of multiple axes of cellular function within intact animals. hardware for light application another necessary factor is hardware (e.g. integrated fiberoptic and solid-state light sources) to allow specific cell types, even deep within the brain, to be controlled in freely behaving animals. most commonly, the latter is now achieved using the fiberoptic-coupled diode technology introduced in 2007, though to avoid use of implanted electrodes, researchers have engineered ways to inscribe a "window" made of zirconia that has been modified to be transparent and implanted in mice skulls, to allow optical waves to penetrate more deeply to stimulate or inhibit individual neurons. to stimulate superficial brain areas such as the cerebral cortex, optical fibers or leds can be directly mounted to the skull of the animal. more deeply implanted optical fibers have been used to deliver light to deeper brain areas. complementary to fiber-tethered approaches, completely wireless techniques have been developed utilizing wirelessly delivered power to headborne leds for unhindered study of complex behaviors in freely behaving organisms. expression of optogenetic actuators optogenetics also necessarily includes the development of genetic targeting strategies such as cell-specific promoters or other customized conditionally-active viruses, to deliver the light-sensitive probes to specific populations of neurons in the brain of living animals (e.g. worms, fruit flies, mice, rats, and monkeys). in invertebrates such as worms and fruit flies some amount of all-trans-retinal (atr) is supplemented with food. a key advantage of microbial opsins as noted above is that they are fully functional without the addition of exogenous co-factors in vertebrates. technique three primary components in the application of optogenetics are as follows (a) identification or synthesis of a light-sensitive protein (opsin) such as channelrhodopsin-2 (chr2), halorhodopsin (nphr), etc… (b) the design of a system to introduce the genetic material containing the opsin into cells for protein expression such as application of cre recombinase or an adeno-associated-virus (c) application of light emitting instruments. the technique of using optogenetics is flexible and adaptable to the experimenter's needs. cation-selective channelrhodopsins (e.g. chr2) are used to excite neurons, anion-conducting channelrhodopsins (e.g. gtacr2) inhibit neuronal activity. combining these tools into a single construct (e.g. bipoles) allows for both inhibition and excitation, depending on the wavelength of illumination. introducing the microbial opsin into a specific subset of cells is challenging. a popular approach is to introduce an engineered viral vector that contains the optogenetic actuator gene attached to a specific promoter such as camkiiα, which is active in excitatory neurons. this allows for some level of specificity, preventing e.g. expression in glia cells. a more specific approach is based on transgenic "driver" mice which express cre recombinase, an enzyme that catalyzes recombination between two lox-p sites, in a specific subset of cells, e.g. parvalbumin-expressing interneurons. by introducing an engineered viral vector containing the optogenetic actuator gene in between two lox-p sites, only the cells producing cre recombinase will express the microbial opsin. this technique has allowed for multiple modified optogenetic actuators to be used without the need to create a whole line of transgenic animals every time a new microbial opsin is needed. after the introduction and expression of the microbial opsin, a computer-controlled light source has to be optically coupled to the brain region in question. light-emitting diodes (leds) or fiber-coupled diode-pumped solid-state lasers (dpss) are frequently used. recent advances include the advent of wireless head-mounted devices that apply leds to the targeted areas and as a result, give the animals more freedom to move. fiber-based approaches can also be used to combine optical stimulation and calcium imaging. this enables researchers to visualize and manipulate the activity of single neurons in awake behaving animals. it is also possible to record from multiple deep brain regions at the same using grin lenses connected via optical fiber to an externally positioned photodetector and photostimulator. technical challenges selective expression one of the main problems of optogenetics is that not all the cells in question may express the microbial opsin gene at the same level. thus, even illumination with a defined light intensity will have variable effects on individual cells. optogenetic stimulation of neurons in the brain is even less controlled as the light intensity drops exponentially from the light source (e.g. implanted