Elevated dietary fat alone is not sufficient to decrease AgRP projections in the paraventricular nucleus of the hypothalamus in mice

Elevated dietary fat alone is not sufficient to decrease AgRP projections in the paraventricular nucleus of the hypothalamus in mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Elevated dietary fat alone is not sufficient to decrease AgRP projections in the paraventricular nucleus of the hypothalamus in mice Selma Yagoub, Robert Chesters, Jonathan Ott, Jiajie Zhu, Lídia Cantacorps, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4358544/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Aug, 2024 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Within the brain, the connections between neurons are constantly changing in response to environmental stimuli. A prime environmental regulator of neuronal activity is diet, and previous work has highlighted changes in hypothalamic connections in response to diets high in dietary fat and elevated sucrose. We sought to determine if the change in hypothalamic neuronal connections was driven primarily by an elevation in dietary fat alone. Analysis was performed in both male and female animals. We measured Agouti-related peptide (AgRP) neuropeptide and Synaptophysin markers in the paraventricular nucleus of the hypothalamus (PVH) in response to an acute 48h high fat diet challenge. Using two image analysis methods described in previous studies, an effect of a high fat diet on AgRP neuronal projections in the PVH of male or female mice was not identified. These results suggest that it may not be dietary fat alone that is responsible for the previously published alterations in hypothalamic connections Future work should focus on deciphering the role of individual macronutrients on neuroanatomical and functional changes. AgRP high fat diet axonal projections paraventricular nucleus of the hypothalamus melanocortin Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Feeding responses are predominantly regulated within the brain via Agouti-related peptide (AgRP) and proopiomelanocortin (POMC) neurons, two neural populations of the central melanocortin system. They are located mainly in the arcuate nucleus of the hypothalamus (ARC) and have opposite actions on regulating feeding. When activated, POMC neurons inhibit food intake whilst AgRP neurons increase it. These neurons also express receptors to metabolic hormones such as insulin, leptin and ghrelin, that allow them to continuously monitor the energy state of the animal and alter their activity to maintain energy homeostasis. In this context, periods of fasting, or prolonged food deprivation are known to decrease circulating levels of leptin and insulin. Fasting also increases the activity of arcuate AgRP neurons [ 1 , 2 ] while refeeding behaviour after 24h fasting is prevented if these AgRP neurons are ablated [ 3 ]. Furthermore, AgRP neuron activity changes in response to energetically dense foods. This change in activity may be dependent on the length of HFD exposure. For example, long term access to high fat diet (HFD) compromises AgRP sensitivity to hormonal signals such as ghrelin or leptin, contributing therefore to the disruption of energy balance and resulting in leptin resistance [ 4 – 6 ]. Moreover, acute 48h exposure to HFD has been shown to cause a loss of AgRP neuron leptin sensitivity [ 7 , 8 ]. Conversely however, a recent electrophysiological study has shown an increase in AgRP neuronal activity upon an acute exposure to HFD for 2 days [ 9 ]. The consequences of long term (greater than 8 weeks) HFD exposure on the function of the hypothalamic melanocortin system has been extensively studied, but predominantly only in male mice. Electrophysiology and fiber photometry studies have shown that AgRP/ Neuropeptide-Y (NPY) neurons become hyperexcitable after 8 weeks of HFD in male mice [ 7 , 10 ]. In females, the same time exposure also induces AgRP neuron hyperexcitability but to a lesser extent because of an elevated baseline firing rate compared to males [ 9 , 10 ]. Moreover, in male mice, it has been shown that 8 weeks of HFD is sufficient to cause a dramatic, 60% decrease in AgRP axonal projections to the paraventricular nucleus of the hypothalamus (PVH), a nucleus where the action of AgRP release on their receptor, the melanocortin receptor 4 (MC4R) neurons is known to be the dominant driver of food intake [ 7 ]. Interestingly, and most surprisingly, short-term HFD exposure, as short as 48h, has been reported to have an even greater effect on the reduction in AgRP neuronal projections, with an 80% decrease in projections observed in the PVH of male mice [ 7 ]. As in the long term HFD exposure, functional changes in AgRP neuronal activity have also been observed following 48h of HFD, with a rapid change in calcium activity seen in response to the presentation of food [ 11 – 13 ] as well as increased neuronal firing of AgRP neurons [ 7 , 14 ]. Additionally, 48h HFD led to an up-regulation of suppressor of cytokine signaling-3 (SOCS3), an inflammatory and insulin signalling hallmark in AgRP but not in POMC neurons in the ARC [ 8 ]. Another study showed that 24h intralipid treatment in the ARC revealed an up-regulation of TNF-α and an increase in the number of astrocytes mirroring neuroinflammation in that nucleus [ 15 ]. Furthermore, hypothalamic proteomics data collected from 72h HFD-exposed male mice highlighted a change in protein spots involved in neuronal remodelling and synaptic plasticity indicating a structural adaptation of the hypothalamus [ 16 ]. Altogether, it seems that in male mice short term HFD exposure presents similar electrophysiological, molecular and structural signature changes in AgRP neurons as long term HFD exposure. Despite the growing evidence pointing toward sex-specific metabolic and central adaptations (e.g. firing rate, neuronal connections) in response to a long term HFD exposure [ 17 – 23 ], the effect of a short term HFD exposure in female mice is still poorly studied in comparison to males. Here, we explored the potential sex-specific adaptative response to an acute (48h) HFD exposure on AgRP axonal projections within the hypothalamus in male and female mice. We specifically targeted dietary fat, and not elevated sucrose as commonly found in HFD, to ensure that the effects would be attributed to increased fat consumption alone. We assessed neuroanatomical changes using both the endogenous AgRP peptide as well as a targeted labeling of a synaptic protein. Image analyses utilized two pipelines designed based on previous literature. Our findings using both image analyses pipelines show no change of AgRP neuronal connections in the PVH after 48h of HFD both in male as well as female mice. Thus, acute elevations in dietary fat alone are not sufficient to modify AgRP axonal architecture in the PVH. Results 48h HFD exposure does not markedly change metabolic parameters in mice . The effects of HFD on AgRP neurons are often attributed the increased dietary fat alone. However, the high fat diets used in previous studies are often confounded by the presence of increased dietary sucrose as well [ 7 , 10 , 24 ]. To overcome this and to specifically study the effects attributed to elevated dietary fat, we utilized a 60% HFD with similar sucrose to animals receiving only a standard diet (STD: 9% kCal from fat). The HFD-exposed group were given access to HFD at the onset of the dark phase (Zeitgeber time (ZT) 12) for 48h until sacrifice (Fig. 1 a). There were no significant changes in body weight after 48h HFD exposure, in either male (Fig. 1 b and d) or female animals (Fig. 1 c and e). In males (Fig. 1 f) but not in females (Fig. 1 g), random fed detection of glycemia was significantly increased after 48h of access to HFD (Males: 18.49+/- SD vs. 42.72+/- SD, p = 0.0161, Females: 19.66+/- SD vs. 68.70+/- SD, p = 0.6614). AgRP neuronal projections in the PVH are unchanged after 48h HFD exposure . It has been shown that HFD exposure induces sex-specific responses particularly in the hypothalamus [ 17 – 19 , 22 , 23 ]. It has been reported that in male mice, 48h of HFD access is sufficient to decrease 80% of AgRP axonal projections to the PVH [ 7 ]. However, no studies have investigated the effect in female mice. In order to compare the effect of a short-term HFD exposure on AgRP projections to the PVH in male and female mice, we used AgRP-IRES-Cre; Synaptophysin-TdTomato adult mice. This mouse model allowed us to assess the synaptic protein, Synaptophysin, specifically in AgRP neuronal projections through detection of TdTomato signal from the Synaptophysin-TdTomato fusion protein expression. Image