Two-photon Uncaging Research Articles

Improved Synthesis of Caged Glutamate and Caging Each Functional Group.

Glutamate is an excitatory neurotransmitter that controls numerous pathways in the brain. Neuroscientists make use of photoremovable protecting groups, also known as cages, to release glutamate with precise spatial and temporal control. Various cage designs have been developed and among the most effective has been the nitroindolinyl caging of glutamate. We, hereby, report an improved synthesis of one of the current leading molecules of caged glutamate, 4-carboxymethoxy-5,7-dinitroindolinyl glutamate (CDNI-Glu), which possesses efficiencies with the highest reported quantum yield of at least 0.5. We present the shortest route, to date, for the synthesis of CDNI-Glu in 4 steps, with a total reaction time of 40 h and an overall yield of 20%. We also caged glutamate at the other two functional groups, thereby, introducing two new cage designs: α-CDNI-Glu and N-CDNI-Glu. We included a study of their photocleavage properties using UV-vis, NMR, as well as a physiology experiment of a two-photon uncaging of CDNI-Glu in acute hippocampal brain slices. The newly introduced cage designs may have the potential to minimize the interference that CDNI-Glu has with the GABAA receptor. We are broadly disseminating this to enable neuroscientists to use these photoactivatable tools.

One- and Two-Photon Uncaging: Carbazole Fused o-Hydroxycinnamate Platform for Dual Release of Alcohols (Same or Different) with Real-Time Monitoring.

A one- and two-photon activated photoremovable protecting group (PRPG) was designed based on a carbazole fused o-hydroxycinnamate platform for the dual (same or different) release of alcohols. The mechanism for the dual release proceeds through a stepwise pathway and also monitors the first and second photorelease in real time by an increase in fluorescence intensity and color change, respectively. Further, its application in staining live neurons and ex vivo imaging with two-photon excitation is shown.

Comparative one- and two-photon uncaging of MNI-glutamate and MNI-kainate on hippocampal CA1 neurons

BackgroundThe light-induced release of neurotransmitters from caging chromophores provides a powerful means to study the underlying receptors in a physiologically relevant context. Surprisingly, most caged neurotransmitters, including the widely used 4-methoxy-7-nitroindolinyl (MNI)-glutamate, show strong antagonism against GABA-A receptors. Kainate has been shown to exhibit a higher efficacy at glutamate receptors compared to glutamate itself. Thus, uncaging of kainate might allow the application of the caged compound at lower, less antagonistic concentrations. New methodsThis study provides a detailed comparison of MNI-glutamate and MNI–kainate uncaging by different modes of one- and two-photon irradiation on hippocampal CA1 pyramidal neurons in acute brain slices. Results/Comparison with existing methodsUnexpectedly, the data revealed that currents in response to MNI-glutamate uncaging were larger compared to MNI-kainate with local one-photon laser uncaging at the soma and two-photon uncaging at the same spines. Furthermore, the direct comparison demonstrates the influence of type of caged agonist and light delivery conditions used for uncaging on the amplitude and kinetic properties of the current response. ConclusionThese findings highlight the importance of experimental design for uncaging experiments and provide a basis for future studies employing one- and two-photon uncaging to understand glutamate-dependent processes. It further provides the first example of two-photon uncaging of kainate at single spines in acute brain slices.

Three-Dimensional Control of DNA Hybridization by Orthogonal Two-Color Two-Photon Uncaging.

We successfully introduced two-photon-sensitive photolabile groups ([7-(diethylamino)coumarin-4-yl]methyl and p-dialkylaminonitrobiphenyl) into DNA strands and demonstrated their suitability for three-dimensional photorelease. To visualize the uncaging, we used a fluorescence readout based on double-strand displacement in a hydrogel and in neurons. Orthogonal two-photon uncaging of the two cages is possible, thus enabling complex scenarios of three-dimensional control of hybridization with light.

Two-photon uncaging, from neuroscience to materials

The use of photolabile protecting groups has been growing in emphasis for decades, in particular because of their numerous applications ranging from organic synthesis to neurosciences. More recently, two-photon sensitive photolabile protecting groups were developed, bringing the advantages (e.g. finer spatial resolution with a deeper tissue penetration) of this nonlinear excitation technique to a photostimulation method. However, the widespread photolabile protecting groups developed for classical one-photon excitation exhibited low two-photon sensitivity. Therefore, the rules of molecular engineering pointed out for the optimization of nonlinear properties of molecular systems for material sciences were applied to this specific field. Consequently, efficient two-photon photolabile protecting groups have been developed. We describe here the recent developments in molecular engineering of two-photon sensitive photolabile protecting groups as well as their application in neurobiology, physiology and biomaterials.

