Light-gated Glutamate Receptor Research Articles

Versatile Optical Control of Voltage-Gated Sodium Channel Function in Engineered HEK293 Cells

Optical control of voltage-gated ion channels offers a potentially flexible and inexpensive alternative to patch clamp systems in large-scale compound library characterization. Toward this end, we have engineered HEK293 cells to express a multi-component system conferring precise iterative control over heterologously expressed sodium channels including Nav1.5 and Nav1.7. In combination with sodium channels, balanced expression of an inwardly-rectifying potassium channel plus the Light-gated Glutamate Receptor (LiGluR) has yielded cell lines that maintain resting potentials around −90 mV and can be rapidly depolarized and repolarized via exposure to millisecond 390 nm and 490 nm light pulses, respectively. Prolonged incremental depolarizations in response to varying 390 nm light pulse duration are also possible due to the “latching” property of LiGluR in the presence of concanavlin A, enabling a range of sodium channel assay modes. Here, we demonstrate light-controlled protocols capable of discriminating among closed-state, inactivated-state, and use-dependent sodium channel inhibitors, and illustrate the use of voltage-step control in measuring channel voltage gating. We also present progress toward pairing these cell lines with voltage-sensitive dyes and extracellular field recording methods compatible with prospective or modified multiwell instrumentation systems.Supported by NIH grant R44 GM087755.

Optical Modulation of Neurotransmission

The computational properties of an isolated neuron have been analyzed in detail by postsynaptic activation with caged compounds. However, new tools are needed to manipulate neurotransmission at individual synapses in order to understand how a neuron integrates physiological stimuli received from presynaptic neurons within a circuit. Here we describe a method to control neurotransmitter exocytosis at the presynaptic compartment by using a light-gated glutamate receptor (LiGluR). In chromaffin cells, LiGluR supports exocytosis by means of a calcium influx that is comparable to voltage-gated calcium channels. Presynaptic expression of LiGluR in hippocampal neurons enables direct and reversible control of neurotransmission with light, and allows modulating the firing rate of the postsynaptic neuron with the wavelength of illumination. This method constitutes an important step toward the determination of the complex transfer function of individual synapses.

Molecular probes and switches for functional analysis of receptors, ion channels and synaptic networks

Molecular probes and switches for functional analysis of receptors, ion channels and synaptic networks

Optical modulation of neurotransmission using calcium photocurrents through the ion channel LiGluR

A wide range of light-activated molecules (photoswitches and phototriggers) have been used to the study of computational properties of an isolated neuron by acting pre and postsynaptically. However, new tools are being pursued to elicit a presynaptic calcium influx that triggers the release of neurotransmitters, most of them based in calcium-permeable Channelrhodopsin-2 mutants. Here we describe a method to control exocytosis of synaptic vesicles through the use of a light-gated glutamate receptor (LiGluR), which has recently been demonstrated that supports secretion by means of calcium influx in chromaffin cells. Expression of LiGluR in hippocampal neurons enables reversible control of neurotransmission with light, and allows modulating the firing rate of the postsynaptic neuron with the wavelength of illumination. This method may be useful for the determination of the complex transfer function of individual synapses.

Colloids as Mobile Substrates for the Implantation and Integration of Differentiated Neurons into the Mammalian Brain

Neuronal degeneration and the deterioration of neuronal communication lie at the origin of many neuronal disorders, and there have been major efforts to develop cell replacement therapies for treating such diseases. One challenge, however, is that differentiated cells are challenging to transplant due to their sensitivity both to being uprooted from their cell culture growth support and to shear forces inherent in the implantation process. Here, we describe an approach to address these problems. We demonstrate that rat hippocampal neurons can be grown on colloidal particles or beads, matured and even transfected in vitro, and subsequently transplanted while adhered to the beads into the young adult rat hippocampus. The transplanted cells have a 76% cell survival rate one week post-surgery. At this time, most transplanted neurons have left their beads and elaborated long processes, similar to the host neurons. Additionally, the transplanted cells distribute uniformly across the host hippocampus. Expression of a fluorescent protein and the light-gated glutamate receptor in the transplanted neurons enabled them to be driven to fire by remote optical control. At 1-2 weeks after transplantation, calcium imaging of host brain slice shows that optical excitation of the transplanted neurons elicits activity in nearby host neurons, indicating the formation of functional transplant-host synaptic connections. After 6 months, the transplanted cell survival and overall cell distribution remained unchanged, suggesting that cells are functionally integrated. This approach, which could be extended to other cell classes such as neural stem cells and other regions of the brain, offers promising prospects for neuronal circuit repair via transplantation of in vitro differentiated, genetically engineered neurons.

Design Of A Potassium Selective, Light-gated Glutamate Receptor

A major goal in molecular neuroscience is to understand structure-function relationships of glutamate receptor ion channels (iGluRs) and their role in behavior. In the mammalian central nervous system, iGluRs form non-selective cation channels that generate depolarizing potentials in fast excitatory synaptic processes. Having earlier introduced the light-activated glutamate receptor LiGuR, we went one step further and applied two types of structure-based design to develop a potassium selective, light-gated ion channel. Simple and fast Monte-Carlo simulations allow the rational design of optically-controlled proteins in general and LiGluR variants in particular. Using homology modeling we modified the transmembrane pore-region of LiGluR to create a potassium selective channel that responds to glutamate and light. We demonstrate that rapid pulses of light can hyperpolarize HEK-293 cells and neurons expressing this novel channel, which can be used as a tool in glutamate receptor screening applications and may enable optical neuronal inhibition.

Remote Control of Neuronal Activity with a Light-Gated Glutamate Receptor

The ability to stimulate select neurons in isolated tissue and in living animals is important for investigating their role in circuits and behavior. We show that the engineered light-gated ionotropic glutamate receptor (LiGluR), when introduced into neurons, enables remote control of their activity. Trains of action potentials are optimally evoked and extinguished by 380 nm and 500 nm light, respectively, while intermediate wavelengths provide graded control over the amplitude of depolarization. Light pulses of 1-5 ms in duration at approximately 380 nm trigger precisely timed action potentials and EPSP-like responses or can evoke sustained depolarizations that persist for minutes in the dark until extinguished by a short pulse of approximately 500 nm light. When introduced into sensory neurons in zebrafish larvae, activation of LiGluR reversibly blocks the escape response to touch. Our studies show that LiGluR provides robust control over neuronal activity, enabling the dissection and manipulation of neural circuitry in vivo.