optical fiber). it remains difficult to target opsin to defined subcellular compartments, e.g. the plasma membrane, synaptic vesicles, or mitochondria. restricting the opsin to specific regions of the plasma membrane such as dendrites, somata or axon terminals provides a more robust understanding of neuronal circuitry. mathematical modelling shows that selective expression of opsin in specific cell types can dramatically alter the dynamical behavior of the neural circuitry. in particular, optogenetic stimulation that preferentially targets inhibitory cells can transform the excitability of the neural tissue, affecting non-transfected neurons as well. kinetics and synchronization the original channelrhodopsin-2 was slower closing than typical cation channels of cortical neurons, leading to prolonged depolarization and calcium influx. many channelrhodopsin variants with more favorable kinetics have since been engineered. a difference between natural spike patterns and optogenetic activation is that pulsed light stimulation produces synchronous activation of expressing neurons, which removes the possibility of sequential activity in the stimulated population. therefore, it is difficult to understand how the cells in the population affected communicate with one another or how their phasic properties of activation relate to circuit function. optogenetic activation has been combined with functional magnetic resonance imaging (ofmri) to elucidate the connectome, a thorough map of the brain's neural connections. precisely timed optogenetic activation is used to calibrate the delayed hemodynamic signal (bold) fmri is based on. light absorption spectrum the opsin proteins currently in use have absorption peaks across the visual spectrum, but remain considerably sensitive to blue light. this spectral overlap makes it very difficult to combine opsin activation with genetically encoded indicators (gevis, gecis, glusnfr, synapto-phluorin), most of which need blue light excitation. opsins with infrared activation would, at a standard irradiance value, increase light penetration and augment resolution through reduction of light scattering. spatial response due to scattering, a narrow light beam to stimulate neurons in a patch of neural tissue can evoke a response profile that is much broader than the stimulation beam. in this case, neurons may be activated (or inhibited) unintentionally. computational simulation tools are used to estimate the volume of stimulated tissue for different wavelengths of light. applications the field of optogenetics has furthered the fundamental scientific understanding of how specific cell types contribute to the function of biological tissues such as neural circuits in vivo. on the clinical side, optogenetics-driven research has led to insights into parkinson's disease and other neurological and psychiatric disorders such as autism, schizophrenia, drug abuse, anxiety, and depression. an experimental treatment for blindness involves a channel rhodopsin expressed in ganglion cells, stimulated with light patterns from engineered goggles. amygdala optogenetic approaches have been used to map neural circuits in the amygdala that contribute to fear conditioning. one such example of a neural circuit is the connection made from the basolateral amygdala to the dorsal-medial prefrontal cortex where neuronal oscillations of 4 hz have been observed in correlation to fear induced freezing behaviors in mice. transgenic mice were introduced with channelrhodoposin-2 attached with a parvalbumin-cre promoter that selectively infected interneurons located both in the basolateral amygdala and the dorsal-medial prefrontal cortex responsible for the 4 hz oscillations. the interneurons were optically stimulated generating a freezing behavior and as a result provided evidence that these 4 hz oscillations may be responsible for the basic fear response produced by the neuronal populations along the dorsal-medial prefrontal cortex and basolateral amygdala. olfactory bulb optogenetic activation of olfactory sensory neurons was critical for demonstrating timing in odor processing and for mechanism of neuromodulatory mediated olfactory guided behaviors (e.g. aggression, mating) in addition, with the aid of optogenetics, evidence has been reproduced to show that the "afterimage" of odors is concentrated more centrally around the olfactory bulb rather than on the periphery where the olfactory receptor neurons would be located. transgenic mice infected with channel-rhodopsin thy1-chr2, were stimulated with a 473 nm laser transcranially positioned over the dorsal section of the olfactory bulb. longer photostimulation of mitral cells in the olfactory bulb led to observations of longer lasting neuronal activity in the region after the