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Title: Mapping Dopaminergic Circuitry Mediating Food Timing on Circadian Times Scales Author: Angel Macias Scheduled meals entrain habitual behavior and physiology—for example, diurnal animals, like humans, do most of their eating during daylight hours. Aside from using sunlight to stay attuned to a 24 hour day, mealtime can also serve as a time cue. Nocturnal animals, such as mice, do most of their feeding during the dark hours and do not show activity during the daylight hours. However, when
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  Title:  Mapping Dopaminergic Circuitry Mediating Food Timing on Circadian Times Scales Author : Angel Macias Scheduled meals entrain habitual behavior and physiology  —  for example, diurnal animals, like humans, do most of their eating during daylight hours. Aside from using sunlight to stay attuned to a 24 hour day, mealtime can also serve as a time cue.  Nocturnal animals, such as mice, do most of their feeding during the dark hours and do not show activity during the daylight hours. However, when mice are fed at the same time daily during the middle of the light period, they will become active in the 2-3 hours  preceding scheduled meal time. This food anticipatory activity (FAA) is displayed in circadian (~24 hr. ) intervals, and mapping the neural circuitry that allows for this  behavior can give further insight into eating disorders, addictive behavior, and influence clinical treatment. Using the mouse model of dopamine depletion, we are attempting to map the critical dopamine inputs that mediate circadian entrainment to feeding. In this mouse model, the gene Tyrosine hydroxylase (TH) , which is required for making dopamine, has been genetically engineered such that it can be deleted by a DNA cutting enzyme called Cre recombinase . Thus, in mice that express Cre in most of their dopaminergic neurons, it is possible to delete the dopamine-producing enzyme rendering these mice severely hypodopaminergic (containing less than 10% of normal dopamine content). We have determined that amongst the hypodopamine (  FloxTH  ;  DAT-cre +) mice there are two different groups  —  one that can predict scheduled meal time and one that cannot. We hypothesize that Cre recombinase  makes different deletions of Tyrosine hydroxylase  in these two groups and we propose to determine where the gene is being retained in both groups of mice. In this manner, we will map the dopamine circuitry, finding the critical population of dopamine neurons that are sufficient to entrain circadian activity rhythms to scheduled feeding. The behavioral aspect of the project was assessed using a computer vision system called HomeCageScan. This program inputs video recording data and provides a measurement of total movement in meters displayed within an isolated time interval. In order to identify the dopaminergic circuitry that mediates the mice’s behavior, w e utilized the Cre-lox recombination system to render the mice hypodopaminergic. We then assessed whether the mice expressed Cre in their dopaminergic neurons using both Polymerase Chain Reaction to amplify their DNA and Electrophoresis to compare the result against a ladder within an agarose gel. Finally, once our video recording data was gathered and the mice were culled, utilizing Immunohistochemistry, we used a Tyrosine Hydroxylase Antibody staining protocol so as to identify any remaining dopamine neurons within the VTA on the brain tissue slices under the microscope. This was done to further assess whether our mice were indeed hypodopaminergic.  Results 020040060080010001200123456789101112131415161718192021222324     H    i   g    h    A   c   t    i   v    i   t   y    (   s   e   c   o   n    d   s    ) #62 0200400600800100012001400123456789101112131415161718192021222324     H    i   g    h    A   c   t    i   v    i   t   y    (   s   e   c   o   n    d   s    ) #63    Figure 1 (#62)  illustrates total activity of a dopamine depleted mouse and Figure 2 (#63) illustrates total activity of a hypodopamine mouse with no FAA o ver a 24 hour period. Grayed lines display the mouse’s activity over separate 24 hour intervals, whereas the blackened line illustrates the average. The black and white scale bars beneath the graph indicate light and dark hours. Scheduled meal was administered at the end of the 24th hour and the total activity with the 2- 3 hours preceding the meal represents the mouse’s FAA. Figure 3 is a d iagram displaying the Ventral Tegmental Area (VTA) located in the midbrain-source of dopamine (left). VTA of a wild type mouse(top right) and the VTA of one of our dopamine depleted mice (bottom left) are compared. Upon observation of both the recorded activity of the mice via video analysis using HomeCageScan and the midbrain tissues containing the Ventral Tegmental Area (VTA) of these Th  floxed mice, all were determined to be hypodopaminergic and about half were found to have entrained to scheduled feeding whereas the other approximate half did not. We predict that Cre  makes different deletions amongst these two groups and we propose to identify where the Th gene is being preserved in each group. In this manner, we will have identified the critical dopamine inputs that are sufficient for circadian entrainment to scheduled feeding.    Bibliography Gallardo, Christian M., Martin Darvas, Mia Oviatt, Chris H. Chang, Mateusz Michalik, Timothy F. Huddy, Emily E. Meyer, Scott A. Shuster, Antonio Aguayo, Elizabeth M. Hill, Karun Kiani, Jonathan Ikpeazu, Johan S. Martinez, Mari Purpura, Andrea  N. Smit, Danica F. Patton, Ralph E. Mistlberger, Richard D. Palmiter, and Andrew D. Steele. Dopamine Receptor 1 Neurons in the Dorsal Striatum Regulate Food Anticipatory Circadian Activity Rhythms in Mice.  ELife  3 (2014): n. pag. Web. Henschen, Charles W., Richard D. Palmiter, and Martin Darvas. Restoration of Dopamine Signaling to the Dorsal Striatum Is Sufficient for Aspects of Active Maternal Behavior in Female Mice.  Endocrinology  154.11 (2013): 4316-327. Web.  
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