Ratiometric Imaging of Catecholamine Neurotransmitters with Nanosensors.

Neurotransmitters are important signaling molecules in the brain and are relevant in many diseases. Measuring them with high spatial and temporal resolutions in biological systems is challenging. Here, we develop a ratiometric fluorescent sensor/probe for catecholamine neurotransmitters on the basis of near-infrared (NIR) semiconducting single wall carbon nanotubes (SWCNTs). Phenylboronic acid (PBA)-based quantum defects are incorporated into them to interact selectively with catechol moieties. These PBA-SWCNTs are further modified with poly(ethylene glycol) phospholipids (PEG-PL) for biocompatibility. Catecholamines, including dopamine, do not affect the intrinsic E11 fluorescence (990 nm) of these (PEG-PL-PBA-SWCNT) sensors. In contrast, the defect-related E11* emission (1130 nm) decreases by up to 35%. Furthermore, this dual functionalization allows tuning selectivity by changing the charge of the PEG polymer. These sensors are not taken up by cells, which is beneficial for extracellular imaging, and they are functional in brain slices. In summary, we use dual functionalization of SWCNTs to create a ratiometric biosensor for dopamine.

Axons of cortical basket cells originating from dendrites develop higher local complexity than axons emerging from basket cell somata.

Neuronal differentiation is regulated by neuronal activity. Here, we analyzed dendritic and axonal growth of Basket cells (BCs) and non-Basket cells (non-BCs) using sparse transfection of channelrhodopsin-YFP and repetitive optogenetic stimulation in slice cultures of rat visual cortex. Neocortical interneurons often display axon-carrying dendrites (AcDs). We found that the AcDs of BCs and non-BCs were, on average, the most complex dendrites. Further, the AcD configuration had an influence on BC axonal development. Axons originating from an AcD formed denser arborizations with more terminal endings within the dendritic field of the parent cell. Intriguingly, this occurred already in unstimulated BCs, and complexity was not increased further by optogenetic stimulation. However, optogenetic stimulation exerted a growth-promoting effect on axons emerging from BC somata. The axons of non-BCs neither responded to the AcD configuration nor to the optogenetic stimulation. The results suggest that the formation of locally dense BC plexuses is regulated by spontaneous activity. Moreover, in the AcD configuration, the AcD and the axon it carries mutually support each other's growth.

Sensitive multicolor indicators for monitoring norepinephrine in vivo.

Genetically encoded indicators engineered from G-protein-coupled receptors are important tools that enable high-resolution in vivo neuromodulator imaging. Here, we introduce a family of sensitive multicolor norepinephrine (NE) indicators, which includes nLightG (green) and nLightR (red). These tools report endogenous NE release in vitro, ex vivo and in vivo with improved sensitivity, ligand selectivity and kinetics, as well as a distinct pharmacological profile compared with previous state-of-the-art GRABNE indicators. Using in vivo multisite fiber photometry recordings of nLightG, we could simultaneously monitor optogenetically evoked NE release in the mouse locus coeruleus and hippocampus. Two-photon imaging of nLightG revealed locomotion and reward-related NE transients in the dorsal CA1 area of the hippocampus. Thus, the sensitive NE indicators introduced here represent an important addition to the current repertoire of indicators and provide the means for a thorough investigation of the NE system.

Next generation genetically encoded fluorescent sensors for serotonin.

We developed a family of genetically encoded serotonin (5-HT) sensors (sDarken) on the basis of the native 5-HT1A receptor and circularly permuted GFP. sDarken 5-HT sensors are bright in the unbound state and diminish their fluorescence upon binding of 5-HT. Sensor variants with different affinities for serotonin were engineered to increase the versatility in imaging of serotonin dynamics. Experiments in vitro and in vivo showed the feasibility of imaging serotonin dynamics with high temporal and spatial resolution. As demonstrated here, the designed sensors show excellent membrane expression, have high specificity and a superior signal-to-noise ratio, detect the endogenous release of serotonin and are suitable for two-photon in vivo imaging.

Structural and compositional diversity in the kainate receptor family.

