ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE V This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. REGULATION OF FEEDING BEHAVIOR BY OPTOGENETIC ACTIVATION OF INPUTS TO LATERAL HYPOTHALAMIC AREA HASSAN S. CHOUDHRY, KULDEEP SHRIVASTAVA, VIKSHAR ATHREYA, MARK A. ROSSI ✵ ABSTRACT The bed nucleus stria terminalis (BNST) and parabrachial nucleus (PBN) are two brain regions in correspondence with the lateral hypothalamic area (LHA) that are responsible for regulating feeding behavior in mice. It is acknowledged that increasing the activity of GABAergic BNST inputs (which re- lease the inhibitory neurotransmitter GABA) to the LHA inhibits LHA glutamate neurons and increases feeding, whereas increasing activity of glutama- tergic PBN inputs (which release the excitatory neu- rotransmitter glutamate) to the LHA excites local glutamate neurons and decreases feeding. Obesity is a prevalent problem in our society, and therefore it is important to understand the reasons behind ex- cessive food intake habits of humans. To address this issue, we tested for effects of activating the BNST-LHA and PBN-LHA pathways in relation to the satiety state of the mice. We stimulated the BNST and PBN inputs to LHA in fasted and fed mice. We tested the effects of Satiety State (fed vs. fasted) and stimulation Frequency (5-40 Hz) on sucrose seeking behavior of two Groups of mice (ChR2 vs YFP con- trols). The BNST-LHA pathway tended to show a general increase in feeding behaviors, while the PBN-LHA pathway did not show a significant effect of activation. From these results, further studies can be conducted to explore more about these neural pathways and the mechanisms underlying feeding behaviors. On a broader scale, these findings can inform future therapeutics that could help prevent unhealthy eating habits and obesity. 1 INTRODUCTION Overeating and obesity are increasing in prevalence among our society. In fact, the 2022 World Obesity Atlas published by the World Obe- sity Federation states that by 2030, approximately 1 billion people will be obese (World Obesity Atlas, 2022). Previous research has suggested that the re- cent rise in obesity, especially in the United States, is due to an increase in eating portions, lack of phys- ical activity, and greater consumption of fats, sugars, and grains (Why are Americans obese, 2022). In or- der to understand why obesity prevalence is contin- uing to increase, further exploration of brain circuits that control feeding is essential. The mouse genome is approximately 99% similar to the human genome (Vandamme, 2014). Moreover, mice and human brains are also quite similar in terms of structure and function. Due to this, we can use mouse models to inform our under- standing of human neurobiology. The lateral hypo- thalamic area (LHA) is a region of the brain that is responsible for controlling feeding behaviors in ro- dents and humans (Rossi et al., 2019). The bed nu- cleus of the stria terminalis (BNST) and the para- brachial nucleus (PBN) are two brain regions that provide inputs to the LHA where they synapse (con- nect) onto glutamatergic neurons (Jennings et al., 2013; Phua et al., 2021). It has previously been dis- covered that selective activation of LHA glutamate neurons halts feeding (Rossi et al., 2019). Addition- ally, activation of inhibitory (GABAergic) inputs from BNST inhibits the LHA glutamate neurons, thereby increasing feeding (Jennings et al., 2013). However, activation of excitatory (glutamatergic) inputs from PBN excites the LHA glutamate neurons, thereby decreasing feeding (Phua et al., 2021). The bidirec- tional nature of selectively activating LHA glutamate neurons to either increase or decrease feeding was demonstrated previously (Stamatakis et al., 2016; Jennings et al., 2013). ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE V While previous research has been con- ducted using stimulation of these pathways, this study highlights the relationship between the BNST- LHA/PBN-LHA pathways and satiety state. We are specifically looking at that to see if the activity differ- entially influences food seeking based on the cur- rent energy needs of the mice. This includes under- standing the factors that influence the ability of stim- ulation of LHA inputs for modifying feeding behav- ior (e.g., hunger, satiety, obesity). Cell-type and pro- jection-specific neuron activation is achieved with Channelrhodopsin-2 (ChR2) proteins. These are light-gated cation channels that open when ex- posed to blue light (Deisseroth & Hegemann, 2017). In turn, these ChR2 channels allow quick