Two-photon Uncaging Research Articles

Switch the click: Ultrafast photochemistry of photoDIBO-OH tracked by time-resolved IR spectroscopy.

Click chemistry refers to selective reactions developed for grafting of bio(macro)molecules in their biological media. Caged click compounds have been employed to spatiotemporally control click reactions. Here, we survey the uncaging of photo-dibenzocyclooctyne-OH (photoDIBO-OH) to its click-chemistry active form DIBO-OH, with particular attention to its conversion timescale and efficiency. Ultraviolet pump-infrared probe experiments reveal a stepwise decarbonylation: first, carbon monoxide (C≡O) is released within 1.8ps, and then, it converts, within 10ps, to DIBO-OH. Completion of uncaging is achieved with an efficiency of ∼50%. A successful demonstration of two-photon uncaging of photoDIBO-OH at long wavelength (700nm) confers enhanced in vivo compatibility and proceeds on the same timescale.

Low throughput screening in neuroscience: using light to study central synapses one at a time.

Neurophotonic approaches have fostered substantial progress in our understanding of the brain by providing an assortment of means to either monitor or manipulate neural processes. Among these approaches, the development of two-photon uncaging provides a useful and flexible approach to manipulate the activity of individual synapses. In this short piece, we explore how this technique has emerged at the intersection of chemistry, optics, and electrophysiology to enable spatially and temporally precise photoactivation for studying functional aspects of synaptic transmission and dendritic integration. We discuss advantages and limitations of this approach, focusing on our efforts to study several functional aspects of glutamate receptors using uncaging of glutamate. Among other advancements, this approach has contributed to further our understanding of the subcellular regulation, trafficking, and biophysical features of glutamate receptors (e.g., desensitization and silent synapse regulation), the dynamics of spine calcium, and the integrative features of dendrites, and how these functions are altered by several forms of plasticity.

Fast Synaptically Activated Calcium and Sodium Kinetics in Hippocampal Pyramidal Neuron Dendritic Spines.

An accurate assessment of the time course, components, and magnitude of postsynaptic currents is important for a quantitative understanding of synaptic integration and signaling in dendritic spines. These parameters have been studied in some detail in previous experiments, primarily using two-photon imaging of [Ca2+]i changes and two-photon uncaging of glutamate. However, even with these revolutionary techniques, there are some missing pieces in our current understanding, particularly related to the time courses of synaptically evoked [Ca2+]i and [Na+]i changes. In new experiments, we used low-affinity, linear Na+ and Ca2+ indicators, laser fluorescence stimulation, and a sensitive camera-based detection system, combined with electrical stimulation and two-photon glutamate uncaging, to extend measurements of these spine parameters. We found that (1) almost all synaptically activated Na+ currents in CA1 hippocampal pyramidal neuron spines in slices from mice of either sex are through AMPA receptors with little Na+ entry through voltage-gated sodium channels (VGSCs) or NMDA receptor channels; (2) a spectrum of sodium transient decay times was observed, suggesting a spectrum of spine neck resistances, even on the same dendrite; (3) synaptically activated [Ca2+]i changes are very fast and are almost entirely because of Ca2+ entry through NMDA receptors at the time when the Mg2+ block is relieved by the fast AMPA-mediated EPSP; (4) the [Ca2+]i changes evoked by uncaging glutamate are slower than the changes evoked by synaptic release, suggesting that the relative contribution of Ca2+ entering through NMDA receptors at rest following uncaging is higher than following electrical stimulation.

Three-branched coumarin derivatives for two-photon uncaging. How does branching influence the efficacy?

In the design of non-linear optical chromophores, the conjugation pathways in multi-branched dyes are known to influence heavily their optical properties. Herein, we investigate this strategy for the design of two-photon (2P) responsive photolabile protecting groups (PPG) by assembling via a triphenylamine core extended coumarinylmethyl derivatives. The experimental study reveals an enhancement of the 2P absorption in the tri-branched compound, but a strikingly different photophysics behaviour resulting in a decrease in bond cleavage efficiency, and aggregation in aqueous acetonitrile. These combined effects results in much poorer 2P uncaging efficiency of the three-branched derivatives. In contrast, the corresponding mono-branched coumarin exhibit very high 2P photochemical efficiency (450 GM).

Involvement of GABAA receptors containing α6 subtypes in antisecretory factor activity on rat cerebellar granule cells studied by two-photon uncaging.

