Interrogating the function of GABAA receptors in the brain with optogenetic pharmacology.

To better understand neural circuits and behavior, microbial opsins have been developed as optogenetic tools for stimulating or inhibiting action potentials with high temporal and spatial precision. However, if we seek a more reductionist understanding of how neuronal circuits operate, we also need high-resolution tools for perturbing the function of synapses. By combining photochemical tools and molecular biology, a wide variety of light-regulated neurotransmitter receptors have been developed, enabling photo-control of excitatory, inhibitory, and modulatory synaptic transmission. Here we focus on photo-control of GABAA receptors, ligand-gated Cl- channels that underlie almost all synaptic inhibition in the mammalian brain. By conjugating a photoswitchable tethered ligand onto a genetically-modified subunit of the GABAA receptor, light-sensitivity can be conferred onto specific isoforms of the receptor. Through gene editing, this attachment site can be knocked into the genome, enabling photocontrol of endogenous GABAA receptors. This strategy can be employed to explore the cell biology and neurophysiology of GABAA receptors. This includes investigating how specific isoforms contribute to synaptic and tonic inhibition and understanding the roles they play in brain development, long-term synaptic plasticity, and learning and memory.

Relocation of an Extrasynaptic GABAA Receptor to Inhibitory Synapses Freezes Excitatory Synaptic Strength and Preserves Memory.

The excitatory synapse between hippocampal CA3 and CA1 pyramidal neurons exhibits long-term potentiation (LTP), a positive feedback process implicated in learning and memory in which postsynaptic depolarization strengthens synapses, promoting further depolarization. Without mechanisms for interrupting positive feedback, excitatory synapses could strengthen inexorably, corrupting memory storage. Here, we reveal a hidden form of inhibitory synaptic plasticity that prevents accumulation of excitatory LTP. We developed a knockin mouse that allows optical control of endogenous α5-subunit-containing γ-aminobutyric acid (GABA)A receptors (α5-GABARs). Induction of excitatory LTP relocates α5-GABARs, which are ordinarily extrasynaptic, to inhibitory synapses, quashing further NMDA receptor activation necessary for inducing more excitatory LTP. Blockade of α5-GABARs accelerates reversal learning, a behavioral test for cognitive flexibility dependent on repeated LTP. Hence, inhibitory synaptic plasticity occurs in parallel with excitatory synaptic plasticity, with the ensuing interruption of the positive feedback cycle of LTP serving as a possible critical early step in preserving memory.

Localizing Proton-Mediated Inhibitory Feedback at the Retinal Horizontal Cell-Cone Synapse with Genetically-Encoded pH Probes.

Lateral inhibition in the vertebrate retina depends on a negative feedback synapse between horizontal cells (HCs) and rod and cone photoreceptors. A change in pH is thought to be the signal for negative feedback, but its spatial profile in the synaptic cleft is unknown. Here we use three different membrane proteins, each fused to the same genetically-encoded pH-sensitive Green Fluorescent Protein (GFP) (pHluorin), to probe synaptic pH in retina from transgenic zebrafish (Danio rerio) of either sex. We used the cone transducin promoter to express SynaptopHluorin (pHluorin on vesicle-associated membrane protein (VAMP2)) or CalipHluorin (pHluorin on an L-type Ca2+ channel) and the HC-specific connexin-55.5 promoter to express AMPApHluorin (pHluorin on an AMPA receptor). Stimulus light led to increased fluorescence of all three probes, consistent with alkalinization of the synaptic cleft. The receptive field size, sensitivity to surround illumination, and response to activation of an alien receptor expressed exclusively in HCs, are consistent with lateral inhibition as the trigger for alkalinization. However, SynaptopHluorin and AMPApHluorin, which are displaced farther from cone synaptic ribbons than CalipHluorin, reported a smaller pH change. Hence, unlike feedforward glutamatergic transmission, which spills over to allow cross talk between terminals in the cone network, the pH change underlying HC feedback is compartmentalized to individual synaptic invaginations within a cone terminal, consistent with private line communication.SIGNIFICANCE STATEMENT Lateral inhibition (LI) is a fundamental feature of information processing in sensory systems, enhancing contrast sensitivity and enabling edge discrimination. Horizontal cells (HCs) are the first cellular substrate of LI in the vertebrate retina, but the synaptic mechanisms underlying LI are not completely understood, despite decades of study. This paper makes a significant contribution to our understanding of LI, by showing that each HC-cone synapse is a "private-line" that operates independently from other HC-cone connections. Using transgenic zebrafish expressing pHluorin, a pH-sensitive GFP variant spliced onto three different protein platforms expressed either in cones or HCs we show that the feedback pH signal is constrained to individual cone terminals, and more stringently, to individual synaptic contact sites within each terminal.

Design of a Highly Bistable Photoswitchable Tethered Ligand for Rapid and Sustained Manipulation of Neurotransmission.

