Investigate func of spec areas of brain?

Lesions problematic, Permanent Not always spec One prob w current approaches to studying brain is that any given spatial location has more than one cell type that might have diff func roles. (projection neurons vs interneurons all destroyed in lesion) How to stimulate only one subset of cells? Pharmocology, used to study contrib. of cell types to behave (receptor agonists and antagonists) Prob: drug not readily reversible, can last long time, with differential effects betw subjects (lots of variables) Optogenetics may solve? Optogenetics, tech that allows rsrchers to use light to manip cells w/in living tiss, perform spatially (target spec group of cell in heterogeneous area of cells)AND temporally (down to milisec range) precise manips on brain. Opsin, gene that encodes light sensitive prot, encode light-responsie prot that directly bind retinal (rhodopsin) Type I, microbial. Most commonly used, form ion chnnls, when activated by light, they open and form ion flux. (fast recovery) Type II, animal. G-prot coupled, found only in higher anims and used mostly for vision (slow recovery) Steps to successfully stimul neurons w light (fast devel field) 1) package opsin w a virus, virus simply funcs to allow opsin to infiltrate cell/genome (plasmid, circular containment of genetic material) 2) put virus into animals brain so opsin will go into cell and get into genome (or at least be expressed in the cell) 3) implant optic fiber into site of viral infection 4) wait few weeks after implantation to allow the protein to be translated and put into membranes of neurons. 5) shine some light on the cell that are infected w virus (fiber optics) 6) compare behave/physiology/other meas when infected cells are being stimul when w/in subject cntrls between subject cntrls microbial opsins are useful bc they combine light sens and ion flux into one gene prot that is encoded by one gene. Perfect for neuro apps? History of optical cntrl in brain Curret optogenics is not first time that scientsts use light to maip nueral antivity. Laser dstr of cells

UV-light-sens-ligand gate chnnl(19960s) Bacterioprhopdosi (2003) 3 families of current optogenetic tools, excitaty, case inflection, deploy I(Depol), etec. When neurons expressins CMR are stimul w blue light, then cells are excited. Inhib, halorhodopsin (eNpHR3.0) inward Cl-pump causes memb hyperhol (inib) Achaoutward proton pum causes optoXR, G-prot-coupled microbial opsins (based on ChR2, regulate intracell signaling) **bi-direc opsins, express both ChR2 and HR in same neuron, can hyper/depol depen on neuron. Limit? How far can shine night in brain? 1mm through brand tissue. How to use this to study brain? Can do, cell type specific targeting, use promoter driven reductions, genes expressed IE, CaMKIIa, spec viruses do not infect cortical interneurons, with this, a subject of cells in given read can be homogenously. Probs w promoter-driven targeting Bc of law that opsin is inserted ito genome, the size for the target pop is very small/expensive. Reliability of expression patter teach otherspecific viral vetor and cell type is uique. Traget sepc cell types? Cre-depen cell type targeting. Use transgenic techs to regulate vius expression. Use recombinase is expessed in certainpopl of cells. Whe cre-spefic opsin is introduced, expression will only be w Cre=fell. Expressions: Usually use transgenic tech to regulae virus info Cre pop expressed in certain popl of cells When Cre recombinase is expressed on in TH (dopamine) neurons, allows expression o ChR2 in only dopamine neurons in VTA Limit of Cre-driver sys? Opsin expression is lim both by spec of cre line and by spec of the virus/promoter If youre targeting popl of cells that is very sparese, it might be diff to get an effect bc less than 100% of possib cells are expressing the opsin, and the light stimul is limited spatially by props of the tiss. Sum of optogenetic tools Excitatory, ChR2 Inhib, HR, Arch OptoXE Bidirectional

Cell targeting Promoter Cre-Depen Using diff comb of these, can cover diver possib.