analyses were performed in accordance with previously literature using both a maximum intensity projection method and sum of slices method [ 7 , 25 , 26 ]. In both analysis pipelines the first step to process the acquired images for analysis is to set a threshold for each image. This thresholding value utilizes an automatic detection of overall signal intensities across the image and uses an algorithm to apply a standardized selection of positively labelled signal to generate a binary image. For each image analysis method, we compared between STD and 48h HFD groups the automatically generated thresholding values applied to AgRP and Synaptophysin-TdTomato images. AgRP and Synaptophysin-TdTomato thresholding values were not significantly different between STD and 48h HFD groups (Supp. Figure 1 , Supp. Table 1 ). As the cellular identity and heterogeneity of the PVH dramatically differs across the anterior to posterior axis, we first, assessed AgRP and Synaptophysin-TdTomato in the PVH anterior (PVHant) (Fig. 2 a) [ 27 ]. This region corresponds to the neuroendocrine compartment of the PVH, a region enriched in receptors for a number of neuropeptide hormones such as MC4R [ 28 , 29 ]. Using the maximum intensity projection method of analysis, the most common approach used in the literature, we did not detect any sex-specific difference in the mean gray value of AgRP and Synaptophysin-TdTomato labeling (Fig. 2 b and d). However, analysis of the Raw Integrated Density of AgRP and Synaptophysin-TdTomato signals did highlight an overall effect of sex, with a significant difference between male and female animals on STD (Fig. 2 c and e, Table 1 ). Contrary to the published literature, we did not detect any significant reduction in AgRP projections after 48h of HFD exposure in the PVHant in neither males nor females, regardless of the analysis method used (Fig. 2 and Table 1 ). Table 1 Two-way ANOVA statistical analysis of data presented in Fig. 2 . Method 1: Maximum intensity projection Panel Source of variation F (DFn, DFd) p -value b) AgRP mean gray value Interaction Sex Diet "F (1, 15) = 3.131" "F (1, 15) = 2.396" "F (1, 15) = 1.134" 0.0971 0.1425 0.3037 c) AgRP Raw Integrated Density Interaction Sex Diet "F (1, 14) = 1.277" "F (1, 14) = 4.895" "F (1, 14) = 1.638" 0.2774 0.0441 * 0.2214 d) Syn-TdTom mean gray value Interaction Sex Diet "F (1, 14) = 2.858" "F (1, 14) = 3.674" "F (1, 14) = 0.9927" 0.1130 0.0759 0.3360 e) Syn-TdTom Raw Integrated Density Interaction Sex Diet "F (1, 14) = 2.000" "F (1, 14) = 6.781" "F (1, 14) = 1.761" 0.1791 0.0208 * 0.2057 Method 2: Sum slices f) AgRP mean gray value Interaction Sex Diet "F (1, 15) = 0.8681" "F (1, 15) = 0.06371" "F (1, 15) = 1.665" 0.3662 0.8041 0.2164 g) AgRP Raw Integrated Density Interaction Sex Diet "F (1, 15) = 1.321" "F (1, 15) = 0.08496" "F (1, 15) = 1.673" 0.2684 0.7747 0.2154 h) Syn-TdTom mean gray value Interaction Sex Diet "F (1, 14) = 0.1469" "F (1, 14) = 0.7393" "F (1, 14) = 0.04054" 0.7072 0.4044 0.8433 i) Syn-TdTom Raw Integrated Density Interaction Sex Diet "F (1, 14) = 4.327e-005" "F (1, 14) = 0.9702" "F (1, 14) = 0.02912" 0.9948 0.3413 0.8669 Next, we assessed AgRP and Synaptophysin projections in the midpoint of the PVH (PVHmid) (Fig. 3 a) [ 27 ]. By applying the maximum intensity projection method of image analysis, the labeling of AgRP and Synaptophysin-TdTomato remained unchanged after 48h access to HFD with no significant difference of AgRP projections between males and females in the mean gray value (Fig. 3 b and d, Table 2 b and d) and Raw Integrated Density (Fig. 3 c and e, Table 2 c and e). The sum slices analysis method also did not detect any difference in AgRP and Synaptophysin labeling (Fig. 3 f-i) and no effect of the diet on the projections (Table 2 f-i). In sum, our results show no effect of 48h HFD exposure on AgRP projections in the PVHmid. Table 2 Two-way ANOVA Statistical analysis of data presented in Fig. 3 . Method 1: Maximum intensity projection Panel Source of variation F (DFn, DFd) p -value b) AgRP mean gray value Interaction Sex Diet "F (1, 15) = 0.04563" "F (1, 15) = 0.4600" "F (1, 15) = 1.184" 0.8337 0.5080 0.2937 c) AgRP Raw Integrated Density Interaction Sex Diet "F (1, 15) = 0.07372" "F (1, 15) = 0.5952" "F (1, 15) = 1.645" 0.7897 0.4524 0.2191 d) Syn-TdTom mean gray value Interaction Sex Diet "F (1, 14) = 0.07452" "F (1, 14) = 0.6688" "F (1, 14) = 0.2914" 0.7889 0.4272 0.5978 e) Syn-TdTom Raw Integrated Density Interaction Sex Diet "F (1, 14) = 0.01304" "F (1, 14) = 0.7729" "F (1, 14) = 0.4367" 0.9107 0.3942 0.5195 Method 2: Sum slices f) AgRP mean gray value Interaction Sex Diet "F (1, 15) = 0.4469" "F (1, 15) = 0.9467" "F (1, 15) = 0.5240" 0.5139 0.3460 0.4803 g) AgRP Raw Integrated Density Interaction Sex Diet "F (1, 15) = 1.286" "F (1, 15) = 0.7140" "F (1, 15) = 0.6582" 0.2745 0.4114 0.4299 h) Syn-TdTom mean gray value Interaction Sex Diet "F (1, 14) = 0.2569" "F (1, 14) = 0.5981" "F (1, 14) = 1.224" 0.6201 0.4522 0.2871 i) Syn-TdTom Raw Integrated Density Interaction Sex Diet "F (1, 14) = 0.01766" "F (1, 14) = 0.4794" "F (1, 14) = 1.628" 0.8962 0.5000 0.2227 Discussion The aim of our study was to investigate sex-specific metabolic and/or neuroanatomical changes in AgRP projections to the PVH in response to acute 48h HFD exposure. We specifically analysed AgRP neuronal projections to the anterior and middle PVH (PVHant and PVHmid), as this is the neuroendocrine compartment of the PVH, and is known to express the MC4R; the main target of AgRP release in this region [ 29 – 31 ]. In accordance with the literature, we did not see an effect of acute 48h HFD exposure on body weight [ 16 , 32 ]. However, blood glucose levels were significantly increased in male mice. Since blood glucose was measured in ad-libitum fed mice, this minor, but significant difference may be due to the consumption of HFD shortly before sacrifice: as the animals were sacrificed after onset of the dark phase (ZT 12), when mice usually consume a large majority of their daily food intake. Next, we investigated the effects of 48h HFD on AgRP projections using an AgRP-Cre; Synaptophysin-TdTomato mouse model specifically labeling these projections. To analyse images of AgRP and Synaptophysin-TdTomato, we applied two methods of neuronal projection analysis published in the literature: the maximum intensity projection and sum slices (described in material and methods section) [ 7 , 25 , 26 ]. In the PVHant and using the maximum intensity method, our analysis of the Raw Integrated Density of AgRP and Synaptophysin-TdTomato revealed a significant sex difference but ultimately no significant effect of the 48h HFD exposure within each sex. This sex effect seems to be driven primarily by a difference within the STD-exposed groups. In the PVHmid, no significant effect of sex nor diet were observed. Methodological factors that can potentially influence the results are: method of thresholding (manual versus automated), what defines the Region of Interest (ROI) drawn on the image, and/or if the same approach to slice analysis is used (single comparable slice versus average of multiple slices). In our study, we standardized the coronal brain slice selected, the ROI used, and selected an automated unbiased thresholding algorithm (ImageJ - Moments). We also quality checked all thresholded binarized images to ensure the accurate inclusion of all positively-labeled pixels. Furthermore, we checked that automatic thresholding values were not significantly different from each other across both sexes and diet groups. While using the maximum intensity projection and sum of slices for confocal image analyses, we observed a higher accuracy between the binarized image and the original image using the maximum intensity projection. It was observed that with the sum of slices method, there was an over estimation of the positive signal, including signal from the background and not specifically the positively labelled neuronal projections. Therefore, we would recommend applying the maximum intensity projection method for analysing neuronal projections. Regarding the region of analysis representing the PVH, we differentiated the anterior and mid PVH to localize and target the effect of HFD on AgRP projections due to the known intra-PVH complexity and likely differing functions of subregions of the PVH [ 24 , 29 , 30 ]. Our results demonstrate that acute exposure to elevated amounts of dietary fat, alone, does not alter AgRP axonal projections to the PVH. This holds true for both males and females. An interesting point that is still open for discussion is the contribution of fat alone, or fat in the presence of elevated sugar content