Molecular profiling and targeting of brain cells and circuits related to energy balance and food reward

Abstract

In the great, grand scheme of things, we're just tiny specks that will one day be forgotten. So it doesn't matter what we did in the past or how we'll be remembered. The only thing that matters is right now, this moment. This one spectacular moment we are sharing together. Molecular profiling and genetic targeting of brain cells RNA sequencing for molecular profiling and classification of brain cells A popular and reliable method to investigate brain heterogeneity is RNA sequencing, which provides information about the whole transcriptome and can be applied to a brain region, a specific cell population or at the single cell level. Many different cell dissociation protocols and sequencing technologies exist, the application of which can vary depending on the brain region, cell abundance, developmental stage and the desired sequencing depth. For example, when researchers desire to sequence a particular cell type, they can use a transgenic animal expressing a fluorescent protein in the cell population of interest, Targeting neural subtypes and circuits with viral vectors Molecular profiling of cells is very useful for identifying novel cell types and gaining insights into their molecular makeup and transcriptional responses to cues. Nevertheless, in order to understand cell types better, we need to gain genetic access to them. A popular method for gene delivery into the brain in order to map cells, visualize brain connectivity and study the function of cell populations and circuits is viral vector technology. Viral vectors are used to target genetic information into cells of choice. To this end, either wild-type or engineered viral capsids are used to "package" the genetic information (transgene) designed in a way to facilitate proper expression of RNA or protein. Few of the most commonly used viral vectors in neuroscience are the adeno-associated virus (AAV), lentivirus (LV), canine adenovirus 2 (Cav2) and rabies. The two former are mostly used for anterograde targeting, while the two latter are used for retrograde targeting of neurons and are extremely useful for brain mapping of circuits. In this thesis we use AAV and Cav2, therefore we will discuss those. Adeno-associated viral vectors (AAVs) AAV is the most popular viral vector system used in neuroscience and gene therapy. The first gene therapy drug, which treats retinal dystrophy, was approved in 2017, while there are at least 20 approved gene therapy treatments available and at least 4000 ongoing clinical trials [25], [26]. Besides the fact that the AAV capsid has limited capacity and can accommodate only up to 5.2 kb of DNA, their use has a lot of advantages. Genes delivered by AAVs do not incorporate into the host genome and rather stay episomal [27], therefore they do not cause insertional mutations that could lead to toxic consequences. AAV capsids Hypothalamus, energy balance and leptin In this thesis we mostly focus on cells located in the hypothalamus (Chapters 1-3) and profile leptin receptor-expressing cells (Chapters 1 & 2). The hypothalamus is loc...