The antisecretory factor (AF) is an endogenous protein that counteracts intestinal hypersecretion and various inflammation conditions in vivo. It has been detected in many mammalian tissues and plasma, but its mechanisms of action are largely unknown. To study the pharmacological action of the AF on different GABAA receptor populations in cerebellar granule cells, we took advantage of the two‐photon uncaging method as this technique allows to stimulate the cell locally in well‐identified plasma membrane parts. We compared the electrophysiological response evoked by releasing a caged GABA compound on the soma, the axon initial segment and neurites before and after administering AF‐16, a 16 amino acids long peptide obtained from the amino‐terminal end of the AF protein. After the treatment with AF‐16, we observed peak current increases of varying magnitude depending on the neuronal region. Thus, studying the effects of furosemide and AF‐16 on the electrophysiological behaviour of cerebellar granules, we suggest that GABAA receptors, containing the α6 subunit, may be specifically involved in the increase of the peak current by AF, and different receptor subtype distribution may be responsible for differences in this increase on the cell.

NIR-Responsive Lysosomotropic Phototrigger: An "AIE + ESIPT" Active Naphthalene-Based Single-Component Photoresponsive Nanocarrier with Two-Photon Uncaging and Real-Time Monitoring Ability.

In recent times, organelle-targeted drug delivery systems have gained tremendous attention due to the site-specific delivery of active drug molecules, resulting in enhanced bioefficacy. In this context, a phototriggered drug delivery system (DDS) for releasing an active molecule is superior, as it provides spatial and temporal control over the release. So far, a near-infrared (NIR) light-responsive organelle-targeted DDS has not yet been developed. Hence, we introduced a two-photon NIR light-responsive lysosome-targeted "AIE + ESIPT" active single-component DDS based on the naphthalene chromophore. The two-photon absorption cross section of our DDS is 142 GM at 850 nm. The DDS was converted into pure organic nanoparticles for biological applications. Our nano-DDS is capable of selective targeting, AIE luminogenic imaging, and drug release within the lysosome. In vitro studies using cancerous cell lines showed that our single-component photoresponsive nanocarrier exhibited enhanced cytotoxicity and real-time monitoring ability of drug release.

One- and Two-Photon Uncaging of Carbon Monoxide (CO) with Real-Time Monitoring: On-Demand Carbazole-Based Dual CO-Releasing Platform to Test over Single and Combinatorial Approaches for the Efficient Regression of Orthotopic Murine Melanoma In Vivo.

Herein, we report three new metal-free, photochemically active single, dual, and combinatorial CORMs (photoCORMs) based on a carbazole-fused 1,3-dioxol-2-one moiety which released one equivalent of CO, two equivalent of CO, and a combination of one equivalent of each CO and anticancer drug upon one- and two-photon excitation, respectively. The photoCORMs exhibited good cellular uptake and real-time monitoring ability of CO uncaging by a color change approach in cancerous B16F10 cells. Interestingly, the cytotoxicity assay on B16F10 cells indicated that the dual photoCORM has increased anticancer activity over the single and combinatorial photoCORMs upon irradiation. Our results also showed that CO could accelerate the effectiveness of the well-known anticancer drug (chlorambucil). Finally, the in vivo evaluation of the dual photoCORM on an established murine melanoma tumor (C57BL/6J mouse model) manifested a significant regression of tumor volume and led to significant improvement (>50%) in the overall survivability.

P-Nitroterphenyl units for near-infrared two-photon uncaging of calcium ions

Near-infrared two-photon (NIR-2P) uncaging reactions are useful for precise investigations of the roles of biologically active compounds in various phenomena. In this study, we synthesized new NIR-2P-responsive chromophores with terphenyl backbones featuring an ethylene glycol tetraacetic acid (EGTA) unit as a Ca2+-selective chelator. The photolabile Ca2+ chelators were synthesized in high yields (83 % for 1a, 45 % for 1b) from commercially available starting materials. One- and two-photon photochemical excitations of the synthesized terphenyl derivatives featuring two different electron-donating groups (dimethylamino (NMe2) and methoxyl (OMe)), which were thermally stable at least 36 h at 37 C in HEPES buffer (pH 7.4), were conducted to clarify the substituent effect on the photophysical and photochemical properties. The NMe2 group, which is a stronger electron-donating group, has led to a higher two-photon cross-section (σ2 = 75 GM at 780 nm) compared to the OMe group (σ2 = 9 GM at 720 nm) in a pH = 7.4 HEPES buffer solution, although the molar extinction coefficient (ε) of the NMe2-substituted one (ε = 10,371 at λmax =363 nm) was lower than that of the OMe-derivative (ε = 14,351 at λmax = 344 nm). The quantum yield (Φ = 0.04) for the decomposition of the NMe2-substituted caged compound was lower than that for the OMe-substituted caged compound (Φ = 0.40). One and two-photon uncaging efficiency values of the OMe substituted compound, ε × Φ = 5740 and σ2 × Φ = 3.6, were higher than those of the NMe2-substituted compound, 415 and 3.0, respectively. Laser flash photolysis experiments clarified the decay rate constants (2.5 × 106 and 1.1 × 102 s−1) of aci-nitro intermediates formed during the uncaging process. A concentration jump of Ca2+ was confirmed in the photolysis of caged calcium compounds newly synthesized in this study, indicating the terphenyl derivatives can be also utilized in physiological experiments. The corresponding acetoxymethyl esters were also prepared for physiological studies.