as well as the cause for synaptic change. The primary study showing a dramatic decrease in AgRP projections to the PVH used a diet high in fat (45%) but also in sugar (17% sucrose) [ 7 ]. It was previously shown that AgRP neurons adjust their response to acute high fat and high sucrose, through electrophysiological and calcium dynamics or even AgRP mRNA levels [ 7 , 10 , 13 , 33 ]. Moreover, a recent publication highlighted electrophysiological changes upon acute dietary exposures appear to be predominantly due to the excess dietary sucrose [ 14 , 34 ], underscoring that there are unique responses of AgRP neurons to macronutrient content in the diet. This evidence raises the question as to the contribution of individual macronutrient contents, separately and/or in combination, to induce synaptic changes within the hypothalamus and the brain generally. Therefore, understanding the role of individual macronutrients acquired through the diet, on aspects of synaptic physiology may give insight into the acute adaptability of neuronal circuits in adjusting to nutritional changes. Materials and methods Animals. AgRP-IRES-Cre male mice (strain AgRPtm1(cre)Lowl/J, Jackson laboratories Stock number:012899) were crossed with ROSA-CAG-LSL-Synaptophysin-tdTomato-WPRE (Ai34D flox/flox) female mice (Strain B6;129S-Gt (RO-SA)26Sortm34.1 (CAG-Syp/tdTomato)Hze/J, Jackson Laboratories, Stock number: 012570) to obtain AgRP-IRES-Cre;Syn-TdTomato flox/wt mice used in our experiments. Animals were group housed (2–3 per cage) with a 12-h light/dark cycle at 22 ± 2°C and 50–70% humidity with ad libitum access to water and control STD (Ssniff # V1534-300; kCals metabolizable energy: 67% carbohydrates; 24% proteins and 9% fat; with 5.3% of total weight coming from crude sugar) unless otherwise stated. Mice were randomly assigned to either STD control or HFD for 48h (HFD introduced at start of the dark phase) (Ssniff #EF acc. D12492 (I) mod; kCals metabolizable energy: 22% carbohydrates, 24% protein and 60% fat; with 9.4% of total weight coming from crude sugar). All animal experiments and procedures were approved by the animal welfare committee (Landesamt für Arbeitsschutz, Verbraucherschutz und Gesundheit; animal ethics application number 2347-36-2021). Experiments were conducted in accordance with the ARRIVE guidelines and the European Directive 2010/63/EU. Metabolic measurements. All measurements were performed during the dark phase at ZT 12. Body weight and food intake were measured at onset of the dark cycle on the first day of the experiment, 24h and 48h later before the sacrifice. Percentage body weight change was calculated as: body weight (g) at 48h x 100/body weight (g) at start of the experiment. Glycemia was assessed at sacrifice by blood collection directly from the right atrium and measured with a glucometer (Contour Care, Ascensia). Immunostaining . Mice were deeply anesthetised with an intra-peritoneal injection of Pentobarbital (400mg/kg) and sacrificed by transcardial perfusion with, first, 60mL of ice cold 1X phosphate-buffered saline (PBS), then 4% paraformaldehyde (PFA, pH 9.5, 3.8% Borate) after onset of the dark phase ZT 12–15. Brains were post fixed in 4% PFA + Borax for 4h then switched to a 20% sucrose solution overnight before being stored at -80°C until usage. Fixed brains were sliced coronally using a freezing sledge microtome (Slide 4004 M, pfm medical, Cat. 400410) to obtain 30µm frozen brain slices that were stored in anti-freeze solution (10% 10X PBS, 20% Ethyl glycol and 20% Glycerol) at -20° C until further use. The day of the staining, slices were washed 3x10min times with 0.02M KPBS, 5min in 0.3% glycine in PBS and 10min 0.03% SDS in PBS followed by a 1h incubation in blocking and permeabilization buffer containing 3% donkey serum in 0.25% Triton-X in PBS. Slices were then incubated with primary antibodies Rabbit anti-AgRP (Phoenix; Cat# H-003-57; dilution 1:4,000) and Goat anti-TdTomato (SICGEN, Cat# AB8181-200; dilution 1:4,000) mixed in SignalStain® Antibody Diluent (CellsSignal; Cat# 8112) for 48h at 4°C plus 2h at room temperature. After washing with 0.02M KPBS (1x5min and 3x10min), slices were incubated with the secondary antibodies Donkey anti-Rabbit (1:500) Alexa Fluor 488-conjugated and Donkey anti-Goat (1:500) Alexa Fluor 546-conjugated diluted in 0.25% Triton-X in 1x KPBS for 1h at room temperature then washed with 0.02M KPBS (1x5min and 3x10min). Brain slices were mounted on Superfrost Plus microscope slides and cover slipped with VECTASHIELD Antifade Mounting Medium with DAPI (Biozol; Cat# VEC-H-1200). Images of the PVHant (distance from Bregma − 0.83mm) and PVHmid (distance from Bregma − 0.95mm), selected based on the mouse brain atlas [ 27 ], were collected using a confocal microscope (Multiphoton Laser Scanning Microscope LSM 780, Zeiss) and Zen (Zeiss) software. 3D Z-stack (z = 17 slices/image, interval = 1.5µm) images were acquired. Two mice were excluded from further image processing and analysis due to obvious germline deletion of the transgene STOP cassette resulting in ubiquitous expression of Synaptophysin-TdTomato protein. Image Processing and Analysis. Images were processed and analyzed with a custom-written ImageJ macro and performed by an experimenter blinded to the experimental conditions. First, a region of interest (ROI) defining the outlines of the PVH was generated (Figs. 2 and 3 a). This ROI was then used in the following two methods: Maximum intensity projection : For each channel, Z-stack images were converted to a 2D image using a maximum intensity projection, and saved opened as individual files. A threshold value was automatically applied to these images using the “Moments” algorithm and a binary image created. The ROI was then overlayed on to the binary image. The workflow is represented in Fig. 4 . Sum slices : For each channel, a sum-slice intensity projection was created: this creates a 2D image by adding together the intensity of each pixel of each slice of the Z-stack at every x-y pixel coordinate in the 2D image space. The middle slice (number 9 out of 17) of the Z-stack was used as the reference slice and the sum-slice image, thresholded using “Moments” algorithm same this was applied to all 17 slices of the Z-stack image and a binary 2D image was saved. The same ROI used for the maximum intensity projection method was applied here. The workflow is represented in Fig. 5 . For both methods, the size of the area, Raw Integrated Density (= sum of the pixels), and mean gray value (= sum of the gray values of all pixels divided by the number of pixels) were measured. Threshold values of the PVH ROI, from both analysis methods, were compared for differences across sexes and diet. All ImageJ scripts are supplied in the Supplementary Methods. All ROIs and confocal images used for the maximum intensity projection and sum slices analyses can be shared upon request. Statistical analysis . All data are represented as mean +/- standard deviation (SD) with each single data point representing an individual mouse. GraphPad Prism 9 was used for all statistical analysis and graphs. An unpaired two-tailed t -test was conducted to analyse the body weight, the percentage change of body weight and blood glucose levels. AgRP projections were analysed with a two-way ANOVA to assess the sex, diet and Interaction of sex and diet. When appropriate, the two-way ANOVA was followed by Bonferroni post-hoc comparison. Threshold values were analyzed using a Mann Whitney comparison. Significant differences were considered when p-value < 0.05. Declarations Acknowledgements We would like to acknowledge all of the animal caretaking staff and veterinary staff of the Max Rubner Laboratory for their support in the maintenance and care of the animals used in this study. Graphical depictions in Figure 1 were generated using BioRender.com. This work was performed with financial support from the Leibniz Association through the Leibniz Competition Best Minds Grant 'BAByMIND’ (J99/2020) to RNL. Additional financial support was provided by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany´s Excellence Strategy – EXC-2049 – 390688087 (NeuroCure) and by the German Center for Diabetes Research (82DZD03D2Y and 82DZD03D03). Author contributions RNL and SY designed the experiment. SY, JO, LC, JZ, and RNL conducted all animal work and contributed to tissue collection. KR performed all genotyping experiments. SY, RC, JO, LC and JZ contributed to tissue slicing, immunostaining and image analysis. RC developed ImageJ scripts for image analyses. SY and RNL analysed the data and wrote the manuscript. All authors edited and critically reviewed the manuscript. RNL acquired the funding for the project and supervised all authors. Data availability statement All data will be made available upon reasonable request to the corresponding author. Additional Information The authors declare no competing interests. References Betley, J.N., et al., Neurons for hunger and thirst transmit a negative-valence teaching signal. Nature, 2015. 