aminopeptidase inhibitor br Genetic methods of opsin gene

Genetic methods of opsin gene delivery to specific neural populations Opsin genes can be selectively expressed in a specific pre-chosen type of neurons in the brain. Let us discuss the main strategies whose efficiency has been proved to achieve in vivo expression. One of the most widespread methods of genetic material delivery is using lentiviruses. Optimal gene aminopeptidase inhibitor time is two weeks after injection. The main advantages of this method are in a high level of gene expression and in the required expression persisting for several years. The disadvantages of using lentiviruses include insufficient specificity and a low level of expression for several cell-specific promoters. Currently, gene material delivery by lentiviruses is used in practically all experiments on mammals. Another common method is delivering the materials by adenoassociated viruses (AAV). The optimal gene expression time is 3 weeks after injection. A less popular method is using Cre-dependent AAV expression systems. The expression time is 3 weeks. The advantages and disadvantages of the second and the third methods are similar to those of the lentiviral method.
Main strategies of optical control by opsins
Tools for regulating biochemical signaling There is one more type of opsins (type II), for example, the photosensitive proteins in mammalian eyes; these proteins are capable of not only inducing a photocurrent at light exposure but of acting as G-coupled protein receptors (GPCRs), and therefore taking part in intracellular signaling. It is possible to control slow inhibition [41] or excitation [42]. Currently a great number of chimeras [43] between vertebrate rhodopsins and GPCR families are being developed that may serve as one-component control tools (among them, dopaminergic, serotonergic and adrenergic receptors which all play an important role in neurotransmission and neuromodulation). These optogenetic tools are called optoXRs, and they allow to control intracellular signaling for studying the behavior of freely moving mice [11].
Selecting light beam parameters and light delivery After opsin expression was achieved in neurons presenting an interest to researchers, the problem of light beam delivery arose. The requirements imposed on the beam\'s parameters vary depending on the conditions of the experiment. For example, beam parameters required for studying fast oscillations in thin brain slices while using several opsins in vitro are different from those for studying the effects of prolonged in vivo stimulation of certain regions of the animal brain [44]. The parameters of photocurrents induced in neurons by light pulses depend on many factors. Some of them are the type of expressed opsin, irradiation wavelength, intensity and duration, and even events that happened before the start of irradiation. If not all channelrhodopsin molecules recover their initial state after the previous exposure, the initial response to a light pulse is reduced.
Methods of recording the experimental data Various methods of measuring experimental parameters are used for optogenetic control. First of all, these are the methods of obtaining and analyzing images using various dyes which include Ca2+-specific dyes (e.g., fura-3, Fluo-5F), and also voltage-sensitive dyes (VSDs, e.g., RH-155). Such methods are effective for measuring the electrical activity in large cell populations ex vivo and in vivo with a high temporal resolution. A two-photon microscope can be used for imaging with Ca2+-specific dyes, since noises from channelrhodopsin photoactivation are virtually absent during two-photon excitation. Voltage-sensitive dyes are lipophilic molecules whose optical absorption depends on membrane potential. Along with high-speed cameras for recording the changes in optical signal, VSD imaging is used, which allows to detect the changes in neural electrical activity with high spatial and temporal resolutions (on the order of µm and ms). The absorption maximum for the RH-155 dye is 700 µm, while excitation peaks for opsins are in the 470–590 µm range. Such a difference in wavelengths allows to simultaneously optically stimulate opsins and to detect images.