Doris L. Fortin

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.

Photochemical control of endogenous ion channels and cellular excitability

Light-activated ion channels provide a precise and noninvasive optical means for controlling action potential firing, but the genes encoding these channels must first be delivered and expressed in target cells. Here we describe a method for bestowing light sensitivity onto endogenous ion channels that does not rely on exogenous gene expression. The method uses a synthetic photoisomerizable small molecule, or photoswitchable affinity label (PAL), that specifically targets K+ channels. PALs contain a reactive electrophile, enabling covalent attachment of the photoswitch to naturally occurring nucleophiles in K+ channels. Ion flow through PAL-modified channels is turned on or off by photoisomerizing PAL with different wavelengths of light. We showed that PAL treatment confers light sensitivity onto endogenous K+ channels in isolated rat neurons and in intact neural structures from rat and leech, allowing rapid optical regulation of excitability without genetic modification.

Photochromic blockers of voltage-gated potassium channels.

Photochromic ligands (PCLs) can be optically switchedbetween isomers that show different biological activities. Assuch, they offer an opportunity to convert ligand-actuatedpathways into light-actuated pathways, thus making it possi-ble to control a wide range of biological processes with light.PCLs have been explored for various classes of targetproteins, including enzymes,

New photochemical tools for controlling neuronal activity

Neurobiology has entered a new era in which optical methods are challenging electrophysiological techniques for their value in measuring and manipulating neuronal activity. This change is occurring largely because of the development of new photochemical tools, some synthesized by chemists and some provided by nature. This review is focused on the three types of photochemical tools for neuronal control that have emerged in recent years. Caged neurotransmitters, including caged glutamate, are synthetic molecules that enable highly localized activation of neurotransmitter receptors in response to light. Natural photosensitive proteins, including channelrhodopsin-2 and halorhodopsin, can be exogenously expressed in neurons and enable rapid photocontrol of action potential firing. Synthetic small molecule photoswitches can bestow light-sensitivity on native or exogenously expressed proteins, including K(+) channels and glutamate receptors, allowing photocontrol of action potential firing and synaptic events. At a rapid pace, these tools are being improved and new tools are being introduced, thanks to molecular biology and synthetic chemistry. The three families of photochemical tools have different capabilities and uses, but they all share in enabling precise and noninvasive exploration of neural function with light.

The behavior of α-synuclein in neurons†

Despite considerable evidence linking α‐synuclein with membranes in vitro, it has proven difficult to demonstrate membrane association of the protein in vivo. α‐Synuclein localizes to the nerve terminal, but biochemical experiments have not revealed a tight association with membranes. To address the dynamics of the protein in live cells, we have used photobleaching and found that α‐synuclein exhibits high mobility, although distinctly less than an entirely soluble protein. Further, neural activity controls the distribution of α‐synuclein, causing its dispersion from the synapse. In addition to the presumed role of α‐synuclein dynamics in synaptic function, changes in its physiological behavior may underlie the pathological changes associated with Parkinsons disease.

Chapter 18 – The Dynamics of α-Synuclein at the Nerve Terminal

Publisher Summary This chapter discusses the underlying dynamics of α-synuclein at the nerve terminal. α-synuclein fibrils are found in abundance in Lewy bodies, a cardinal pathological lesion found in most cases of Parkinsons disease (PD). α-synuclein can adopt multiple structural conformations depending on its environment, some of which are capable of converting into key intermediates in the assembly of Lewy bodies. Controlling the appearance of these pathogenic intermediates may prevent or at least slow down PD. Under normal circumstances, α-synuclein interacts with synaptic vesicle membranes, localizing to the nerve terminal where it has been proposed to regulate neurotransmitter release. Upon membrane binding, α-synuclein undergoes a major conformational change, from relatively unstructured to highly α-helical. Neuronal activity induces the dissociation of the protein from synaptic vesicle membranes and its dispersion from the synapse. The link between membrane association and conformation indicates that the dynamics of α-synuclein probably has an important role in the pathogenesis of PD. This chapter, while explaining the dynamics of α-synuclein, also elaborates in detail the membrane binding in vitro as well as the membrane interactions in vivo.