The kainate receptors (KARs) are members of the ionotropic glutamate receptor family and assemble into tetramers from a pool of five subunit types (GluK1-5). Each subunit confers distinct functional properties to a receptor, but the compositional and stoichiometric diversity of KAR tetramers is not well understood. To address this, we first solve the structure of the GluK1 homomer, which enables a systematic assessment of structural compatibility among KAR subunits. Next, we analyze single-cell RNA sequencing data, which reveal extreme diversity in the combinations of two or more KAR subunits co-expressed within the same cell. We then investigate the composition of individual receptor complexes using single-molecule fluorescence techniques and find that di-heteromers assembled from GluK1, GluK2, or GluK3 can form with all possible stoichiometries, while GluK1/K5, GluK2/K5, and GluK3/K5 can form 3:1 or 2:2 complexes. Finally, using three-color single-molecule imaging, we discover that KARs can form tri- and tetra-heteromers.

Splicing and editing of ionotropic glutamate receptors: a comprehensive analysis based on human RNA-Seq data.

Ionotropic glutamate receptors (iGluRs) play key roles for signaling in the central nervous system. Alternative splicing and RNA editing are well-known mechanisms to increase iGluR diversity and to provide context-dependent regulation. Earlier work on isoform identification has focused on the analysis of cloned transcripts, mostly from rodents. We here set out to obtain a systematic overview of iGluR splicing and editing in human brain based on RNA-Seq data. Using data from two large-scale transcriptome studies, we established a workflow for the de novo identification and quantification of alternative splice and editing events. We detected all canonical iGluR splice junctions, assessed the abundance of alternative events described in the literature, and identified new splice events in AMPA, kainate, delta, and NMDA receptor subunits. Notable events include an abundant transcript encoding the GluA4 amino-terminal domain, GluA4-ATD, a novel C-terminal GluD1 (delta receptor 1) isoform, GluD1-b, and potentially new GluK4 and GluN2C isoforms. C-terminal GluN1 splicing may be controlled by inclusion of a cassette exon, which shows preference for one of the two acceptor sites in the last exon. Moreover, we identified alternative untranslated regions (UTRs) and species-specific differences in splicing. In contrast, editing in exonic iGluR regions appears to be mostly limited to ten previously described sites, two of which result in silent amino acid changes. Coupling of proximal editing/editing and editing/splice events occurs to variable degree. Overall, this analysis provides the first inventory of alternative splicing and editing in human brain iGluRs and provides the impetus for further transcriptome-based and functional investigations.

GluN2B but Not GluN2A for Basal Dendritic Growth of Cortical Pyramidal Neurons.

NMDA receptors are important players for neuronal differentiation. We previously reported that antagonizing NMDA receptors with APV blocked the growth-promoting effects evoked by the overexpression of specific calcium-permeable or flip-spliced AMPA receptor subunits and of type I transmembrane AMPA receptor regulatory proteins which both exclusively modify apical dendritic length and branching of cortical pyramidal neurons. These findings led us to characterize the role of GluN2B and GluN2A for dendritogenesis using organotypic cultures of rat visual cortex. Antagonizing GluN2B with ifenprodil and Ro25-6981 strongly impaired basal dendritic growth of supra- and infragranular pyramidal cells at DIV 5-10, but no longer at DIV 15-20. Growth recovered after washout, and protein blots revealed an increase of synaptic GluN2B-containing receptors as indicated by a enhanced phosphorylation of the tyrosine 1472 residue. Antagonizing GluN2A with TCN201 and NVP-AAM077 was ineffective at both ages. Dendrite growth of non-pyramidal interneurons was not altered. We attempted to overexpress GluN2A and GluN2B. However, although the constructs delivered currents in HEK cells, there were neither effects on dendrite morphology nor an enhanced sensitivity to NMDA. Further, co-expressing GluN1-1a and GluN2B did not alter dendritic growth. Visualization of overexpressed, tagged GluN2 proteins was successful after immunofluorescence for the tag which delivered rather weak staining in HEK cells as well as in neurons. This suggested that the level of overexpression is too weak to modify dendrite growth. In summary, endogenous GluN2B, but not GluN2A is important for pyramidal cell basal dendritic growth during an early postnatal time window.

Subunit-selective iGluR antagonists can potentiate heteromeric receptor responses by blocking desensitization.