Calcium Uncaging with Visible Light.

We have designed a nitroaromatic photochemical protecting group that absorbs visible light in the violet-blue range. The chromophore is a dinitro derivative of bisstyrylthiophene (or BIST) that absorbs light very effectively (ε440 = 66,000 M(-1) cm(-1) and two-photon cross section of 350 GM at 775 nm). We developed a "caged calcium" molecule by conjugation of BIST to a Ca(2+) chelator that upon laser flash photolysis rapidly releases Ca(2+) in <0.2 ms. Using the patch-clamp method the optical probe, loaded with Ca(2+), was delivered into acutely isolated mouse cardiac myocytes, where either one- and two-photon uncaging of Ca(2+) induced highly local or cell-wide physiological Ca(2+) signaling events.

One and Two-Photon Calcium Uncaging with Visible Light in Cardiac Myocytes

In cardiomyocytes a small amount of Ca2+ enters the cytoplasm upon depolarization and initiates Ca2+-induced Ca2+ release from the sarcoplasmic reticulum (SR). Spontaneous SR Ca2+ release events can remain highly localized or initiate Ca2+ waves that propagate through the cell. A powerful optical method for spatio-temporal control of [Ca2+]i is one- and two-photon uncaging. We have designed a nitroaromatic photochemical protecting group absorbing visible light very efficiently in the violet to blue range. The chromophore is a dinitro derivative of bisstyrylthiophene bearing two EGTA chelators (BIST-2EGTA). BIST-2EGTA has an absorption maximum at 440 nm, quantum yield of photolysis of 0.23, extinction coefficient at 440 nm of 66,000 M−1cm−1 and binds Ca2+ with high affinity (Kd 84 nM at pH 7.2). Fluorescence imaging with a confocal microscope in point scan mode revealed that rhod-FF showed a rapid change in signal (<200 µs) when BIST-2EGTA was photolyzed using two-photon excitation at 810 nm. Compared to DM-nitrophen the fluorescence signal from BIST-2EGTA was ∼13.7x larger when photolyzed under the same conditions and could be elicited on physiologically relevant time-scales (<1 ms). The efficiency of BIST-2EGTA to create changes in [Ca2+]i that are useful for physiological studies was examined in whole-cell patch-clamped cardiomyocytes loaded with BIST-2EGTA and rhod-2 or X-rhod-5F. Short (1 ms) two-photon excitation at 810 nm produced localized Ca2+ releases that were considerably larger than those produced by BIST-2EGTA photolysis in caffeine-treated cells, suggesting active SR Ca2+ release. Uncaging for longer periods triggered intracellular Ca2+ “mini-waves” or propagating Ca2+ waves. Cells displayed normal excitation-contraction coupling, implying that BIST-2EGTA is nontoxic. One-photon uncaging at 405 nm also produced striking Ca2+ waves. BIST-2EGTA has a set of properties that enables highly efficient and fast control of Ca2+inside living cells using visible light.

Targeted pruning of a neuron's dendritic tree via femtosecond laser dendrotomy.

Neurons are classified according to action potential firing in response to current injection. While such firing patterns are shaped by the composition and distribution of ion channels, modelling studies suggest that the geometry of dendritic branches also influences temporal firing patterns. Verifying this link is crucial to understanding how neurons transform their inputs to output but has so far been technically challenging. Here, we investigate branching-dependent firing by pruning the dendritic tree of pyramidal neurons. We use a focused ultrafast laser to achieve highly localized and minimally invasive cutting of dendrites, thus keeping the rest of the dendritic tree intact and the neuron functional. We verify successful dendrotomy via two-photon uncaging of neurotransmitters before and after dendrotomy at sites around the cut region and via biocytin staining. Our results show that significantly altering the dendritic arborisation, such as by severing the apical trunk, enhances excitability in layer V cortical pyramidal neurons as predicted by simulations. This method may be applied to the analysis of specific relationships between dendritic structure and neuronal function. The capacity to dynamically manipulate dendritic topology or isolate inputs from various dendritic domains can provide a fresh perspective on the roles they play in shaping neuronal output.

Design and synthesis of a new chromophore, 2-(4-nitrophenyl)benzofuran, for two-photon uncaging using near-IR light.

