Optogenetics: Controlling Neurons with Light
There are many obstacles for uncovering the physiological and pathophysiological mechanism of nervous system. One of most diffiult challenges is theheterogeneity of cell types which come with billions of synaptic connections. Neuronsare among the most challenging cells to fiure out their working principles which willhelp our understanding of the brain. During the past decades, specifi light-sensitiveproteins and molecules helped scientists to develop an important technical approachcalled “Optogenetics,” with which precise inhibition or activation of neural pathwaysin nervous system can be achieved temporally and spatially using light. Interestingly,these light sensitive proteins come from mostly unicellular organisms such as algaeand bacteria. Combination with genetically engineered tools like adeno-associated viruses and ion-gated channels like channelrhodopsins, halorhodopsins and archaerhodopsins, the activity of neurons can be manipulated by excitation and silencing. In thisarticle, I reviewed basic principles of optogenetics to provide the reader with currentupdates.
Brain is one of the most complex organs in ourbody and difficult to understand how nervous system works properly under normal conditions. Upto decade ago, neuronal circuits have been mainlyprobed by traditional electric and magnetic stimulations that are impossible to investigate selectively specific subtypes of neurons under physiological and pathological conditions. Other techniques,such as lesion studies that do not offer any chanceto neuronal selectivity, or microinjection of neurotransmitters like dopamine, glutamate, serotonin etc., are limited to spatially and temporallyconstrained applications in neuroscience studiesuntil the appearance of optogenetics techniques which give researchers important details regarding not only for specific neuronal activities butalso for neuronal receptors (1,2). Optogenetics wasinitially used within the context of neuroscienceto describe the approach of using light to drive orsilence neuronal activity in the intact, living brainin wild type or transgenic animals, for instance,mice or rats (3). Optogenetics composed of two important research fields that are optics (light) whichare used to activate or inhibit neurons, thanks tospecific light-sensitive rhodopsins such as channelorhodopsin-2 (ChR2), halorohodosin (NpHR),archaerhodopsin (Arch), and genetic modificationswhich are used to synthesis of various kinds of rhodopsins by using viral approaches such as adeno-associated virus (AAV). The success of optogenetics in neuroscience has taken attention of manyneuroscientists and engineers in other fields, andnow the definition of optogenetics has expanded toincluding the general field of biotechnology (2,4-6).In this review, I will briefly explain the most fundamentals of optogenetics.
1) LIGHT SENSITIVE PROTEINS
a) Channelrhodopsins (ChRs)
Channelrhodopsins (ChRs) are light-gated ionchannels found in a unicellular alga (Chlamydomonas reinhardtii) (7-9). The use of microbial opsin to control the activity of neurons utilizechannelrhodopsin-2 (ChR2), one of two channelrhodopsins have by this alga (10). The most obvious and important feature of ChR2 is a light-gatednonspecific cation channel which, when illuminated with blue light, opens and permits the passageof cations (positively charged sodium and calciumions) and the subsequent depolarization of the cell(8,9). In 2005, ChR2 was introduced into hippocampal neurons in petri dish, and control neuronalspiking activity with fine temporal precision (10).Very brief (millisecond level) pulses of blue lightmay be used to induce single action potentials inChR2-expressing neurons, and neuronal spikingactivity driven by the activation of this opsin canbe controlled with high precision. This preliminaryexperiments of the usefulness of ChR2 for the control of neural activity was immediately followed bya number of reports and scientific papers confirming its function in neurons (11,12) and usefulnessfor investigate basic questions in neuroscience(13-15). ChR2 has subsequently been transferredfrom in vitro to in vivo experiments, to optimizeexpression and photocurrent in mammalian systems (13,16). After these pionnering reports, theoptogenetic toolbox has greatly become indispensible for neuroscientists, and many different opsinswith a variety of spectral, temporal, and conduc tive features have been discovered or engineered(17-19).
b) Halorhodopsin (NpHR)
like activation of neurons, inhibition of neuronalactivity is critical for understanding the mechanism of neural networks, and might complementexcitatory tools by allowing researchers to investigate the individual circuit components. One ofthe most efficient and widely used inhibitory opsins, NpHR, is a halorhodopsin from the archaeonNatronomonas pharaonis (20,21). NpHR pumpschloride ions into the cell upon light activation,resulting in hyperpolarization. With an excitationmaximum at 590 nm, eNpHR3.0 can be stimulatedby green, yellow, or red light
c) Archaerhodopsins (Arch)
Proton pumps might also be used to inhibit neurons through hyperpolarization, by pumping protons like (H+ ions) out of the cell, and have somefeatures that make them another option to chloride pumps, which include fast recovery from inactivation and high light-driven currents. Arch(archaerhodopsin-3 from Halorubrum sodomense),is proton pumps that provide strong efficiency ininhibition of neurons (22-24)
2) OPSIN EXPRESSION
To control specific neural circuit with optogenetics,one of the most crucial approaches to take consideration is the targeting specific neurons in brain.There are so many ways to target subpopulationsof neurons such cell body, axonal terminations (25).Genetically modified experimental animal models(mostly mice and rats) that express the enzyme Crerecombinase (Cre) under the transcriptional control of a specific gene are typically used to targetneuronal subpopulations. For instance, vesiculargamma aminobutyric acid transporter (VGAT)-Cremice express Cre only in inhibitory neurons thatexpress VGAT. Many different transgenic rodentlines with stable and heritable expression of Creare commercially available through Jackson Laboratory (www.jax.org), Charles River Laboratories(www.criver.com) and other breeding facilities,provide to researchers to target and manipulate avariety of different neuronal subpopulations (26).In order to provide anatomically local specificity ofopsin expression, it is necessary to make stereotaxic injections of viral vectors encoding these proteinsin the brain regions of interest. Cre is an importantenzyme that catalyzes site-specific recombinationbetween two LoxP sites, and modern Cre-drivenviral vectors are constructed with “double-floxed”genes encoding the various types of opsin, causingtargetted gene expression only in transfected cellsthat have Cre. A fluorescent tag is also encoded inthe viral construct such as green fluorescent protein (GFP), allowing for postmortem histologicalconfirmation of gene expression in the targeted celltype and brain region. Cre-inducible adeno-associated viruses (AAVs) are commercially availablefrom Addgene (wwww.addgene.org), North Carolina University-Vector Core (https://www.med.unc.edu). These viruses are genetically engineered,therefore, replication deficient and it is not knownto cause disease in humans. The numerous typesof AAV strains (e.g., AAV 2, 5) have unique transfection features in brain; hence, it is important tocontrol efficiency of the viral vectors for proper expression in the targeted brain region. After virusis injected to targeted brain area, at least 3 weeksare recommended prior to beginning experiments,in order to allow enough time for opsin expressionin neurons (27)
Optogenetics has changed the way of neuroscienceto new horizons, and has produced a new generation of experiments that dissect the causal roles ofspecific neural network components in physiological and pathological conditions. It has been used toincrease our understanding of the neural circuitsunderlying psychiatric and neurological disorders(28), addiction (29), Parkinson’s disease (30), obsessive compulsive disorder (31), social behavior(32) and reward (33), and many others (3). Thereis still an explosion in the development of new generation optogenetic tools, both through discoveryin nature and engineering in laboratories. Thecoming years should see exciting progress in thedevelopment and application of these tools to deconstruct the neural networks underlying normalbehavior and their dysfunction in psychiatric andneurological diseases.
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