Two-photon uncaging of bioactive thiols in live cells at wavelengths above 800 nm.

Photoactivatable protecting groups (PPGs) are useful for a broad range of applications ranging from biology to materials science. In chemical biology, induction of biological processes via photoactivation is a powerful strategy for achieving spatiotemporal control. The importance of cysteine, glutathione, and other bioactive thiols in regulating protein structure/activity and cell redox homeostasis makes modulation of thiol activity particularly useful. One major objective for enhancing the utility of photoactivatable protecting groups (PPGs) in living systems is creating PPGs with longer wavelength absorption maxima and efficient two-photon (TP) absorption. Toward these objectives, we developed a carboxyl- and dimethylamine-functionalized nitrodibenzofuran PPG scaffold (cDMA-NDBF) for thiol photoactivation, which has a bathochromic shift in the one-photon absorption maximum from λmax = 315 nm with the unfunctionalized NDBF scaffold to λmax = 445 nm. While cDMA-NDBF-protected thiols are stable in the presence of UV irradiation, they undergo efficient broad-spectrum TP photolysis at wavelengths as long as 900 nm. To demonstrate the wavelength orthogonality of cDMA-NDBF and NDBF photolysis in a biological setting, caged farnesyltransferase enzyme inhibitors (FTI) were prepared and selectively photoactivated in live cells using 850-900 nm TP light for cDMA-NDBF-FTI and 300 nm UV light for NDBF-FTI. These experiments represent the first demonstration of thiol photoactivation at wavelengths above 800 nm. Consequently, cDMA-NDBF-caged thiols should have broad applicability in a wide range of experiments in chemical biology and materials science.

Presynaptic NMDARs cooperate with local spikes toward GABA release from the reciprocal olfactory bulb granule cell spine.

In the rodent olfactory bulb the smooth dendrites of the principal glutamatergic mitral cells (MCs) form reciprocal dendrodendritic synapses with large spines on GABAergic granule cells (GC), where unitary release of glutamate can trigger postsynaptic local activation of voltage-gated Na+-channels (Navs), that is a spine spike. Can such single MC input evoke reciprocal release? We find that unitary-like activation via two-photon uncaging of glutamate causes GC spines to release GABA both synchronously and asynchronously onto MC dendrites. This release indeed requires activation of Navs and high-voltage-activated Ca2+-channels (HVACCs), but also of NMDA receptors (NMDAR). Simulations show temporally overlapping HVACC- and NMDAR-mediated Ca2+-currents during the spine spike, and ultrastructural data prove NMDAR presence within the GABAergic presynapse. This cooperative action of presynaptic NMDARs allows to implement synapse-specific, activity-dependent lateral inhibition, and thus could provide an efficient solution to combinatorial percept synthesis in a sensory system with many receptor channels.

Local Postsynaptic Signaling on Slow Time Scales in Reciprocal Olfactory Bulb Granule Cell Spines Matches Asynchronous Release.