521 (7551): p. 180-185. Nakajima, K., et al., Gs-coupled GPCR signalling in AgRP neurons triggers sustained increase in food intake. Nat Commun, 2016. 7 : p. 10268. 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Bouret, Maternal obesity-induced endoplasmic reticulum stress causes metabolic alterations and abnormal hypothalamic development in the offspring. PLoS Biol, 2020. 18 (3): p. e3000296. Franklin, K.P., G. , Paxinos and Franklin's the Mouse Brain in Stereotaxic Coordinates. 2019. 5th Edition : p. 376. Biag, J., et al., Cyto- and chemoarchitecture of the hypothalamic paraventricular nucleus in the C57BL/6J male mouse: a study of immunostaining and multiple fluorescent tract tracing. J Comp Neurol, 2012. 520 (1): p. 6-33. Li, C., et al., Defined Paraventricular Hypothalamic Populations Exhibit Differential Responses to Food Contingent on Caloric State. Cell Metab, 2019. 29 (3): p. 681-694 e5. Biddinger, J.E., et al., Leptin suppresses development of GLP-1 inputs to the paraventricular nucleus of the hypothalamus. Elife, 2020. 9 . Simmons, D.M. and L.W. Swanson, Comparison of the spatial distribution of seven types of neuroendocrine neurons in the rat paraventricular nucleus: toward a global 3D model. J Comp Neurol, 2009. 516 (5): p. 423-41. Melhorn, S.J., et al., Acute exposure to a high-fat diet alters meal patterns and body composition. Physiol Behav, 2010. 99 (1): p. 33-9. Cansell, C., et al., Dietary fat exacerbates postprandial hypothalamic inflammation involving glial fibrillary acidic protein‐positive cells and microglia in male mice. Glia, 2020. 69 (1): p. 42-60. Lorch, C.M., et al., Sucrose overconsumption impairs AgRP neuron dynamics and promotes palatable food intake. Cell Rep, 2024. 43 (2): p. 113675. Additional Declarations No competing interests reported. Supplementary Files Yagoubetal.Supplementarymaterial.pdf Cite Share Download PDF Status: Published Journal Publication published 29 Aug, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 20 May, 2024 Reviews received at journal 18 May, 2024 Reviews received at journal 17 May, 2024 Reviewers agreed at journal 08 May, 2024 Reviewers agreed at journal 08 May, 2024 Reviewers invited by journal 07 May, 2024 Editor assigned by journal 07 May, 2024 Editor invited by journal 06 May, 2024 Submission checks completed at journal 06 May, 2024 First submitted to journal 02 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Nutrition","correspondingAuthor":false,"prefix":"","firstName":"Robert","middleName":"","lastName":"Chesters","suffix":""},{"id":300311787,"identity":"a22b6b55-b6a1-452b-ab59-b22953771f20","order_by":2,"name":"Jonathan Ott","email":"","orcid":"","institution":"German Institute for Human Nutrition","correspondingAuthor":false,"prefix":"","firstName":"Jonathan","middleName":"","lastName":"Ott","suffix":""},{"id":300311789,"identity":"9150a66b-a119-4f48-9817-10aea305a8c8","order_by":3,"name":"Jiajie Zhu","email":"","orcid":"","institution":"German Institute for Human Nutrition","correspondingAuthor":false,"prefix":"","firstName":"Jiajie","middleName":"","lastName":"Zhu","suffix":""},{"id":300311790,"identity":"c8d92e1f-dfa1-46c2-a35d-dcb84e0220bc","order_by":4,"name":"Lídia Cantacorps","email":"","orcid":"","institution":"German Institute for Human Nutrition","correspondingAuthor":false,"prefix":"","firstName":"Lídia","middleName":"","lastName":"Cantacorps","suffix":""},{"id":300311791,"identity":"d1eff2f6-5ce4-4480-958b-4debd7af3a5a","order_by":5,"name":"Katrin Ritter","email":"","orcid":"","institution":"German Institute for Human Nutrition","correspondingAuthor":false,"prefix":"","firstName":"Katrin","middleName":"","lastName":"Ritter","suffix":""},{"id":300311792,"identity":"d103f4eb-915c-4b4a-a94f-7aae7eaaedae","order_by":6,"name":"Rachel Lippert","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+ElEQVRIiWNgGAWjYHACNgaGAgYZNgjHhrGN+QADAw9BLQYMPBAtCWmMbWwJRGqBsBMOMzYQ0mLefvjZgw9ALXzSvQcfV/44L9vHBnTZmwrcWmTOpJkbzgA5TOZcsuGZhNvGbWxsCYxzzuDWIsGQwybNA9IikWMm2ZBwO7FNvseAmbcNjxb+N2zSfyBazH82JJxLbGPj/8DM+w+PFgmgLQxQWxgbEg4AtfAwMPM24NPyzEyyx0AC7BfJhrRkkF8MDs45hs9hyc8kflTYyMnP7j34scHGTnZ+G/PDB29qcGuBhwKDBFJcHCCoAaKLQIyPglEwCkbByAUAApZDgefjgYMAAAAASUVORK5CYII=","orcid":"","institution":"German Institute for Human Nutrition","correspondingAuthor":true,"prefix":"","firstName":"Rachel","middleName":"","lastName":"Lippert","suffix":""}],"badges":[],"createdAt":"2024-05-02 10:30:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4358544/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4358544/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-70870-0","type":"published","date":"2024-08-29T15:58:15+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":56478654,"identity":"ee2f90e8-2c3e-4d54-a64a-119a72f98c8e","added_by":"auto","created_at":"2024-05-14 17:52:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":528687,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4358544/v1/d2345cf5cbad91c8f40ae439.png"},{"id":56476546,"identity":"089b68f4-4ac2-457f-942b-9c7bb604714a","added_by":"auto","created_at":"2024-05-14 17:44:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1241970,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4358544/v1/a8431caf48d604f26b1680bd.png"},{"id":56476575,"identity":"a6a8dd20-504a-4cf5-b965-48a8c44a5ef6","added_by":"auto","created_at":"2024-05-14 17:44:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1220787,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4358544/v1/0df81cca63ee3d00f0da9ba7.png"},{"id":56476576,"identity":"43d56df6-9841-4502-bb88-03f66de18517","added_by":"auto","created_at":"2024-05-14 17:44:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1106496,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4358544/v1/5bf8784bcfe8c0eab19c7868.png"},{"id":56476543,"identity":"db0fa5be-34ea-4259-8997-461bbe9b4454","added_by":"auto","created_at":"2024-05-14 17:44:30","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1463941,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4358544/v1/e88c17adb5c28731c8b3771b.png"},{"id":63821119,"identity":"e60333b7-0003-4045-aaf3-23eb4f7635aa","added_by":"auto","created_at":"2024-09-02 16:12:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6625546,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4358544/v1/4c9baf18-c9b0-4dbd-8013-79866762d57f.pdf"},{"id":56476545,"identity":"406e9506-ab53-40c6-8d38-45df6ad46975","added_by":"auto","created_at":"2024-05-14 17:44:30","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":335906,"visible":true,"origin":"","legend":"","description":"","filename":"Yagoubetal.Supplementarymaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4358544/v1/32ef24409fea11487487c878.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eElevated dietary fat alone is not sufficient to decrease AgRP projections in the paraventricular nucleus of the hypothalamus in mice\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFeeding responses are predominantly regulated within the brain via Agouti-related peptide (AgRP) and proopiomelanocortin (POMC) neurons, two neural populations of the central melanocortin system. They are located mainly in the arcuate nucleus of the hypothalamus (ARC) and have opposite actions on regulating feeding. When activated, POMC neurons inhibit food intake whilst AgRP neurons increase it. These neurons also express receptors to metabolic hormones such as insulin, leptin and ghrelin, that allow them to continuously monitor the energy state of the animal and alter their activity to maintain energy homeostasis. In this context, periods of fasting, or prolonged food deprivation are known to decrease circulating levels of leptin and insulin. Fasting also increases the activity of arcuate AgRP neurons [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] while refeeding behaviour after 24h fasting is prevented if these AgRP neurons are ablated [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Furthermore, AgRP neuron activity changes in response to energetically dense foods. This change in activity may be dependent on the length of HFD exposure. For example, long term access to high fat diet (HFD) compromises AgRP sensitivity to hormonal signals such as ghrelin or leptin, contributing therefore to the disruption of energy balance and resulting in leptin