A new chromophore, 2-(4-nitrophenyl)benzofuran (NPBF), was designed for two-photon (TP) uncaging using near-IR light. The TP absorption (TPA) cross-sections of the newly designed NPBF chromophore were determined to be 18 GM at 720 nm and 54 GM at 740 nm in DMSO. The TP uncaging reaction of a caged benzoate with the NPBF chromophore quantitatively produced benzoic acid with an efficiency (δu) of ∼5.0 GM at 740 nm. The TP fragmentation of an EGTA unit was observed with δu = 16 GM. This behavior makes the new chromophore a promising TP photoremovable protecting group for physiological studies.

Local Postsynaptic Voltage-Gated Sodium Channel Activation in Dendritic Spines of Olfactory Bulb Granule Cells

Neuronal dendritic spines have been speculated to function as independent computational units, yet evidence for active electrical computation in spines is scarce. Here we show that strictly local voltage-gated sodium channel (Nav) activation can occur during excitatory postsynaptic potentials in the spines of olfactory bulb granule cells, which we mimic and detect via combined two-photon uncaging of glutamate and calcium imaging in conjunction with whole-cell recordings. We find that local Nav activation boosts calcium entry into spines through high-voltage-activated calcium channels and accelerates postsynaptic somatic depolarization, without affecting NMDA receptor-mediated signaling. Hence, Nav-mediated boosting promotes rapid output from the reciprocal granule cell spine onto the lateral mitral cell dendrite and thus can speed up recurrent inhibition. This striking example of electrical compartmentalization both adds to the understanding of olfactory network processing and broadens the general view of spine function.

Cooperative dyads for two-photon uncaging.

A series of dyads that combine a photolabile protecting group (PPG) 4,5-dimethoxy-2-nitrobenzyl and different bis-donor or bis-acceptor dissymmetric chromophores acting as two-photon (2P) absorbers were synthesized. Even for low energy transfer efficiency from the 2PA subunit to the uncaging one, improvement of the 2P uncaging sensitivity in the NIR is achieved as compared to isolated PPG. Moreover enhancement of the 2PA response is achieved by tuning the electronic dissymmetry of the 2PA subunit and the arrangement of the complementary subunits in the dyads.

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.

Lab-on-a-brain: implantable micro-optical fluidic devices for neural cell analysis in vivo.

The high-resolution imaging of neural cells in vivo has brought about great progress in neuroscience research. Here, we report a novel experimental platform, where the intact brain of a living mouse can be studied with the aid of a surgically implanted micro-optical fluidic device; acting as an interface between neurons and the outer world. The newly developed device provides the functions required for the long-term and high-resolution observation of the fine structures of neurons by two-photon laser scanning microscopy and the microfluidic delivery of chemicals or drugs directly into the brain. A proof-of-concept experiment of single-synapse stimulation by two-photon uncaging of caged glutamate and observation of dendritic spine shrinkage over subsequent days demonstrated a promising use for the present technology.

Activity-Dependent Structural Plasticity of Perisynaptic Astrocytic Domains Promotes Excitatory Synapse Stability

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. 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. 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.

Impact of subthreshold membrane potential on synaptic responses at dendritic spines of layer 5 pyramidal neurons in the prefrontal cortex

Glutamatergic inputs onto cortical pyramidal neurons are received and initially processed at dendritic spines. AMPA and NMDA receptors generate both synaptic potentials and calcium (Ca) signals in the spine head. These responses can in turn activate a variety of Ca, sodium (Na), and potassium (K) channels at spines. In principle, the roles of these receptors and channels can be strongly regulated by the subthreshold membrane potential. However, the impact of different receptors and channels has usually been studied at the level of dendrites. Much less is known about their influence at spines, where synaptic transmission and plasticity primarily occur. Here we examine single-spine responses in the basal dendrites of layer 5 pyramidal neurons in the mouse prefrontal cortex. Using two-photon microscopy and two-photon uncaging, we first show that synaptic potentials and Ca signals differ at resting and near-threshold potentials. We then determine how subthreshold depolarizations alter the contributions of AMPA and NMDA receptors to synaptic responses. We show that voltage-sensitive Ca channels enhance synaptic Ca signals but fail to engage small-conductance Ca-activated K (SK) channels, which require greater numbers of inputs. Finally, we establish how the subthreshold membrane potential controls the ability of voltage-sensitive Na channels and K channels to influence synaptic responses. Our findings reveal how subthreshold depolarizations promote electrical and biochemical signaling at dendritic spines by regulating the contributions of multiple glutamate receptors and ion channels.