depolariza- tion (influx of positively charged ions into the cell) of neurons, which is important (along with repolariza- tion) for driving action potentials to communicate electrical signals between neurons. Ultimately, un- derstanding the role that these pathways play in regulating feeding behavior can help us understand more of the neurobiology underlying eating disor- ders and obesity. In addition to observing the effect of activa- tion of neural pathways on consummatory behav- iors, this paper is also interested in exploring how satiety influences the ability of LHA inputs to control eating. We hypothesize that the optogenetic stimu- lation (activation of ChR2 channels) of PBN inputs to the LHA will excite glutamate neurons and decrease food intake, while the activation of BNST inputs to the LHA will suppress glutamate neurons and in- crease food intake. Motivational state will influence the effect of stimulation because hypothalamic glu- tamate neurons will be suppressed when the mice are hungry, but they will be excited when the mice are satiated. It is believed that before obesity, the LHA glut cells act as a brake on feeding (Rossi et al., 2019). Specifically, these cells are excited after a mouse starts eating and the magnitude of their re- sponses is negatively associated with feeding Thus, it is believed that these cells act as a brake on feed- ing. During diet-induced obesity, these cells be- come hypoactive, so the removal of that brake may be facilitating overeating (Rossi et al., 2019). This study intends to build upon previous studies by utilizing numerous stimulation frequencies to ob- serve the effect of satiety state on feeding habits of mice and the role that ChR2 plays in these neural pathways compared to when it is absent (YFP). 2 METHODOLOGY ANIMAL CARE AND SURGERIES The subjects of this experiment are male and female C57BL6J wild type mice for the BNST and Vglut2-Cre mice for the PBN (Jackson Labora- tories). At 3 weeks old, the mice were weaned to fol- low standard animal welfare methods. The mice were set on a 12 hour light-dark cycle with all tests being performed during light hours. All experi- ments received approval from the Rutgers Univer- sity Institutional Animal Care and Use Committee. All experiments were also conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. A virus injection surgery was performed for two different viruses on the mice at ~6 weeks of age. To target the BNST-LHA pathway, AAV-hSyn-ChR2- YFP (AAV: adenosine associated virus; hSyn: human synapsin promoter) or AAV-hSyn-YFP were injected into the BNST. To target the PBN-LHA pathway, the Cre-dependent viruses AAV-hSyn-DIO-ChR2-YFP (DIO: double inverted operator) or AAV-hSyn-DIO- YFP were injected into the PBN. The purpose of the control was to provide justification that if there was to be a difference between the results from the two viruses, then that difference would be attributed to the ChR2 (not the AAV virus or external light source from the optogenetic experiment soon to be dis- cussed). The virus was injected into the PBN or BNST (PBN coordinates: AP-5.05mm, ML+/-1.25mm, DV- 2.82mm; BNST coordinates: AP+15mm, ML+/- 0.90mm, DV-3.82mm) of the mice depending on the targeted area of study: 10 BNST-ChR2 mice (6 males, 4 females); 7 BNST-YFP mice (5 males, 2 fe- males); 5 PBN-ChR2 mice (2 males; 3 females); 2 PBN-YFP mice (1 male, 1 female) - further research will consist of more PBN mice experiments. After the virus was injected, two optic fibers were surgically implanted bilaterally above the LHA region to allow selective activation of axon terminal signals from the BNST and PBN. ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE V BEHAVIORAL PARADIGMS Three weeks after the surgeries were com- pleted, a custom head-fixed behavior set-up was used to track the mice’s consumption of sucrose. This set-up restrained the mouse in a tube attached to a stand to lock the mouse in place, which pre- vented the mouse from moving but allowed it to vol- untarily consume sucrose solutions. Activation of the BNST-LHA or PBN-LHA pathways was made possible by sending 473 nm laser light at different frequencies to the optic fibers placed in the LHA. These different frequencies would reveal the effect of stimulation frequency on how motivated the mice were in licking the sucrose. As a result, the BNST and PBN pathways are activated to test their effects on the consummatory behavior of the mice. Drops of 10% sucrose solution were randomly delivered through a spout positioned directly in front of the mice, and licks at the spout were recorded with a custom lickometer. During each session, a total of 50 trials (sucrose reward deliveries) were performed on each mouse. 