resistance [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Moreover, acute 48h exposure to HFD has been shown to cause a loss of AgRP neuron leptin sensitivity [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Conversely however, a recent electrophysiological study has shown an increase in AgRP neuronal activity upon an acute exposure to HFD for 2 days [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe consequences of long term (greater than 8 weeks) HFD exposure on the function of the hypothalamic melanocortin system has been extensively studied, but predominantly only in male mice. Electrophysiology and fiber photometry studies have shown that AgRP/ Neuropeptide-Y (NPY) neurons become hyperexcitable after 8 weeks of HFD in male mice [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In females, the same time exposure also induces AgRP neuron hyperexcitability but to a lesser extent because of an elevated baseline firing rate compared to males [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Moreover, in male mice, it has been shown that 8 weeks of HFD is sufficient to cause a dramatic, 60% decrease in AgRP axonal projections to the paraventricular nucleus of the hypothalamus (PVH), a nucleus where the action of AgRP release on their receptor, the melanocortin receptor 4 (MC4R) neurons is known to be the dominant driver of food intake [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Interestingly, and most surprisingly, short-term HFD exposure, as short as 48h, has been reported to have an even greater effect on the reduction in AgRP neuronal projections, with an 80% decrease in projections observed in the PVH of male mice [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. As in the long term HFD exposure, functional changes in AgRP neuronal activity have also been observed following 48h of HFD, with a rapid change in calcium activity seen in response to the presentation of food [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] as well as increased neuronal firing of AgRP neurons [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Additionally, 48h HFD led to an up-regulation of suppressor of cytokine signaling-3 (SOCS3), an inflammatory and insulin signalling hallmark in AgRP but not in POMC neurons in the ARC [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Another study showed that 24h intralipid treatment in the ARC revealed an up-regulation of TNF-α and an increase in the number of astrocytes mirroring neuroinflammation in that nucleus [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Furthermore, hypothalamic proteomics data collected from 72h HFD-exposed male mice highlighted a change in protein spots involved in neuronal remodelling and synaptic plasticity indicating a structural adaptation of the hypothalamus [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Altogether, it seems that in male mice short term HFD exposure presents similar electrophysiological, molecular and structural signature changes in AgRP neurons as long term HFD exposure.\u003c/p\u003e \u003cp\u003eDespite the growing evidence pointing toward sex-specific metabolic and central adaptations (e.g. firing rate, neuronal connections) in response to a long term HFD exposure [\u003cspan additionalcitationids=\"CR18 CR19 CR20 CR21 CR22\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], the effect of a short term HFD exposure in female mice is still poorly studied in comparison to males. Here, we explored the potential sex-specific adaptative response to an acute (48h) HFD exposure on AgRP axonal projections within the hypothalamus in male and female mice. We specifically targeted dietary fat, and not elevated sucrose as commonly found in HFD, to ensure that the effects would be attributed to increased fat consumption alone. We assessed neuroanatomical changes using both the endogenous AgRP peptide as well as a targeted labeling of a synaptic protein. Image analyses utilized two pipelines designed based on previous literature. Our findings using both image analyses pipelines show no change of AgRP neuronal connections in the PVH after 48h of HFD both in male as well as female mice. Thus, acute elevations in dietary fat alone are not sufficient to modify AgRP axonal architecture in the PVH.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cem\u003e48h HFD exposure does not markedly change metabolic parameters in mice\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe effects of HFD on AgRP neurons are often attributed the increased dietary fat alone. However, the high fat diets used in previous studies are often confounded by the presence of increased dietary sucrose as well [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. To overcome this and to specifically study the effects attributed to elevated dietary fat, we utilized a 60% HFD with similar sucrose to animals receiving only a standard diet (STD: 9% kCal from fat). The HFD-exposed group were given access to HFD at the onset of the dark phase (Zeitgeber time (ZT) 12) for 48h until sacrifice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). There were no significant changes in body weight after 48h HFD exposure, in either male (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb and d) or female animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec and e). In males (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef) but not in females (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg), random fed detection of glycemia was significantly increased after 48h of access to HFD (Males: 18.49+/- SD vs. 42.72+/- SD, p\u0026thinsp;=\u0026thinsp;0.0161, Females: 19.66+/- SD vs. 68.70+/- SD, p\u0026thinsp;=\u0026thinsp;0.6614).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eAgRP neuronal projections in the PVH are unchanged after 48h HFD exposure\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIt has been shown that HFD exposure induces sex-specific responses particularly in the hypothalamus [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. It has been reported that in male mice, 48h of HFD access is sufficient to decrease 80% of AgRP axonal projections to the PVH [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, no studies have investigated the effect in female mice. In order to compare the effect of a short-term HFD exposure on AgRP projections to the PVH in male and female mice, we used AgRP-IRES-Cre; Synaptophysin-TdTomato adult mice. This mouse model allowed us to assess the synaptic protein, Synaptophysin, specifically in AgRP neuronal projections through detection of TdTomato signal from the Synaptophysin-TdTomato fusion protein expression. Image analyses were performed in accordance with previously literature using both a maximum intensity projection method and sum of slices method [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In both analysis pipelines the first step to process the acquired images for analysis is to set a threshold for each image. This thresholding value utilizes an automatic detection of overall signal intensities across the image and uses an algorithm to apply a standardized selection of positively labelled signal to generate a binary image. For each image analysis method, we compared between STD and 48h HFD groups the automatically generated thresholding values applied to AgRP and Synaptophysin-TdTomato images. AgRP and Synaptophysin-TdTomato thresholding values were not significantly different between STD and 48h HFD groups (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Supp. Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAs the cellular identity and heterogeneity of the PVH dramatically differs across the anterior to posterior axis, we first, assessed AgRP and Synaptophysin-TdTomato in the PVH anterior (PVHant) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. This region corresponds to the neuroendocrine compartment of the PVH, a region enriched in receptors for a number of neuropeptide hormones such as MC4R [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Using the maximum intensity projection method of analysis, the most common approach used in the literature, we did not detect any sex-specific difference in the mean gray value of AgRP and Synaptophysin-TdTomato labeling (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb and d). However, analysis of the Raw Integrated Density of AgRP and Synaptophysin-TdTomato signals did highlight an overall effect of sex, with a significant difference between male and female animals on STD (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec and e, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Contrary to the published literature, we did not detect any significant reduction in AgRP projections after 48h of HFD exposure in the PVHant in neither males nor females, regardless of the analysis method used (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eand Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTwo-way ANOVA statistical analysis of data presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003eMethod 1: Maximum intensity projection\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePanel\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSource of variation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF (DFn, DFd)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eb) AgRP mean gray value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInteraction\u003c/p\u003e \u003cp\u003eSex\u003c/p\u003e \u003cp\u003eDiet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\"F (1, 15)\u0026thinsp;=\u0026thinsp;3.131\"\u003c/p\u003e \u003cp\u003e\"F (1, 15)\u0026thinsp;=\u0026thinsp;2.396\"\u003c/p\u003e \u003cp\u003e\"F (1, 15)\u0026thinsp;=\u0026thinsp;1.134\"\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0971\u003c/p\u003e \u003cp\u003e0.1425\u003c/p\u003e \u003cp\u003e0.3037\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ec) AgRP Raw Integrated Density\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInteraction\u003c/p\u003e \u003cp\u003eSex\u003c/p\u003e \u003cp\u003eDiet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\"F (1, 14)\u0026thinsp;=\u0026thinsp;1.277\"\u003c/p\u003e \u003cp\u003e\"F (1, 14)\u0026thinsp;=\u0026thinsp;4.895\"\u003c/p\u003e \u003cp\u003e\"F (1, 14)\u0026thinsp;=\u0026thinsp;1.638\"\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.2774\u003c/p\u003e \u003cp\u003e0.0441\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e \u003cp\u003e0.2214\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ed) Syn-TdTom mean gray value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInteraction\u003c/p\u003e \u003cp\u003eSex\u003c/p\u003e \u003cp\u003eDiet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\"F (1, 14)\u0026thinsp;=\u0026thinsp;2.858\"\u003c/p\u003e \u003cp\u003e\"F (1, 14)\u0026thinsp;=\u0026thinsp;3.674\"\u003c/p\u003e \u003cp\u003e\"F (1, 14)\u0026thinsp;=\u0026thinsp;0.9927\"\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1130\u003c/p\u003e \u003cp\u003e0.0759\u003c/p\u003e \u003cp\u003e0.3360\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ee) Syn-TdTom Raw Integrated Density\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInteraction\u003c/p\u003e \u003cp\u003eSex\u003c/p\u003e \u003cp\u003eDiet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\"F (1, 14)\u0026thinsp;=\u0026thinsp;2.000\"\u003c/p\u003e \u003cp\u003e\"F (1, 14)\u0026thinsp;=\u0026thinsp;6.781\"\u003c/p\u003e \u003cp\u003e\"F (1, 14)\u0026thinsp;=\u0026thinsp;1.761\"\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1791\u003c/p\u003e \u003cp\u003e0.0208\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e \u003cp\u003e0.2057\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMethod 2: Sum slices\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ef) AgRP mean gray value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInteraction\u003c/p\u003e \u003cp\u003eSex\u003c/p\u003e \u003cp\u003eDiet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\"F (1, 15)\u0026thinsp;=\u0026thinsp;0.8681\"\u003c/p\u003e \u003cp\u003e\"F (1, 15)\u0026thinsp;=\u0026thinsp;0.06371\"\u003c/p\u003e \u003cp\u003e\"F (1, 15)\u0026thinsp;=\u0026thinsp;1.665\"\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.3662\u003c/p\u003e \u003cp\u003e0.8041\u003c/p\u003e \u003cp\u003e0.2164\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eg) AgRP Raw Integrated Density\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInteraction\u003c/p\u003e \u003cp\u003eSex\u003c/p\u003e \u003cp\u003eDiet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\"F (1, 15)\u0026thinsp;=\u0026thinsp;1.321\"\u003c/p\u003e \u003cp\u003e\"F (1, 15)\u0026thinsp;=\u0026thinsp;0.08496\"\u003c/p\u003e \u003cp\u003e\"F (1, 15)\u0026thinsp;=\u0026thinsp;1.673\"\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.2684\u003c/p\u003e \u003cp\u003e0.7747\u003c/p\u003e \u003cp\u003e0.2154\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eh) Syn-TdTom mean gray value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInteraction\u003c/p\u003e \u003cp\u003eSex\u003c/p\u003e \u003cp\u003eDiet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\"F (1, 14)\u0026thinsp;=\u0026thinsp;0.1469\"\u003c/p\u003e \u003cp\u003e\"F (1, 14)\u0026thinsp;=\u0026thinsp;0.7393\"\u003c/p\u003e \u003cp\u003e\"F (1, 14)\u0026thinsp;=\u0026thinsp;0.04054\"\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.7072\u003c/p\u003e \u003cp\u003e0.4044\u003c/p\u003e \u003cp\u003e0.8433\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ei) Syn-TdTom Raw Integrated Density\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInteraction\u003c/p\u003e \u003cp\u003eSex\u003c/p\u003e \u003cp\u003eDiet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\"F (1, 14)\u0026thinsp;=\u0026thinsp;4.327e-005\"\u003c/p\u003e \u003cp\u003e\"F (1, 14)\u0026thinsp;=\u0026thinsp;0.9702\"\u003c/p\u003e \u003cp\u003e\"F (1, 14)\u0026thinsp;=\u0026thinsp;0.02912\"\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.9948\u003c/p\u003e \u003cp\u003e0.3413\u003c/p\u003e \u003cp\u003e0.8669\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eNext, we assessed AgRP and Synaptophysin projections in the midpoint of the PVH (PVHmid) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. By applying the maximum intensity projection method of image analysis, the labeling of AgRP and Synaptophysin-TdTomato remained unchanged after 48h access to HFD with no significant difference of AgRP projections between males and females in the mean gray value (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb and d, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb and d) and Raw Integrated Density (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec and e, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec and e). The sum slices analysis method also did not detect any difference in AgRP and Synaptophysin labeling (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef-i) and no effect of the diet on the projections (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef-i). In sum, our results show no effect of 48h HFD exposure on AgRP projections in the PVHmid.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTwo-way ANOVA Statistical analysis of data presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003eMethod 1: Maximum intensity projection\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePanel\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSource of variation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF (DFn, DFd)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eb) AgRP mean gray value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInteraction\u003c/p\u003e \u003cp\u003eSex\u003c/p\u003e \u003cp\u003eDiet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\"F (1, 15)\u0026thinsp;=\u0026thinsp;0.04563\"\u003c/p\u003e \u003cp\u003e\"F (1, 15)\u0026thinsp;=\u0026thinsp;0.4600\"\u003c/p\u003e \u003cp\u003e\"F (1, 15)\u0026thinsp;=\u0026thinsp;1.184\"\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.8337\u003c/p\u003e \u003cp\u003e0.5080\u003c/p\u003e \u003cp\u003e0.2937\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ec) AgRP Raw Integrated Density\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInteraction\u003c/p\u003e \u003cp\u003eSex\u003c/p\u003e \u003cp\u003eDiet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\"F (1, 15)\u0026thinsp;=\u0026thinsp;0.07372\"\u003c/p\u003e \u003cp\u003e\"F (1, 15)\u0026thinsp;=\u0026thinsp;0.5952\"\u003c/p\u003e \u003cp\u003e\"F (1, 15)\u0026thinsp;=\u0026thinsp;1.645\"\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.7897\u003c/p\u003e \u003cp\u003e0.4524\u003c/p\u003e \u003cp\u003e0.2191\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ed) Syn-TdTom mean gray value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInteraction\u003c/p\u003e \u003cp\u003eSex\u003c/p\u003e \u003cp\u003eDiet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\"F (1, 14)\u0026thinsp;=\u0026thinsp;0.07452\"\u003c/p\u003e \u003cp\u003e\"F (1, 14)\u0026thinsp;=\u0026thinsp;0.6688\"\u003c/p\u003e \u003cp\u003e\"F (1, 14)\u0026thinsp;=\u0026thinsp;0.2914\"\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.7889\u003c/p\u003e \u003cp\u003e0.4272\u003c/p\u003e \u003cp\u003e0.5978\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ee) Syn-TdTom Raw Integrated Density\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInteraction\u003c/p\u003e \u003cp\u003eSex\u003c/p\u003e \u003cp\u003eDiet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\"F (1, 14)\u0026thinsp;=\u0026thinsp;0.01304\"\u003c/p\u003e \u003cp\u003e\"F (1, 14)\u0026thinsp;=\u0026thinsp;0.7729\"\u003c/p\u003e \u003cp\u003e\"F (1, 14)\u0026thinsp;=\u0026thinsp;0.4367\"\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.9107\u003c/p\u003e \u003cp\u003e0.3942\u003c/p\u003e \u003cp\u003e0.5195\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMethod 2: Sum slices\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ef) AgRP mean gray value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInteraction\u003c/p\u003e \u003cp\u003eSex\u003c/p\u003e \u003cp\u003eDiet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\"F (1, 15)\u0026thinsp;=\u0026thinsp;0.4469\"\u003c/p\u003e \u003cp\u003e\"F (1, 15)\u0026thinsp;=\u0026thinsp;0.9467\"\u003c/p\u003e \u003cp\u003e\"F (1, 15)\u0026thinsp;=\u0026thinsp;0.5240\"\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5139\u003c/p\u003e \u003cp\u003e0.3460\u003c/p\u003e \u003cp\u003e0.4803\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eg) AgRP Raw Integrated Density\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInteraction\u003c/p\u003e \u003cp\u003eSex\u003c/p\u003e \u003cp\u003eDiet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\"F (1, 15)\u0026thinsp;=\u0026thinsp;1.286\"\u003c/p\u003e \u003cp\u003e\"F (1, 15)\u0026thinsp;=\u0026thinsp;0.7140\"\u003c/p\u003e \u003cp\u003e\"F (1, 15)\u0026thinsp;=\u0026thinsp;0.6582\"\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.2745\u003c/p\u003e \u003cp\u003e0.4114\u003c/p\u003e \u003cp\u003e0.4299\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eh) Syn-TdTom mean gray value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInteraction\u003c/p\u003e \u003cp\u003eSex\u003c/p\u003e \u003cp\u003eDiet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\"F (1, 14)\u0026thinsp;=\u0026thinsp;0.2569\"\u003c/p\u003e \u003cp\u003e\"F (1, 14)\u0026thinsp;=\u0026thinsp;0.5981\"\u003c/p\u003e \u003cp\u003e\"F (1, 14)\u0026thinsp;=\u0026thinsp;1.224\"\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.6201\u003c/p\u003e \u003cp\u003e0.4522\u003c/p\u003e \u003cp\u003e0.2871\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ei) Syn-TdTom Raw Integrated Density\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInteraction\u003c/p\u003e \u003cp\u003eSex\u003c/p\u003e \u003cp\u003eDiet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\"F (1, 14)\u0026thinsp;=\u0026thinsp;0.01766\"\u003c/p\u003e \u003cp\u003e\"F (1, 14)\u0026thinsp;=\u0026thinsp;0.4794\"\u003c/p\u003e \u003cp\u003e\"F (1, 14)\u0026thinsp;=\u0026thinsp;1.628\"\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.8962\u003c/p\u003e \u003cp\u003e0.5000\u003c/p\u003e \u003cp\u003e0.2227\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe aim of our study was to investigate sex-specific metabolic and/or neuroanatomical changes in AgRP projections to the PVH in response to acute 48h HFD exposure. We specifically analysed AgRP neuronal projections to the anterior and middle PVH (PVHant and PVHmid), as this is the neuroendocrine compartment of the PVH, and is known to express the MC4R; the main target of AgRP release in this region [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In accordance with the literature, we did not see an effect of acute 48h HFD exposure on body weight [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. However, blood glucose levels were significantly increased in male mice. Since blood glucose was measured in \u003cem\u003ead-libitum\u003c/em\u003e fed mice, this minor, but significant difference may be due to the consumption of HFD shortly before sacrifice: as the animals were sacrificed after onset of the dark phase (ZT 12), when mice usually consume a large majority of their daily food intake.\u003c/p\u003e \u003cp\u003eNext, we investigated the effects of 48h HFD on AgRP projections using an AgRP-Cre; Synaptophysin-TdTomato mouse model specifically labeling these projections. To analyse images of AgRP and Synaptophysin-TdTomato, we applied two methods of neuronal projection analysis published in the literature: the maximum intensity projection and sum slices (described in material and methods section) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In the PVHant and using the maximum intensity method, our analysis of the Raw Integrated Density of AgRP and Synaptophysin-TdTomato revealed a significant sex difference but ultimately no significant effect of the 48h HFD exposure within each sex. This sex effect seems to be driven primarily by a difference within the STD-exposed groups. In the PVHmid, no significant effect of sex nor diet were observed.\u003c/p\u003e \u003cp\u003eMethodological factors that can potentially influence the results are: method of thresholding (manual versus automated), what defines the Region of Interest (ROI) drawn on the image, and/or if the same approach to slice analysis is used (single comparable slice versus average of multiple slices). In our study, we standardized the coronal brain slice selected, the ROI used, and selected an automated unbiased thresholding algorithm (ImageJ - Moments). We also quality checked all thresholded binarized images to ensure the accurate inclusion of all positively-labeled pixels. Furthermore, we checked that automatic thresholding values were not significantly different from each other across both sexes and diet groups. While using the maximum intensity projection and sum of slices for confocal image analyses, we observed a higher accuracy between the binarized image and the original image using the maximum intensity projection. It was observed that with the sum of slices method, there was an over estimation of the positive signal, including signal from the background and not specifically the positively labelled neuronal projections. Therefore, we would recommend applying the maximum intensity projection method for analysing neuronal projections. Regarding the region of analysis representing the PVH, we differentiated the anterior and mid PVH to localize and target the effect of HFD on AgRP projections due to the known intra-PVH complexity and likely differing functions of subregions of the PVH [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur results demonstrate that acute exposure to elevated amounts of dietary fat, alone, does not alter AgRP axonal projections to the PVH. This holds true for both males and females. An interesting point that is still open for discussion is the contribution of fat alone, or fat in the presence of elevated sugar content as well as the cause for synaptic change. The primary study showing a dramatic decrease in AgRP projections to the PVH used a diet high in fat (45%) but also in sugar (17% sucrose) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. It was previously shown that AgRP neurons adjust their response to acute high fat and high sucrose, through electrophysiological and calcium dynamics or even AgRP mRNA levels [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Moreover, a recent publication highlighted electrophysiological changes upon acute dietary exposures appear to be predominantly due to the excess dietary sucrose [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], underscoring that there are unique responses of AgRP neurons to macronutrient content in the diet. This evidence raises the question as to the contribution of individual macronutrient contents, separately and/or in combination, to induce synaptic changes within the hypothalamus and the brain generally. Therefore, understanding the role of individual macronutrients acquired through the diet, on aspects of synaptic physiology may give insight into the acute adaptability of neuronal circuits in adjusting to nutritional changes.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e \u003cem\u003eAnimals.