In the vertebrate olfactory bulb (OB), axonless granule cells (GC) mediate self- and lateral inhibitory interactions between mitral/tufted cells via reciprocal dendrodendritic synapses. Locally triggered release of GABA from the large reciprocal GC spines occurs on both fast and slow time scales, possibly enabling parallel processing during olfactory perception. Here we investigate local mechanisms for asynchronous spine output. To reveal the temporal and spatial characteristics of postsynaptic ion transients, we imaged spine and adjacent dendrite Ca2 +- and Na+-signals with minimal exogenous buffering by the respective fluorescent indicator dyes upon two-photon uncaging of DNI-glutamate in OB slices from juvenile rats. Both postsynaptic fluorescence signals decayed slowly, with average half durations in the spine head of t1/2_Δ[Ca2 +]i ∼500 ms and t1/2_Δ[Na+]i ∼1,000 ms. We also analyzed the kinetics of already existing data of postsynaptic spine Ca2 +-signals in response to glomerular stimulation in OB slices from adult mice, either WT or animals with partial GC glutamate receptor deletions (NMDAR: GluN1 subunit; AMPAR: GluA2 subunit). In a large subset of spines the fluorescence signal had a protracted rise time (average time to peak ∼400 ms, range 20 to >1,000 ms). This slow rise was independent of Ca2 + entry via NMDARs, since similarly slow signals occurred in ΔGluN1 GCs. Additional Ca2 + entry in ΔGluA2 GCs (with AMPARs rendered Ca2 +-permeable), however, resulted in larger ΔF/Fs that rose yet more slowly. Thus GC spines appear to dispose of several local mechanisms to promote asynchronous GABA release, which are reflected in the time course of mitral/tufted cell recurrent inhibition.

Useful Caged Compounds for Cell Physiology.

Light has been instrumental in the study of living cells since its use helped in their discovery in the late 17th century. Further, combining chemical technology with light microscopy was an essential part of the Nobel Prize for Physiology in 1906. Such landmark scientific findings involved passive observation of cells. However, over the past 50 years, a "second use" of light has emerged in cell physiology, namely one of rational control. The seminal method for this emerged in late 1970s with the invention of caged compounds. This was the point when "caged compounds" were defined as optical probes in which the active functionality of a physiological signaling molecule was blocked with a photochemical protecting group. Caged compounds are analogous to prodrugs; in both, the activity of the effector is latent. However, caged compounds, unlike prodrugs, use a trigger that confers the power of full temporal and spatial manipulation of the effects of release of its latent biological cargo. Light is distinct because it is bio-orthogonal, passes through living tissue (even into the cell interior), and initiates rapid release of the "caged" biomolecule. Further, because light can be directed to broad areas or focused to small points, caged compounds offer an array of timing scenarios for physiologists to dissect virtually any type of cellular process.The collaborative interaction between chemists and physiologists plays a fundamental role in the development of caged compounds. First, the physiologists must define the problem to be addressed; then, with the help of chemists, decide if a caged compound would be useful. For this, structure-activity relationships of the potential optical probe and receptor must be determined. If rational targets seem feasible, synthetic organic chemistry is used to make the caged compound. The crucial property of prephotolysis bio-inertness relies on physiological or biochemical assays. Second, detailed optical characterization of the caged compound requires the skill of photochemists because the rate and efficiency of uncaging are also crucial properties for a useful caged compound. Often, these studies reveal limitations in the caged compound which has been developed; thus, chemists and physiologists use their abilities for iterative development of even more powerful optical probes. A similar dynamic will be familiar to scientists in the pharmaceutical industry. Therefore, caged compound development provides an excellent training framework for (young) chemists both intellectually and professionally. In this Account, I draw on my long experience in the field of making useful caged compounds for cell physiology by showing how each probe I have developed has been defined by an important physiological problem. Fundamental to this process has been my initial training by the pioneers in aromatic photochemistry, Derek Bryce-Smith and Andrew Gilbert. I discuss making a range of "caged calcium" probes, ones which went on to be the most widely used of all caged compounds. Then, I describe the development of caged neurotransmitters for two-photon uncaging microscopy. Finally, I survey recent work on making new photochemical protecting groups for wavelength orthogonal, two-color, and ultraefficient two-photon uncaging.

Bidirectional in vivo structural dendritic spine plasticity revealed by two-photon glutamate uncaging in the mouse neocortex

Most excitatory synapses in the brain form on dendritic spines. Two-photon uncaging of glutamate is widely utilized to characterize the structural plasticity of dendritic spines in brain slice preparations in vitro. In the present study, glutamate uncaging was used to investigate spine plasticity, for the first time, in vivo. A caged glutamate compound was applied to the surface of the mouse visual cortex in vivo, revealing the successful induction of spine enlargement by repetitive two-photon uncaging in a magnesium free solution. Notably, this induction occurred in a smaller fraction of spines in the neocortex in vivo (22%) than in hippocampal slices (95%). Once induced, the time course and mean long-term enlargement amplitudes were similar to those found in hippocampal slices. However, low-frequency (1–2 Hz) glutamate uncaging in the presence of magnesium caused spine shrinkage in a similar fraction (35%) of spines as in hippocampal slices, though spread to neighboring spines occurred less frequently than it did in hippocampal slices. Thus, the structural plasticity may occur similarly in the neocortex in vivo as in hippocampal slices, although it happened less frequently in our experimental conditions.