Synthesis and biological evaluation of bis-CNB-GABA, a photoactivatable neurotransmitter with low receptor interference and chemical two-photon uncaging properties.

Photoactivatable “caged” neurotransmitters allow optical control of neural tissue with high spatial and temporal precision. However, the development of caged versions of the chief vertebrate inhibitory neurotransmitter, γ-amino butyric acid (GABA), has been limited by the propensity of caged GABAs to interact with GABA receptors. We describe herein the synthesis and application of a practically useful doubly caged GABA analog, termed bis-α-carboxy-2-nitrobenzyl-GABA (bis-CNB-GABA). Uncaging of bis-CNB-GABA evokes inward GABAergic currents in cerebellar molecular layer interneurons with rise times of 2 ms, comparable to flash duration. Response amplitudes depend on the square of flash intensity, as expected for a chemical two-photon uncaging effect. Importantly, prior to uncaging, bis-CNB-GABA is inactive at the GABAA receptor, evoking no changes in holding current in voltage-clamped neurons and showing an IC50 of at least 2.5 mM as measured using spontaneous GABAergic synaptic currents. Bis-CNB-GABA is stable in solution, with an estimated half-life of 98 days in the light. We expect that bis-CNB-GABA will prove to be an effective tool for high-resolution chemical control of brain circuits.

Light-Induced Remodeling of Physically Crosslinked Hydrogels Using Near-IR Wavelengths.

Poly([6-bromo-7-hydroxycoumarin-4-yl]methyl methacrylate)-based triblock copolymers were synthesized by first preparing PMAA-PEG-PMAA triblocks using ATRP. 6-Bromo-4-chloromethyl-7-hydroxycoumarin was conjugated to PMAA-PEG-PMAA using 1,8-diazabicycloundec-7-ene (DBU). Rheological measurements were conducted using a HR-2 rheometer with a UV illumination accessory (TA Instruments). Single-photon uncaging was performed as previously described.20 Two-photon uncaging was performed using a LSM (Zeiss) equipped with a Ti: Sapphire laser (Coherent). All values reported as mean ± std. dev. unless otherwise stated. See Supporting Information for experimental details.

Vasodilation byin vivoactivation of astrocyte endfeet via two-photon calcium uncaging as a strategy to prevent brain ischemia

Decreased cerebral blood flow causes brain ischemia and plays an important role in the pathophysiology of many neurodegenerative diseases, including Alzheimer's disease and vascular dementia. In this study, we photomodulated astrocytes in the live animal by a combination of two-photon calcium uncaging in the astrocyte endfoot and in vivo imaging of neurovasculature and astrocytes by intravital two-photon microscopy after labeling with cell type specific fluorescent dyes. Our study demonstrates that photomodulation at the endfoot of a single astrocyte led to a 25% increase in the diameter of a neighboring arteriole, which is a crucial factor regulating cerebral microcirculation in downstream capillaries. Two-photon uncaging in the astrocyte soma or endfoot near veins does not show the same effect on microcirculation. These experimental results suggest that infrared photomodulation on astrocyte endfeet may be a strategy to increase cerebral local microcirculation and thus prevent brain ischemia.

Wavelength-Selective One- and Two-Photon Uncaging of GABA

We have synthesized photolabile 7-diethylamino coumarin (DEAC) derivatives of γ-aminobutyric acid (GABA). These caged neurotransmitters efficiently release GABA using linear or nonlinear excitation. We used a new DEAC-based caging chromophore that has a vinyl acrylate substituent at the 3-position that shifts the absorption maximum of DEAC to about 450 nm and thus is named "DEAC450". DEAC450-caged GABA is photolyzed with a quantum yield of 0.39 and is highly soluble and stable in physiological buffer. We found that DEAC450-caged GABA is relatively inactive toward two-photon excitation at 720 nm, so when paired with a nitroaromatic caged glutamate that is efficiently excited at such wavelengths, we could photorelease glutamate and GABA around single spine heads on neurons in brain slices with excellent wavelength selectivity using two- and one-photon photolysis, respectively. Furthermore, we found that DEAC450-caged GABA could be effectively released using two-photon excitation at 900 nm with spatial resolution of about 3 μm. Taken together, our experiments show that the DEAC450 caging chromophore holds great promise for the development of new caged compounds that will enable wavelength-selective, two-color interrogation of neuronal signaling with excellent subcellular resolution.

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.