Ionotropic glutamate receptors (iGluRs) are key molecules for synaptic signaling in the central nervous system, which makes them promising drug targets. Intensive efforts are being devoted to the development of subunit-selective ligands, which should enable more precise pharmacologic interventions while limiting the effects on overall neuronal circuit function. However, many AMPA and kainate receptor complexes in vivo are heteromers composed of different subunits. Despite their importance, little is known about how subunit-selective ligands affect the gating of heteromeric iGluRs, namely their activation and desensitization properties. Using fast ligand application experiments, we studied the effects of competitive antagonists that block glutamate from binding at part of the four subunits. We found that UBP-310, a kainate receptor antagonist with high selectivity for GluK1 subunits, reduces the desensitization of GluK1/GluK2 heteromers and fully abolishes the desensitization of GluK1/GluK5 heteromers. This effect is mirrored by subunit-selective agonists and heteromeric receptors that contain binding-impaired subunits, as we show for both kainate and GluA2 AMPA receptors. These findings are consistent with a model in which incomplete agonist occupancy at the four receptor subunits can provide activation without inducing desensitization. However, we did not detect significant steady-state currents during UBP-310 dissociation from GluK1 homotetramers, indicating that antagonist dissociation proceeds in a nonuniform and cooperativity-driven manner, which disfavors nondesensitizing occupancy states. Besides providing mechanistic insights, these results have direct implications for the use of subunit-selective antagonists in neuroscience research and envisioned therapeutic interventions.

Quinoxalinedione deprotonation is important for glutamate receptor binding.

Quinoxalinediones are an important class of competitive antagonists at ionotropic glutamate receptors (iGluRs), where they are widely used to block α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptor responses. In this study we utilize two prototypic quinoxalinedione antagonists, namely DNQX and CNQX, which quench the intrinsic fluorescence of the ligand binding domain (LBD), to perform in vitro binding assays. We find that binding of DNQX and CNQX at the AMPA receptor GluA2 LBD is strongly pH dependent, whereas glutamate binding is not affected by pH. We also show that the deprotonation of DNQX, CNQX and other quinoxalinediones (NBQX and YM90K) occurs close to physiological pH, which can be explained by the lactam-lactim tautomerization of the quinoxalinedione scaffold. Analysis of our binding data indicates that quinoxalinedione deprotonation is a key requirement for binding, as we find a >100-fold higher affinity for binding of the monoanionic form compared to the neutral form. This suggests a large electrostatic contribution to the interaction with a conserved arginine residue located in the binding pocket of iGluRs. The strong pH dependence of quinoxalinedione binding, which has not previously been reported, is relevant for structure-function studies, but also for the use of quinoxalinediones in physiological experiments and envisioned therapeutic applications.

Glutamatergic Signaling in the Central Nervous System: Ionotropic and Metabotropic Receptors in Concert.

Glutamate serves as both the mammalian brain's primary excitatory neurotransmitter and as a key neuromodulator to control synapse and circuit function over a wide range of spatial and temporal scales. This functional diversity is decoded by two receptor families: ionotropic glutamate receptors (iGluRs) and metabotropic glutamate receptors (mGluRs). The challenges posed by the complexity and physiological importance of each of these subtypes has limited our appreciation and understanding of how these receptors work in concert. In this review, by comparing both receptor families with a focus on their crosstalk, we argue for a more holistic understanding of neural glutamate signaling.

Astroglial Glutamate Signaling and Uptake in the Hippocampus.

Astrocytes have long been regarded as essentially unexcitable cells that do not contribute to active signaling and information processing in the brain. Contrary to this classical view, it is now firmly established that astrocytes can specifically respond to glutamate released from neurons. Astrocyte glutamate signaling is initiated upon binding of glutamate to ionotropic and/or metabotropic receptors, which can result in calcium signaling, a major form of glial excitability. Release of so-called gliotransmitters like glutamate, ATP and D-serine from astrocytes in response to activation of glutamate receptors has been demonstrated to modulate various aspects of neuronal function in the hippocampus. In addition to receptors, glutamate binds to high-affinity, sodium-dependent transporters, which results in rapid buffering of synaptically-released glutamate, followed by its removal from the synaptic cleft through uptake into astrocytes. The degree to which astrocytes modulate and control extracellular glutamate levels through glutamate transporters depends on their expression levels and on the ionic driving forces that decrease with ongoing activity. Another major determinant of astrocytic control of glutamate levels could be the precise morphological arrangement of fine perisynaptic processes close to synapses, defining the diffusional distance for glutamate, and the spatial proximity of transporters in relation to the synaptic cleft. In this review, we will present an overview of the mechanisms and physiological role of glutamate-induced ion signaling in astrocytes in the hippocampus as mediated by receptors and transporters. Moreover, we will discuss the relevance of astroglial glutamate uptake for extracellular glutamate homeostasis, focusing on how activity-induced dynamic changes of perisynaptic processes could shape synaptic transmission at glutamatergic synapses.