The role of beta-actin mRNA localization in single dendritic spines studied by two-photon uncaging

The role of beta-actin mRNA localization in single dendritic spines studied by two-photon uncaging

Parsing Out the Variability of Transmission at Central Synapses Using Optical Quantal Analysis.

Properties of synaptic release dictates the core of information transfer in neural circuits. Despite decades of technical and theoretical advances, distinguishing bona fide information content from the multiple sources of synaptic variability remains a challenging problem. Here, we employed a combination of computational approaches with cellular electrophysiology, two-photon uncaging of MNI-Glutamate and imaging at single synapses. We describe and calibrate the use of the fluorescent glutamate sensor iGluSnFR and found that its kinetic profile is close to that of AMPA receptors, therefore providing several distinct advantages over slower methods relying on NMDA receptor activation (i.e., chemical or genetically encoded calcium indicators). Using an array of statistical methods, we further developed, and validated on surrogate data, an expectation-maximization algorithm that, by biophysically constraining release variability, extracts the quantal parameters n (maximum number of released vesicles) and p (unitary probability of release) from single-synapse iGluSnFR-mediated transients. Together, we present a generalizable mathematical formalism which, when applied to optical recordings, paves the way to an increasingly precise investigation of information transfer at central synapses.

Dendrimer Conjugation Enables Multiphoton Chemical Neurophysiology Studies with an Extended π-Electron Caging Chromophore.

We have developed a caged neurotransmitter using an extended π-electron chromophore for efficient multiphoton uncaging on living neurons. Widely studied in a chemical context, such chromophores are inherently bioincompatible due to their highly lipophilic character. Attachment of two polycarboxylate dendrimers, a method we call "cloaking", to a bisstyrylthiophene (or BIST) core effectively transformed the chromophore into a water-soluble optical probe, whilst maintaining the high two-photon absorption of over 500 GM. Importantly, the cloaked caged compound was biologically inert at the high concentrations required for multiphoton chemical physiology. Thus, in contrast to non-cloaked BIST compounds, the BIST-caged neurotransmitter can be safely delivered onto neurons in acutely isolated brain slices, thereby enabling high-resolution two-photon uncaging without any side effects. We expect that our cloaking method will enable the development of new classes of cell-compatible photolabile probes using a wide variety of extended π-electron caging chromophores.

5-HT2A receptor activation enhances NMDA receptor-mediated glutamate responses through Src kinase in the dendrites of rat jaw-closing motoneurons.

5-HT increases the excitability of brainstem and spinal motoneurons, including the jaw-closing motoneurons, by depolarizing the membrane potential and decreasing the medium-duration afterhyperpolarization. In this study, we focused on how 5-HT enhances postsynaptic glutamatergic responses in the dendrites of the jaw-closing motoneurons. We demonstrate that 5-HT augments glutamatergic signalling by enhancing the function of the GluN2A-containing NMDA receptor (NMDAR) through the activation of 5-HT2A receptors (5-HT2A Rs) and Src kinase. To enhance glutamatergic responses, activation of the 5-HT2A Rs must occur within ∼60μm of the location of the glutamate responses. 5-HT inputs to the jaw-closing motoneurons can significantly vary their input-output relationship, which may contribute to wide-range regulation of contractile forces of the jaw-closing muscles. Various motor behaviours are modulated by 5-HT. Although the masseter (jaw-closing) motoneurons receive both glutamatergic and serotonergic inputs, it remains unclear how 5-HT affects the glutamatergic inputs to the motoneuronal dendrites. We examined the effects of 5-HT on postsynaptic responses evoked by single- or two-photon uncaging of caged glutamate (glutamate responses) to the dendrites of masseter motoneurons in postnatal day 2-5 rats of either sex. Application of 5-HT induced membrane depolarization and enhanced the glutamate-response amplitude. This enhancement was mimicked by the 5-HT2A receptor (5-HT2A R) agonist and was blocked by the 5-HT2A/2C R antagonist. However, neither the 5-HT2B R nor the 5-HT2C R agonists altered glutamate responses. Blockade of the NMDA receptors (NMDARs), but not AMPA receptors, abolished the 5-HT-induced enhancement. Furthermore, the selective antagonist for the GluN2A subunit abolished the 5-HT-induced enhancement. 5-HT increased GluN2A phosphorylation, while the Src kinase inhibitor reduced the 5-HT-induced enhancement and GluN2A phosphorylation. When exposure to the 5-HT2A R agonist was targeted to the dendrites, the enhancement of glutamate responses was restricted to the loci of the dendrites near the puff loci. Electron microscopic immunohistochemistry revealed that both the NMDARs and the 5-HT2A Rs were close to each other in the same dendrite. These results suggest that activation of dendritic 5-HT2A Rs enhances the function of local GluN2A-containing NMDARs through Src kinase. Such enhancement of the glutamate responses by 5-HT may contribute to wide-range regulation of contractile forces of the jaw-closing muscles.