photostimulation had ceased, meaning the olfactory sensory system is able to undergo long term changes and recognize differences between old and new odors. nucleus accumbens optogenetics, freely moving mammalian behavior, in vivo electrophysiology, and slice physiology have been integrated to probe the cholinergic interneurons of the nucleus accumbens by direct excitation or inhibition. despite representing less than 1% of the total population of accumbal neurons, these cholinergic cells are able to control the activity of the dopaminergic terminals that innervate medium spiny neurons (msns) in the nucleus accumbens. these accumbal msns are known to be involved in the neural pathway through which cocaine exerts its effects, because decreasing cocaine-induced changes in the activity of these neurons has been shown to inhibit cocaine conditioning. the few cholinergic neurons present in the nucleus accumbens may prove viable targets for pharmacotherapy in the treatment of cocaine dependence prefrontal cortex cages for rat equipped with optogenetic led commutators which permit in vivo study of animal behavior during optogenetic stimulations. in vivo and in vitro recordings from the university of colorado, boulder optophysiology laboratory of donald c. cooper ph.d. showing individual camkii aav-chr2 expressing pyramidal neurons within the prefrontal cortex that demonstrated high fidelity action potential output with short pulses of blue light at 20 hz (figure 1). motor cortex in vivo repeated optogenetic stimulation in healthy animals was able to eventually induce seizures. this model has been termed optokindling. piriform cortex in vivo repeated optogenetic stimulation of pyramidal cells of the piriform cortex in healthy animals was able to eventually induce seizures. in vitro studies have revealed a loss of feedback inhibition in the piriform circuit due to impaired gaba synthesis. heart optogenetics was applied on atrial cardiomyocytes to end spiral wave arrhythmias, found to occur in atrial fibrillation, with light. this method is still in the development stage. a recent study explored the possibilities of optogenetics as a method to correct for arrythmias and resynchronize cardiac pacing. the study introduced channelrhodopsin-2 into cardiomyocytes in ventricular areas of hearts of transgenic mice and performed in vitro studies of photostimulation on both open-cavity and closed-cavity mice. photostimulation led to increased activation of cells and thus increased ventricular contractions resulting in increasing heart rates. in addition, this approach has been applied in cardiac resynchronization therapy (crt) as a new biological pacemaker as a substitute for electrode based-crt. lately, optogenetics has been used in the heart to defibrillate ventricular arrhythmias with local epicardial illumination, a generalized whole heart illumination or with customized stimulation patterns based on arrhythmogenic mechanisms in order to lower defibrillation energy. spiral ganglion optogenetic stimulation of the spiral ganglion in deaf mice restored auditory activity. optogenetic application onto the cochlear region allows for the stimulation or inhibition of the spiral ganglion cells (sgn). in addition, due to the characteristics of the resting potentials of sgn's, different variants of the protein channelrhodopsin-2 have been employed such as chronos, catch and f-chrimson. chronos and catch variants are particularly useful in that they have less time spent in their deactivated states, which allow for more activity with less bursts of blue light emitted. additionally, using engineered red-shifted channels as f-chrimson allow for stimulation using longer wavelengths, which decreases the potential risks of phototoxicity in the long term without compromising gating speed. the result being that the led producing the light would require less energy and the idea of cochlear prosthetics in association with photo-stimulation, would be more feasible. brainstem optogenetic stimulation of a modified red-light excitable channelrhodopsin (reachr) expressed in the facial motor nucleus enabled minimally invasive activation of motoneurons effective in driving whisker movements in mice. one novel study employed optogenetics on the dorsal raphe nucleus to both activate and inhibit dopaminergic release onto the ventral tegmental area. to produce activation transgenic mice were infected with channelrhodopsin-2 with a th-cre promoter and to produce inhibition the hyperpolarizing opsin nphr was added onto the th-cre promoter. results showed that optically activating dopaminergic neurons led to an increase in social interactions, and their inhibition decreased the need to socialize only after a period of isolation. visual system studying the visual