A family of photoswitchable NMDA receptors.

NMDA receptors, which regulate synaptic strength and are implicated in learning and memory, consist of several subtypes with distinct subunit compositions and functional properties. To enable spatiotemporally defined, rapid and reproducible manipulation of function of specific subtypes, we engineered a set of photoswitchable GluN subunits ('LiGluNs'). Photo-agonism of GluN2A or GluN2B elicits an excitatory drive to hippocampal neurons that can be shaped in time to mimic synaptic activation. Photo-agonism of GluN2A at single dendritic spines evokes spine-specific calcium elevation and expansion, the morphological correlate of LTP. Photo-antagonism of GluN2A alone, or in combination with photo-antagonism of GluN1a, reversibly blocks excitatory synaptic currents, prevents the induction of long-term potentiation and prevents spine expansion. In addition, photo-antagonism in vivo disrupts synaptic pruning of developing retino-tectal projections in larval zebrafish. By providing precise and rapidly reversible optical control of NMDA receptor subtypes, LiGluNs should help unravel the contribution of specific NMDA receptors to synaptic transmission, integration and plasticity.

A Toolkit for Orthogonal and in vivo Optical Manipulation of Ionotropic Glutamate Receptors.

The ability to optically manipulate specific neuronal signaling proteins with genetic precision paves the way for the dissection of their roles in brain function, behavior, and disease. Chemical optogenetic control with photoswitchable tethered ligands (PTLs) enables rapid, reversible and reproducible activation or block of specific neurotransmitter-gated receptors and ion channels in specific cells. In this study, we further engineered and characterized the light-activated GluK2 kainate receptor, LiGluR, to develop a toolbox of LiGluR variants. Low-affinity LiGluRs allow for efficient optical control of GluK2 while removing activation by native glutamate, whereas variant RNA edited versions enable the synaptic role of receptors with high and low Ca(2+) permeability to be assessed and spectral variant photoswitches provide flexibility in illumination. Importantly, we establish that LiGluR works efficiently in the cortex of awake, adult mice using standard optogenetic techniques, thus opening the door to probing the role of specific synaptic receptors and cellular signals in the neural circuit operations of the mammalian brain in normal conditions and in disease. The principals developed in this study are widely relevant to the engineering and in vivo use of optically controllable proteins, including other neurotransmitter receptors.

Controlling ionotropic and metabotropic glutamate receptors with light: principles and potential.

Light offers unique advantages for studying and manipulating biomolecules and the cellular processes that they control. Optical control of ionotropic and metabotropic glutamate receptors has garnered significant interest, since these receptors are central to signaling at neuronal synapses and only optical approaches provide the spatial and temporal resolution required to directly probe receptor function in cells and tissue. Following the classical method of glutamate photo-uncaging, recently developed methods have added other forms of remote control, including those with high molecular specificity and genetic targeting. These tools open the door to the direct optical control of synaptic transmission and plasticity, as well as the probing of native receptor function in intact neural circuits.

Restoration of visual function by expression of a light-gated mammalian ion channel in retinal ganglion cells or ON-bipolar cells.

Most inherited forms of blindness are caused by mutations that lead to photoreceptor cell death but spare second- and third-order retinal neurons. Expression of the light-gated excitatory mammalian ion channel light-gated ionotropic glutamate receptor (LiGluR) in retinal ganglion cells (RGCs) of the retina degeneration (rd1) mouse model of blindness was previously shown to restore some visual functions when stimulated by UV light. Here, we report restored retinal function in visible light in rodent and canine models of blindness through the use of a second-generation photoswitch for LiGluR, maleimide-azobenzene-glutamate 0 with peak efficiency at 460 nm (MAG0(460)). In the blind rd1 mouse, multielectrode array recordings of retinal explants revealed robust and uniform light-evoked firing when LiGluR-MAG0(460) was targeted to RGCs and robust but diverse activity patterns in RGCs when LiGluR-MAG0(460) was targeted to ON-bipolar cells (ON-BCs). LiGluR-MAG0(460) in either RGCs or ON-BCs of the rd1 mouse reinstated innate light-avoidance behavior and enabled mice to distinguish between different temporal patterns of light in an associative learning task. In the rod-cone dystrophy dog model of blindness, LiGluR-MAG0(460) in RGCs restored robust light responses to retinal explants and intravitreal delivery of LiGluR and MAG0(460) was well tolerated in vivo. The results in both large and small animal models of photoreceptor degeneration provide a path to clinical translation.