Coincidence Detection within the Excitable Rat Olfactory Bulb Granule Cell Spines.

In the mammalian olfactory bulb, the inhibitory axonless granule cells (GCs) feature reciprocal synapses that interconnect them with the principal neurons of the bulb, mitral, and tufted cells. These synapses are located within large excitable spines that can generate local action potentials (APs) upon synaptic input ("spine spike"). Moreover, GCs can fire global APs that propagate throughout the dendrite. Strikingly, local postsynaptic Ca2+ entry summates mostly linearly with Ca2+ entry due to coincident global APs generated by glomerular stimulation, although some underlying conductances should be inactivated. We investigated this phenomenon by constructing a compartmental GC model to simulate the pairing of local and global signals as a function of their temporal separation Δt. These simulations yield strongly sublinear summation of spine Ca2+ entry for the case of perfect coincidence Δt = 0 ms. Summation efficiency (SE) sharply rises for both positive and negative Δt. The SE reduction for coincident signals depends on the presence of voltage-gated Na+ channels in the spine head, while NMDARs are not essential. We experimentally validated the simulated SE in slices of juvenile rat brain (both sexes) by pairing two-photon uncaging of glutamate at spines and APs evoked by somatic current injection at various intervals Δt while imaging spine Ca2+ signals. Finally, the latencies of synaptically evoked global APs and EPSPs were found to correspond to Δt ≈ 10 ms, explaining the observed approximately linear summation of synaptic local and global signals. Our results provide additional evidence for the existence of the GC spine spike.SIGNIFICANCE STATEMENT Here we investigate the interaction of local synaptic inputs and global activation of a neuron by a backpropagating action potential within a dendritic spine with respect to local Ca2+ signaling. Our system of interest, the reciprocal spine of the olfactory bulb granule cell, is known to feature a special processing mode, namely, a synaptically triggered action potential that is restricted to the spine head. Therefore, coincidence detection of local and global signals follows different rules than in more conventional synapses. We unravel these rules using both simulations and experiments and find that signals coincident within ≈±7 ms around 0 ms result in sublinear summation of Ca2+ entry because of synaptic activation of voltage-gated Na+ channels within the spine.

Two-Photon Uncaging of Glutamate.

Two-photon microscopy produces the excited singlet state of a chromophore with wavelengths approximately double that used for normal excitation. Two photons are absorbed almost simultaneously, via a virtual state, and this makes the excitation technique inherently non-linear. It requires ultra-fast lasers to deliver the high flux density needed to access intrinsically very short lived intermediates, and in combination with lenses of high numerical aperture, this confines axial excitation highly. Since the two-photon excitation volume is similar to a large spine head, the technique has been widely used to study glutamatergic transmission in brain slices. Here I describe the principles of two-photon uncaging of glutamate and provide a practical guide to its application.

Ultrastructural, Molecular and Functional Mapping of GABAergic Synapses on Dendritic Spines and Shafts of Neocortical Pyramidal Neurons.

The location of GABAergic synapses on dendrites is likely key for neuronal integration. In particular, inhibitory inputs on dendritic spines could serve to selectively veto or modulate individual excitatory inputs, greatly expanding the computational power of individual neurons. To investigate this, we have undertaken a combined functional, molecular, and ultrastructural mapping of the location of GABAergic inputs onto dendrites of pyramidal neurons from upper layers of juvenile mouse somatosensory cortex. Using two-photon uncaging of GABA, intracellular labeling with gerphyrin intrabodies, and focused ion beam milling with scanning electron microscopy, we find that most (96-98%) spines lack GABAergic synapses, although they still display GABAergic responses, potentially due to extrasynaptic GABA receptors. We conclude that GABAergic inputs, in practice, contact dendritic shafts and likely control clusters of excitatory inputs, defining functional zones on dendrites.