‐nitrobenyl (CNB), dimethoxynitrobenzyl (DMNB), 4‐methoxy‐7‐nitroindolinyl (MNI) and 4‐carboxymethoxy‐7‐nitroindolinyl (CDNI)]. We show that recently developed caging chromophores [rutheniumbipyridial (RuBi) and 7‐diethylaminocoumarin (DEAC)450] that are photolyzed with blue light (~ 430–480 nm range) can be combined with traditional nitroaromatic caged compounds to enable two‐color optical probing of neuronal function. For example, one‐photon uncaging of either RuBi‐GABA or DEAC450‐GABA with a 473‐nm laser is facile, and can block nonlinear currents (dendritic spikes or action potentials) evoked by two‐photon uncaging of CDNI‐Glu at 720 nm. We also show that two‐photon uncaging of DEAC450‐Glu and CDNI‐GABA at 900 and 720 nm, respectively, can be used to fire and block action potentials. Our experiments illustrate that recently developed chromophores have taken uncaging out of the ‘monochrome era’, in which it has existed since 1978, so as to enable multichromic interrogation of neuronal function with single‐synapse precision.