Photoswitching of cell surface receptors using tethered ligands.

Optical probing and manipulation of cellular signaling has revolutionized biological studies ranging from isolated cells to intact tissues in the live animal. A promising avenue of optical manipulation is Chemical Optogenetics (or Optogenetic Pharmacology), an approach for engineering specific proteins to be rapidly and reversibly switched on and off with light. The approach employs synthetic photoswitched ligands, which can be reversibly photo-isomerized to toggle back and forth between two conformations in response to two wavelengths of light. We focus here on the photoswitched tethered ligand (PTL) approach in which the PTL is covalently attached in a site-directed manner to a signaling protein. For this a ligand anchoring site is introduced at a location which allows the ligand to dock only in one of the light-controlled conformations, thus enabling liganding to be rapidly switched. The ligand can be an agonist, antagonist or an active site (or pore) blocker. In principle, orthogonal chemistries of attachment would make PTL anchoring completely unique. However, extremely high specificity of remote control is also obtained by cysteine attachment because of the ligand specificity and precise geometric requirements for liganding. We describe here the design of light-gated ionotropic and metabotropic glutamate receptors, the selection of a site for cysteine placement, the method for PTL attachment, and a detailed protocol of photoswitching experiments in cultured cells. These descriptions can guide applications of Chemical Optogenetics to other receptors and serve as a starting point for use in more complex preparations.

Tethered ligands reveal glutamate receptor desensitization depends on subunit occupancy.

Cell signaling is often mediated by the binding of multiple ligands to multisubunit receptors. The probabilistic nature and sometimes slow rate of binding encountered with diffusible ligands can impede attempts to determine how the ligand occupancy controls signaling in such protein complexes. We describe a solution to this problem that uses a photoswitched tethered ligand as a 'ligand clamp' to induce rapid and stable binding and unbinding at defined subsets of subunits. We applied the approach to study gating in ionotropic glutamate receptors (iGluRs), ligand-gated ion channels that mediate excitatory neurotransmission and plasticity at glutamatergic synapses in the brain. We probed gating in two kainate-type iGluRs, GluK2 homotetramers and GluK2-GluK5 heterotetramers. Ultrafast (submillisecond) photoswitching of an azobenzene-based ligand on specific subunits provided a real-time measure of gating and revealed that partially occupied receptors can activate without desensitizing. The findings have implications for signaling by locally released and spillover glutamate.

A red-shifted, fast-relaxing azobenzene photoswitch for visible light control of an ionotropic glutamate receptor.

The use of azobenzene photoswitches has become a dependable method for rapid and exact modulation of biological processes and material science systems. The requirement of ultraviolet light for azobenzene isomerization is not ideal for biological systems due to poor tissue penetration and potentially damaging effects. While modified azobenzene cores with a red-shifted cis-to-trans isomerization have been previously described, they have not yet been incorporated into a powerful method to control protein function: the photoswitchable tethered ligand (PTL) approach. We report the synthesis and characterization of a red-shifted PTL, L-MAG0460, for the light-gated ionotropic glutamate receptor LiGluR. In cultured mammalian cells, the LiGluR+L-MAG0460 system is activated rapidly by illumination with 400-520 nm light to generate a large ionic current. The current rapidly turns off in the dark as the PTL relaxes thermally back to the trans configuration. The visible light excitation and single-wavelength behavior considerably simplify use and should improve utilization in tissue.

The Brain Prize 2013: the optogenetics revolution.

The 2013 Grete Lundbeck European Brain Research Prize was awarded to Ernst Bamberg, Edward Boyden, Karl Deisseroth, Peter Hegemann, Gero Miesenböck, and Georg Nagel 'for their invention and refinement of optogenetics'. Why optogenetics? And why this sextet? To appreciate why, we turn first to some of the core questions of neuroscience and the technical difficulties that long obstructed their resolution.

Bringing optogenetics to the synapse.

In this issue of Neuron, Lin et al. (2013) describe InSynC, an optogenetic approach that utilizes chromophore-assisted light inactivation (CALI) to inactivate presynaptic neurotransmitter release proteins VAMP2 and synaptophysin. InSynC selectively reduces synaptic transmission in illuminated regions in vitro and in vivo.