# The Impact of Optogenetics on Regenerative Medicine
## 2 Molecular Pathways in Optogenetics  
Optogenetics is a technique that uses that uses specific wavelengths as an activator of specific neural activities, and the proteins as a pathway for this activation. When light stimulates these proteins, such interaction rapidly influences specific ions across the cell membrane. Precisely, to fully modulate and control specific neural populations, light-sensitive genes are inserted and stimulated to act on specific transmembrane channels [2]. Optogenetic neuromodulation is useful to highlight several pathophysiological mechanisms and to explain the function of specific neural networks. Neuromodulation acts on opsins, a family of proteins susceptible to luminous stresses. Neuromodulation can be performed by using viral vectors that infect such neurons carrying specific proteins: these proteins will be activated and will be finally able to modulate specific areas located in the brain [5]. The light-sensitive proteins most commonly used for neural activation and inhibition are respectively Channelrhodopsin-2 and Halorhodopsin. Channelrhodopsin-2 is activated with blue light (~472 nm), and it excites the target neurons by the opening of cation channels. On the other hand, Halorodopsin is activated with yellow light (~590 nm), and it inhibits neural activity by the activation of the chloride pump (Figure 1). With the combined use of these proteins and specific light wavelengths, it is possible to study the behavior of a specific area of the human brain in different conditions [6]. The first trials involving optogenetic techniques were carried out in the field of ophthalmology [7]. These trials mainly involved the Opsins family. Opsins can be classified into two macro-classes: type I (microbial) and type II (animal) Opsins [8]. Type I Opsins can be found in algae and prokaryotes. Type II Opsins are receptor proteins, able to be coupled with other different types of proteins. The difference between the two types of Opsins is related to how the water molecules are positioned in the protein chains. The discovery of natural forms of protein chains has allowed researchers to study the optogenetics method in depth [9]. A well-known example of type II Opsins is the rhodopsin, also known as GPCR (G-Protein-Coupled receptor). Rhodopsin, under luminous stresses, activates hundreds of G downstream of proteins and provides different physiological responses (pupillary constriction, circadian rhythm, etc.). There are  
Figure 1: Neuromodulation with Optogenetic technique: blue light activates ChR2 with an excitatory effect, while yellow light activates NpHR with an inhibitory effect. different G protein subfamilies: Gt-coupled opsins, Gi/o-coupled opsins, encephalopsin, and TMT (Teleost Multiple Tissue) opsins (Gq-coupled opsin, Gi-coupled neuroppsin, cnidarian Gs-coupled opsin) [10]. These G-proteins have a very important feature, used for optogenetic applications: the bistability. Indeed, G-proteins are able to convert the chromophores associated with them into a function of the absorbed light wavelength [11]. Among the numerous proteins used in the manipulation of the neuronal membrane potential, the Channelrhodopsin-2 (ChR2) has the peculiarity to work as light-gated ion channels [12]. Its structure can be easily changed with specific aminoacids that allow different cellular responses, obtained in response to the time of exposure to a specific light. [PERSON] et al. [13] observed that proteins exposed to the pulsed blue light generated a potential action by muscle cells. This experimental finding could suggest a large-scale control of muscle contractions, through the optogenetics technique. Another protein successfully used in optogenetics is the Halorhodopsins (NpHR). In vitro and in vivo experiments reported that the NpHR, stimulated with yellow light (-590 nm), modifies the cell membrane potential in the opposite direction, thus increasing its absolute value. NpHR is also activated by the blue light, and it can inhibit or activate neurons based on the time of exposure to the blue light [14].