25 of these trials included optoge- netic stimulation, while 25 of them did not. Optical stimulation was 3 s in duration (5 ms pulses at 5-40 Hz) and occurred either immediately before (BNST group) or after (PBN group) reward delivery. BNST stimulation occurred before reward delivery be- cause since BNST stimulation is hypothesized to in- crease feeding, stimulation after reward delivery would not produce clear results due to a ceiling ef- fect (already maximized licking). PBN stimulation oc- curred after reward delivery because since PBN stimulation is hypothesized to decrease feeding, stimulation before reward delivery would not pro- duce clear results due to a floor effect (already min- imized licking). The rate of licking during optical stimulation was quantified. After the behavior experiment was success- fully completed, the mice were then transcardially perfused for histological analysis. Mice were eu- thanized with pentobarbital and 20 mL of phos- phate-buffered saline (PBS) and paraformaldehyde (PFA) were perfused through their circulatory sys- tem to fix the brain tissue (made rigid in order to eventually splice the brain). The purpose of this was to ensure a clearer image under the confocal microscope when doing histological analysis later in the process. HISTOLOGY After the perfusion was completed, virus ex- pression and fiber placement were confirmed. To do this, we sliced the brains at 35 µm with a cryostat, mounted them on microscope slides, and imaged the fluorescence with confocal microscopy. Both a tile scan and z-stack were performed to create im- ages of the brain to include the BNST, PBN, or LHA. Histological analysis allowed us to confirm that the BNST and PBN showed expression because of ChR2 or YFP, and that the optic fibers were correctly im- planted in the LHA. All viruses had the YFP fluoro- phore, so all virus expression was verified by visual- izing YFP in the areas of interest. DAPI was utilized, which is a blue stain that allows us to see all cell nu- clei. It is used to visualize the cells/tissue because otherwise the tissue would be invisible with fluores- cence-based confocal microscopy. DATA ANALYSIS AND STATISTICS Head-fixed behavior data was analyzed us- ing custom Python code via Jupyter Notebook. Op- tically evoked licks were measured in accordance with their respective frequencies (BNST mice: 5Hz, 10Hz, 20Hz, and 40Hz; PBN mice: 10Hz and 20Hz). The frequencies were chosen because they span the range of the natural firing of the BNST cells. Data for the BNST mice were recorded during the pre-re- ward epoch of 0-3 seconds before stimulation, while PBN data were recorded during the post-reward epoch of 0-3 seconds after stimulation. GraphPad Prism was used to create grouped graphs of the data and for statistical analyses. Two-way ANOVAs with corrections for Sadik post hoc tests were per- formed to determine significance. A Grubbs’ test was performed to test for statistical outliers in the data. 3 RESULTS HISTOLOGICAL ANALYSIS To validate the viral injections and expres- sion of ChR2 within the BNST and placement of op- tic fibers in the LHA, we performed histological ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE V analysis with confocal microscopy. The image shows that the surgeries were performed correctly with vi- rus expression in the BNST and the presence of op- tic fibers in the LHA (FIGURE 1). While DAPI was used for the BNST and LHA during the experiment, we did not include it in the LHA image below because the background of the green channel (how the YFP is seen) was high enough to visualize fiber place- ments. In concise terms, the virus was injected cor- rectly into the BNST and the optic fibers were im- planted correctly into the LHA. To validate the viral injections and expres- sion of ChR2 within the PBN and placement of optic fibers in the LHA, we performed histological analysis with confocal microscopy. The image shows that the surgeries were performed correctly with virus ex- pression in the PBN and the presence of optic fibers in the LHA (FIGURE 1). While DAPI was used for the PBN and LHA during the experiment, we did not in- clude it in the LHA image below because the back- ground of the green channel (how the YFP is seen) was high enough to visualize fiber placements. In concise terms, the virus was injected correctly into the PBN and the optic fibers were implanted cor- rectly into the LHA. FIGURE 1: Confocal Microscope Image of BNST and LHA. Left panel shows the ChR2-eYFP injection site in BNST. Right panel shows optical fiber placement above the LHA. BNST: bed nucleus of the stria terminalis, ac: anterior commissure, EP: en- topeduncular nucleus, LHA: lateral hypothalamic area, f: fornix, 3V: third ventricle. ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE V FIGURE 2: Confocal Microscope Image of PBN and LHA. Left panel shows the ChR2-eYFP injection site in PBN. Right panel shows optical fiber placement above the LHA. PBN: parabrachial nucleus, LPB: lateral parabrachial nucleus, SCP: superior cerebellar peduncle, mPB: medial parabrachial nucleus, 4/5Cb: lobules 4 & 5 of the cerebellar vermis, EP: entopeduncular nucleus, LHA: lateral hypothalamic area. OPTOGENETIC ACTIVATION OF THE BNST-LHA PATHWAY To determine if the inputs from the BNST and PBN will affect motivated behavior, we utilized a custom-head fixed behavior set-up. It was not sup- ported that motivation can influence feeding behav- iors of mice due to satiety states (FIGURE 3A, B). Both of these figures did not show a main effect from the Satiety State or an overall interaction between Sa- tiety State and Frequency. The results showed that increasing the frequency made a significant effect on the licking behaviors of the ChR2 mice (FIGURE 3A). Furthermore, the results show that the ChR2 group licked significantly more due to activation of the BNST pathway in both the fed and fasted states re- spectively (FIGURE 3C, D). This supports our hypothe- sis that activation of the BNST would cause an in- crease in consummatory behavior. However, in- creasing the frequency alone did not cause a signif- icant difference in licking for fed mice (FIGURE 3C), but it did for fasted mice (FIGURE 3D). Overall, there was an interaction between Group and Frequency for fed mice (FIGURE 3C), with the Sidak post hoc test con- firming a significant difference at 20Hz (FIGURE 3C). However, the fasted mice did not show an interac- tion between Group and Frequency (FIGURE 3D). There were no outliers detected from the statistical analysis. In concise terms, optogenetic activation of the BNST-LHA pathway resulted in an increase in feeding behaviors of the mice. OPTOGENETIC ACTIVATION OF THE PBN-LHA PATHWAY Results were also observed for the PBN pathway (FIGURE 4). We hypothesized that the satiety state would cause a significant effect on the food in- take of mice. This was supported by the observed interaction between Satiety State and Frequency of the ChR2 fasted mice compared to the fed mice (FIG- URE 4A), but there was no interaction for the YFP mice (FIGURE 4B). Part of our hypothesis, however, was not supported: activation of the PBN pathway failed to decrease food intake in mice. However, there was no significant effect of Group and there was no in- teraction between Group and Frequency (FIGURE 4C, D). Additionally, the Sidak post hoc test revealed that when the mice were fed, 20Hz stimulation re- duces their licking (FIGURE 4C). However, when the mice were fasted, there was no significant effect of Frequency (FIGURE 4D). There were no outliers de- tected from the statistical analysis. In concise terms, optogenetic activation of the PBN-LHA pathway did not have a significant effect on mice feeding behav- iors. ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE V FIGURE 3: Behavior Data of BNST-LHA Pathway. A) We performed a two-way ANOVA test (Satiety State x Frequency). There is no main effect of Satiety State: F(1.0, 9.0) = 0.01, p = 0.93. There is a main effect of Frequency: F(1.3, 11.6) = 11.16, p = 0.004. There is no interaction between Satiety State and Frequency: F(1.5, 13.7) = 1.00, p = 0.37. B) We performed a two-way ANOVA test (Satiety State x Frequency). There is no main effect of Satiety State: F(1.0, 6.0) = 5.67, p = 0.05. There is no main effect of Frequency: F(1.4, 8.7) = 0.65, p = 0.50. There is no interaction between Satiety State and Frequency: F(1.5, 9.3) = 1.55, p = 0.26. C) We performed a two-way ANOVA test (Group x Frequency). There is a main effect of Group: F(1, 15) = 7.09, p = 0.02. There is no main effect of Frequency: F(1.3, 19.2) = 3.42, p = 0.07. There is an interaction between Group and Frequency: F(3, 45) = 3.54 p = 0.02. Sidak post hoc test revealed a difference at 20Hz. D) We performed a two-way ANOVA test (Group x Frequency). There is a main effect of Group: F(1, 15) = 7.00, p = 0.02. There is an effect of Frequency: F(2.5,37.4)=5.55, p=0.005. There is no interaction between Group and Frequency: F(3,45)=1.97, p=0.13. All groups (A-D) were tested for po- tential outliers. A Grubbs’ test was performed with alpha = 0.05, but no outliers were detected. Note: * Indicates the frequency at which the overall interaction between Group and Frequency occurs in accordance with the Sidak test. Note: ** Indicates a main effect of Group. ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE V FIGURE 4: Behavior Data of PBN-LHA Pathway. A) We performed a two-way ANOVA test (Satiety State x Frequency). There is no main effect of Satiety State: F(1, 4) = 6.17, p = 0.07. There is no main effect of Frequency: F(1, 4) = 0.003, p = 0.96. There is an interaction between Satiety State and Frequency: F(1, 4) = 23.11, p = 0.01. Sidak post hoc test revealed a difference at 20Hz. B) We performed a two-way ANOVA test (Satiety State x Frequency). There is no main effect of Satiety State: F(1, 1) = 4.18, p = 0.29. There is no main effect of Frequency: F(1, 1) = 25.28, p = 0.13. There is no interaction between Satiety State and Frequency: F(1, 1) = 50.16, p = 0.09. C) We performed a two-way ANOVA test (Group x Frequency). There is no main effect of Group: F(1, 5) = 1.68, p = 0.26. There is no main effect of Frequency: F(1, 5) = 1.99, p = 0.22. There is no interaction between Group and Frequency: F(1, 5) = 2.49, p = 0.18. D) We performed a two-way ANOVA test (Group x Frequency). There is no main effect of Group: F(1, 5) = 1.64, p = 0.26. There is a main effect of Frequency: F(1, 5) = 27.79, p = 0.003. There is no interaction between Group and Frequency: F(1, 5) = 0.005, p = 0.95. All groups (A-D) were tested for potential outliers. A Grubbs’ test was performed with alpha = 0.05, but no outliers were detected. Note: * Indicates the frequency at which the overall interaction between Satiety State and Frequency occurs in accordance with the Sidak test. ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE V 4 DISCUSSION Overall, the results showed that the PBN in- puts to the LHA may not be as relevant for feeding, but activation of BNST inputs to the LHA did influ- ence consummatory licking. Specifically, activation of the BNST-LHA pathway caused a general in- crease in licking, which is similar to what was found previously (Jennings et al., 2013). While previous lit- erature showed activation of the PBN-LHA pathway to cause a decrease in feeding (Phua et al., 2021), we did not observe an effect of stimulation on licking. FIGURE 5 shows a schematic representa- tion of the cell projections from the BNST and PBN to the LHA, while also showing the GABAergic and glutamatergic inputs from the BNST and PBN re- gions respectively. FIGURE 5: Schematic of BNST and PBN cell projections to LHA. Figure 5 is a schematic of the cell input projections from the BNST and PBN inputs to the LHA. GABAergic inputs were from the BNST to the LH, while glutamatergic inputs were from the PBN to the LHA. The BNST, PBN, and LHA regions are each labeled. Stimulated cells were differentiated from non-stimulated cells, while projections towards the LHA were differentiated from projections towards other regions. A key was also included. Note: While the BNST and PBN does contain a mixture of both GABAergic and glutamatergic inputs, the BNST primarily con- tains GABAergic inputs and the PBN primarily contains glutamatergic inputs (viral expression was not restricted to solely GA- BAergic or glutamatergic inputs) . In most cases for both the BNST and PBN mice, increasing the frequency of stimulation caused an increase in licking behavior. There were also effects observed when changing the frequency, which was that increasing the stimulation frequency tended to cause an increase in sucrose licking. How- ever, since many of the significant differences in feeding behavior increases revealed from post hoc tests occurred at a frequency of 20Hz but not as much with 40Hz, it is reasonable to believe that re- peatedly increasing the frequency of light will not automatically increase feeding behaviors. This could be because 40 Hz stimulation is too high for the neurons to follow and could be detrimental for their health, which could ultimately decrease the rate of consummatory licking behavior. It is possible that licking cannot exceed the physical limits set by the central pattern generators. It was also observed that the ChR2 mice tended to lick more than the YFP mice after stimu- lating the BNST-LHA pathway (the CHR2 mice did lick more for the PBN-LHA pathway, but the differ- ence was not statistically significant). This could be due to the function of ChR2, which allows ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE V stimulation of inputs to drive action potentials (Deis- seroth & Hegemann, 2017). Furthermore, this can potentially imply that stimulation of the BNST-LHA pathway causes sucrose seeking. There tended to be more feeding when the mice were hungry (fasted) compared to when they were fed, which can imply that utilizing optogenetics to stimulate path- ways to mimic neuronal activity in action may not be the only factor responsible for differences in feed- ing behaviors. This suggests that motivation (hun- ger) plays a role in the ability of BNST inputs to LHA to influence feeding. This idea can be relevant for human health and disease because it can show how feelings of hunger can cause people to eat more when they have food in front of them, rather than when they are already full. While obese mice were not focused on in this study, our findings support the hypothesis that fed mice were less sensitive to stimulation, while fasted mice were hypersensitive to stimulation. One limitation of the study was that there were only two frequencies tested for the PBN mice (10 Hz and 20Hz). This could be limiting because it may not be as justified to predict a trend based upon the results. Testing more frequencies would allow for more reliable results for the PBN pathway mice. Another potential limiting factor is that since the surgeries were manually performed for both in- jecting the virus and implanting the optic fibers, it is possible that there could be potential variability in the data that is caused by variability in the location of the fibers and/or virus. In summary, activation of the BNST-LHA pathway showed an increase in feeding, while acti- vation of the PBN-LHA pathway did not have a sig- nificant effect on feeding behaviors. Future experi- ments will be needed to address the synaptic mech- anisms behind the BNST-LHA and PBN-LHA path- ways for why activation of these pathways results in certain effects. Also, further trials will be performed on the PBN-LHA pathway for more stimulation fre- quencies to see if new results are found. Addition- ally, future experiments can entail more satiety states, such as testing mice in a thirsty state. One reason for this would be to test whether the effects that we see are specific to hunger. Also, we can observe whether the mice respond differently to non-caloric rewards such as water. Therefore, this could result in understand- ing whether the BNST-LHA or PBN-LHA pathways have functions that are specific to regulating food intake, or if they have more general functions re- lated to all consummatory behaviors. The use of these mouse models will consequently help us de- velop a greater understanding of human neurobiol- ogy∎ 5 ACKNOWLEDGEMENTS I would like to thank the Child Health Insti- tute for its continued support of scientific research and for providing the resources necessary to con- duct this research. 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Journal of Pharmacy and Bioallied Sciences, 6(1), 2. https://doi.org/10.1126/science.aan5544 https://doi.org/10.1126/science.1241812 https://doi.org/10.1126/sciadv.abe4323 https://doi.org/10.1126/science.aax1184 https://doi.org/10.1523/JNEUROSCI.1202-15.2016 ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE V HTTPS://DOI.ORG/10.4103/0975-7406.124301 [7] World Obesity Atlas 2022. World Obesity Federation. (2022). Retrieved November 25, 2022, from HTTPS://WWW.WORLDOBESITY.ORG/RESOURCES/RESOURCE- LIBRARY/WORLD-OBESITY-ATLAS-2022 https://doi.org/10.4103/0975-7406.124301 https://www.worldobesity.org/resources/resource-library/world-obesity-atlas-2022 https://www.worldobesity.org/resources/resource-library/world-obesity-atlas-2022 ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE V Hassan Choudhry is an undergraduate student in the Honors College at Rut- gers University New Brunswick. His interest in research started at Biotechnol- ogy High School, where he conducted experiments on Daphnia magna. At Rut- gers, he has been doing research for over a year under his principal investiga- tor Dr. Mark Rossi. Located at the Child Health Institute of New Jersey, the Rossi Lab aims to study neural circuits that regulate food intake and obesity. Outside of research, Hassan is involved within the Rutgers community as a chemistry teacher intern, Penji tutor, and serves as the Biology major representative for the School of Environmental and Biological Sciences Governing Council. As a current pre-medical student, he aspires to go to medical school with the hopes of becoming a doctor in the future. Hassan can be contacted at HCHOUDHRY03@GMAIL.COM.