Cre-driven optogenetics in the heterogeneous genetic panorama of the VTA Ste ´ fano Pupe 1, 2, 3 and A ˚ sa Walle ´ n-Mackenzie 1, 2 1 Uppsala University, Department of Neuroscience, Unit of Functional Neurobiology, Husargatan 3, Box 593, S-751 24 Uppsala, Sweden 2 Uppsala University, Department of Comparative Physiology, Norbyva ¨ gen 18A, S-752 36 Uppsala, Sweden 3 Federal University of Rio Grande do Norte, Brain Institute, Av Nascimento de Castro 2155, 59056-450 Natal, Brazil The selectivity of optogenetics commonly relies on ge- netic promoters to manipulate specific populations of neurons through the use of Cre-driver lines. All studies performed in the ventral tegmental area (VTA) so far have utilized promoters present in groups of cells that release dopamine (DA), GABA, or glutamate. However, neurons that co-release neurotransmitters and variabil- ities within groups of neurons that release the same neurotransmitter present challenges when evaluating the results. Further complexity is introduced by ectopic expression patterns often occurring in transgenic Cre- drivers. New perspectives could be unfolded by identi- fying and selecting different types of promoter for driv- ing the Cre recombinase. Here, we discuss some promising candidates and highlight the advantages or disadvantages of different methods for creating novel transgenic lines. Addressing diversity in the VTA with optogenetics For several generations, neuroscientists have been puzzled by the striking anatomical and functional characteristics of the ventral midbrain. Multiple findings have demonstrat- ed how DA-releasing neurons of this region, in particular of the VTA, are essential for mechanisms of reward proces- sing [1,2], mainly via their extensive projections to the nucleus accumbens (NAc) [3]. Midbrain DAergic neurons are also a major component in the mechanisms of addiction and other disorders associated with reward dysfunction [4], and display a characteristic adaptation in response to some drugs of abuse, such as cocaine [5]. Besides these essential and more established roles for DA neurons of the VTA, there is growing awareness of several other functions in which this area is also involved. Through its connections with structures such as the hippocampus, prefrontal cor- tex, amygdala, and the hypothalamus, the VTA influences spatial learning [6], social behaviors [7], fear [8], food consumption [9], and even analgesia [10]. This consider- able diversity is likely due to the heterogeneity of the VTA, the complexity of which is being constantly updated [3,11]. Several studies over the past few years have shown that the VTA contains not only DAergic, but also GABAer- gic and glutamatergic neurons, as well as neurons that release two of these classical neurotransmitters [12]. The VTA has received considerable attention lately with the advent of optogenetics. This method confers the advan- tage of depolarizing or hyperpolarizing a selected group of neurons in a fast, precise, and reversible manner by the activation of light-sensitive proteins, also known as opsins Review Glossary Cre-Lox system: a system, discovered in bacteriophages, that has been adapted for use in genetic editing for producing transgenic animals (e.g., mice and zebrafish). It is based on the interaction of a Cre recombinase with so- called Lox sites that can be introduced into any DNA sequence [88]. Depending on the orientation of a pair of these Lox sites, the DNA sequence flanked by these Lox sites (‘floxed’) will either be excised or inverted in the presence of the Cre recombinase. Flp-Frt system: a system that is analogous to the Cre-Lox system, but where the Flp recombinase, originating from Saccharomyces cerevisiae, interacts with Frt sites introduced into DNA sequences to be targeted [89]. Opsins: ChR2 and Archaeorhodopsin (Arch) are two common opsins utilized in optogenetics. Discovered in algae, these proteins promote the inward flow of positively charged ions (ChR2), or pump protons out of the cell (Arch) when exposed to light of a certain wavelength (420–510 nm for ChR2, 520–630 nm for Arch). When expressed in neurons, these opsins bind to the neuronal membrane and, by shining light onto them, researchers can regulate neuronal firing. The opsins respond with millisecond precision, allowing for fast and reversible control. Promoter sequences: these DNA sequences are most commonly located upstream of the transcriptional start site of each gene and contain binding sites for a range of transcription factors whose binding defines the conditions for transcriptional activation, also known as gene expression. Gene expression gives rise to mRNA, which in turn translates into protein, and both mRNA and protein can be detected within the cell by a range of molecular techniques. When gene expression is unique to a specific cell type, it is often referred to as a molecular marker for this cell type. Activation of a promoter defines where and when transcription of the gene starts; that is, the spatial and temporal specificity of the gene expression. Parts of promoters or transcriptional enhancing sequences, ‘‘enhancers’’, can also be found within genes, such as placed within intronic sequences. Selective expression of opsins: for opsins to target only a subset of neurons, their sequences are usually inserted, flanked by lox sites, into adeno- associated viruses (Figure 1C, main text). These viruses are then injected into the desired brain area, and sometimes coupled with an optic fiber so that light can shine there directly (Figure 1D, main text). The key factor in determining specificity will be whether the cell expresses Cre, a factor that is determined by the choice of the promoter for the Cre-driver line. 0166-2236/ ß 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tins.2015.04.005 Corresponding author: Walle ´n-Mackenzie, A ˚ . (asa.mackenzie@neuro.uu.se, asa.mackenzie@ebc.uu.se). Keywords: ventral tegmental area; reward; aversion; dopamine; glutamate; GABA; transgenics. TINS-1142; No. of Pages 12 Trends in Neurosciences xx (2015) 1–12 1