Photoswitchable neurotransmitter receptors are powerful tools for precise manipulation of neural signaling. However, their applications for slow or long-lasting biological events are constrained by fast thermal relaxation of cis-azobenzene. We address this issue by modifying the ortho positions of azobenzene used in the tethered ligand. In cultured cells and intact brain tissue, conjugating inhibitory neurotransmitter receptors with one of the derivatives, dMPC1, allows bidirectional receptor control with 380 and 500 nm light. Moreover, the receptors can be locked in either an active or an inactive state in darkness after a brief pulse of light. This strategy thus enables both rapid and sustained manipulation of neurotransmission, allowing optogenetic interrogation of neural functions over a broad range of time scales.

Synaptophysin 1 Clears Synaptobrevin 2 from the Presynaptic Active Zone to Prevent Short-Term Depression.

Release site clearance is an important process during synaptic vesicle (SV) recycling. However, little is known about its molecular mechanism. Here we identify self-assembly of exocytosed Synaptobrevin 2 (Syb2) and Synaptophysin 1 (Syp1) by homo- and hetero-oligomerization into clusters as key mechanisms mediating release site clearance for preventing cis-SNARE complex formation at the active zone (AZ). In hippocampal neurons from Syp1 knockout mice, neurons expressing a monomeric Syb2 mutant, or after acute block of the ATPase N-ethylmaleimide-sensitive factor (NSF), responsible for cis-SNARE complex disassembly, we found strong frequency-dependent short-term depression (STD), whereas retrieval of Syb2 by compensatory endocytosis was only affected weakly. Defects in Syb2 endocytosis were stimulus- and frequency-dependent, indicating that Syp1 is not essential for Syb2 retrieval, but for its efficient clearance upstream of endocytosis. Our findings identify an SV protein as a release site clearance factor.

Crystal structure and molecular imaging of the Nav channel ß3 subunit indicates a trimeric assembly.

The vertebrate sodium (Nav) channel is composed of an ion-conducting α subunit and associated ß subunits. Here, we report the crystal structure of the human ß3 subunit immunoglobulin (Ig) domain, a functionally important component of Nav channels in neurons and cardiomyocytes. Surprisingly, we found that the ß3 subunit Ig domain assembles as a trimer in the crystal asymmetric unit. Analytical ultracentrifugation confirmed the presence of Ig domain monomers, dimers, and trimers in free solution, and atomic force microscopy imaging also detected full-length ß3 subunit monomers, dimers, and trimers. Mutation of a cysteine residue critical for maintaining the trimer interface destabilized both dimers and trimers. Using fluorescence photoactivated localization microscopy, we detected full-length ß3 subunit trimers on the plasma membrane of transfected HEK293 cells. We further show that ß3 subunits can bind to more than one site on the Nav 1.5 α subunit and induce the formation of α subunit oligomers, including trimers. Our results suggest a new and unexpected role for the ß3 subunits in Nav channel cross-linking and provide new structural insights into some pathological Nav channel mutations.

Critical role of FLRT1 phosphorylation in the interdependent regulation of FLRT1 function and FGF receptor signalling.

BACKGROUND: Fibronectin leucine rich transmembrane (FLRT) proteins have dual properties as regulators of cell adhesion and potentiators of fibroblast growth factor (FGF) mediated signalling. The mechanism by which the latter is achieved is still unknown and is the subject of this investigation. PRINCIPAL FINDINGS: Here we show that FLRT1 is a target for tyrosine phosphorylation mediated by FGFR1 and implicate a non-receptor Src family kinase (SFK). We identify the target tyrosine residues in the cytoplasmic domain of FLRT1 and show that these are not direct substrates for Src kinase suggesting that the SFK may exert effects via potentiation of FGFR1 kinase activity. We show that whilst FLRT1 expression results in a ligand-dependent elevation of MAP kinase activity, a mutant version of FLRT1, defective as an FGFR1 kinase substrate (Y3F-FLRT1), has the property of eliciting ligand-independent chronic activation of the MAP kinase pathway which is suppressed by pharmacological inhibition of either FGFR1 or Src kinase. Functional investigation of FGFR1 and FLRT1 signalling in SH-SY5Y neuroblastoma cells reveals that FLRT1 alone acts to induce a multi-polar phenotype whereas the combination of FLRT1 and FGFR activation, or expression of Y3F-FLRT1, acts to induce neurite outgrowth via MAPK activation. Similar results were obtained in a dendrite outgrowth assay in primary hippocampal neurons. We also show that FGFR1, FLRT1 and activated Src are co-localized and this complex is trafficked toward the soma of the cell. The presence of Y3F-FLRT1 rather than FLRT1 resulted in prolonged localization of this complex within the neuritic arbour. CONCLUSIONS: This study shows that the phosphorylation state of FLRT1, which is itself FGFR1 dependent, may play a critical role in the potentiation of FGFR1 signalling and may also depend on a SFK-dependent phosphorylation mechanism acting via the FGFR. This is consistent with an 'in vivo' role for FLRT1 regulation of FGF signalling via SFKs. Furthermore, the phosphorylation-dependent futile cycle mechanism controlling FGFR1 signalling is concurrently crucial for regulation of FLRT1-mediated neurite outgrowth.