\u003c/em\u003e AgRP-IRES-Cre male mice (strain AgRPtm1(cre)Lowl/J, Jackson laboratories Stock number:012899) were crossed with ROSA-CAG-LSL-Synaptophysin-tdTomato-WPRE \u003csup\u003e(Ai34D flox/flox)\u003c/sup\u003e female mice (Strain B6;129S-Gt (RO-SA)26Sortm34.1 (CAG-Syp/tdTomato)Hze/J, Jackson Laboratories, Stock number: 012570) to obtain AgRP-IRES-Cre;Syn-TdTomato \u003csup\u003eflox/wt\u003c/sup\u003e mice used in our experiments. Animals were group housed (2\u0026ndash;3 per cage) with a 12-h light/dark cycle at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and 50\u0026ndash;70% humidity with \u003cem\u003ead libitum\u003c/em\u003e access to water and control STD (Ssniff # V1534-300; kCals metabolizable energy: 67% carbohydrates; 24% proteins and 9% fat; with 5.3% of total weight coming from crude sugar) unless otherwise stated. Mice were randomly assigned to either STD control or HFD for 48h (HFD introduced at start of the dark phase) (Ssniff #EF acc. D12492 (I) mod; kCals metabolizable energy: 22% carbohydrates, 24% protein and 60% fat; with 9.4% of total weight coming from crude sugar). All animal experiments and procedures were approved by the animal welfare committee (Landesamt f\u0026uuml;r Arbeitsschutz, Verbraucherschutz und Gesundheit; animal ethics application number 2347-36-2021). Experiments were conducted in accordance with the ARRIVE guidelines and the European Directive 2010/63/EU.\u003c/p\u003e \u003cp\u003e \u003cem\u003eMetabolic measurements.\u003c/em\u003e All measurements were performed during the dark phase at ZT 12. Body weight and food intake were measured at onset of the dark cycle on the first day of the experiment, 24h and 48h later before the sacrifice. Percentage body weight change was calculated as: body weight (g) at 48h x 100/body weight (g) at start of the experiment. Glycemia was assessed at sacrifice by blood collection directly from the right atrium and measured with a glucometer (Contour Care, Ascensia).\u003c/p\u003e \u003cp\u003e \u003cem\u003eImmunostaining\u003c/em\u003e. Mice were deeply anesthetised with an intra-peritoneal injection of Pentobarbital (400mg/kg) and sacrificed by transcardial perfusion with, first, 60mL of ice cold 1X phosphate-buffered saline (PBS), then 4% paraformaldehyde (PFA, pH 9.5, 3.8% Borate) after onset of the dark phase ZT 12\u0026ndash;15. Brains were post fixed in 4% PFA\u0026thinsp;+\u0026thinsp;Borax for 4h then switched to a 20% sucrose solution overnight before being stored at -80\u0026deg;C until usage. Fixed brains were sliced coronally using a freezing sledge microtome (Slide 4004 M, pfm medical, Cat. 400410) to obtain 30\u0026micro;m frozen brain slices that were stored in anti-freeze solution (10% 10X PBS, 20% Ethyl glycol and 20% Glycerol) at -20\u0026deg; C until further use. The day of the staining, slices were washed 3x10min times with 0.02M KPBS, 5min in 0.3% glycine in PBS and 10min 0.03% SDS in PBS followed by a 1h incubation in blocking and permeabilization buffer containing 3% donkey serum in 0.25% Triton-X in PBS. Slices were then incubated with primary antibodies Rabbit anti-AgRP (Phoenix; Cat# H-003-57; dilution 1:4,000) and Goat anti-TdTomato (SICGEN, Cat# AB8181-200; dilution 1:4,000) mixed in SignalStain\u0026reg; Antibody Diluent (CellsSignal; Cat# 8112) for 48h at 4\u0026deg;C plus 2h at room temperature. After washing with 0.02M KPBS (1x5min and 3x10min), slices were incubated with the secondary antibodies Donkey anti-Rabbit (1:500) Alexa Fluor 488-conjugated and Donkey anti-Goat (1:500) Alexa Fluor 546-conjugated diluted in 0.25% Triton-X in 1x KPBS for 1h at room temperature then washed with 0.02M KPBS (1x5min and 3x10min). Brain slices were mounted on Superfrost Plus microscope slides and cover slipped with VECTASHIELD Antifade Mounting Medium with DAPI (Biozol; Cat# VEC-H-1200). Images of the PVHant (distance from Bregma \u0026minus;\u0026thinsp;0.83mm) and PVHmid (distance from Bregma \u0026minus;\u0026thinsp;0.95mm), selected based on the mouse brain atlas [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], were collected using a confocal microscope (Multiphoton Laser Scanning Microscope LSM 780, Zeiss) and Zen (Zeiss) software. 3D Z-stack (z\u0026thinsp;=\u0026thinsp;17 slices/image, interval\u0026thinsp;=\u0026thinsp;1.5\u0026micro;m) images were acquired. Two mice were excluded from further image processing and analysis due to obvious germline deletion of the transgene STOP cassette resulting in ubiquitous expression of Synaptophysin-TdTomato protein.\u003c/p\u003e \u003cp\u003e \u003cem\u003eImage Processing and Analysis.\u003c/em\u003e Images were processed and analyzed with a custom-written ImageJ macro and performed by an experimenter blinded to the experimental conditions. First, a region of interest (ROI) defining the outlines of the PVH was generated (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). This ROI was then used in the following two methods:\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eMaximum intensity projection\u003c/span\u003e: For each channel, Z-stack images were converted to a 2D image using a maximum intensity projection, and saved opened as individual files. A threshold value was automatically applied to these images using the \u0026ldquo;Moments\u0026rdquo; algorithm and a binary image created. The ROI was then overlayed on to the binary image. The workflow is represented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eSum slices\u003c/span\u003e: For each channel, a sum-slice intensity projection was created: this creates a 2D image by adding together the intensity of each pixel of each slice of the Z-stack at every x-y pixel coordinate in the 2D image space. The middle slice (number 9 out of 17) of the Z-stack was used as the reference slice and the sum-slice image, thresholded using \u0026ldquo;Moments\u0026rdquo; algorithm same this was applied to all 17 slices of the Z-stack image and a binary 2D image was saved. The same ROI used for the maximum intensity projection method was applied here. The workflow is represented in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor both methods, the size of the area, Raw Integrated Density (=\u0026thinsp;sum of the pixels), and mean gray value (=\u0026thinsp;sum of the gray values of all pixels divided by the number of pixels) were measured. Threshold values of the PVH ROI, from both analysis methods, were compared for differences across sexes and diet. All ImageJ scripts are supplied in the Supplementary Methods. All ROIs and confocal images used for the maximum intensity projection and sum slices analyses can be shared upon request.\u003c/p\u003e \u003cp\u003e \u003cem\u003eStatistical analysis\u003c/em\u003e. All data are represented as mean +/- standard deviation (SD) with each single data point representing an individual mouse. GraphPad Prism 9 was used for all statistical analysis and graphs. An unpaired two-tailed \u003cem\u003et\u003c/em\u003e-test was conducted to analyse the body weight, the percentage change of body weight and blood glucose levels. AgRP projections were analysed with a two-way ANOVA to assess the sex, diet and Interaction of sex and diet. When appropriate, the two-way ANOVA was followed by Bonferroni post-hoc comparison. Threshold values were analyzed using a Mann Whitney comparison. Significant differences were considered when p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to acknowledge all of the animal caretaking staff and veterinary staff of the Max Rubner Laboratory for their support in the maintenance and care of the animals used in this study. Graphical depictions in Figure 1 were generated using BioRender.com.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis work was performed with financial support from the Leibniz Association through the Leibniz Competition Best Minds Grant \u0026apos;BAByMIND\u0026rsquo; (J99/2020) to RNL. Additional financial support was provided by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany\u0026acute;s Excellence Strategy \u0026ndash; EXC-2049 \u0026ndash; 390688087 (NeuroCure) and by the German Center for Diabetes Research (82DZD03D2Y and 82DZD03D03). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNL and SY designed the experiment. SY, JO, LC, JZ, and RNL conducted all animal work and contributed to tissue collection. KR performed all genotyping experiments. SY, RC, JO, LC and JZ contributed to tissue slicing, immunostaining and image analysis. RC developed ImageJ scripts for image analyses. SY and RNL analysed the data and wrote the manuscript. All authors edited and critically reviewed the manuscript. RNL acquired the funding for the project and supervised all authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data will be made available upon reasonable request to the corresponding author.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAdditional Information\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBetley, J.N., et al., \u003cem\u003eNeurons for hunger and thirst transmit a negative-valence teaching signal.\u003c/em\u003e Nature, 2015. \u003cstrong\u003e521\u003c/strong\u003e(7551): p. 180-185.\u003c/li\u003e\n\u003cli\u003eNakajima, K., et al., \u003cem\u003eGs-coupled GPCR signalling in AgRP neurons triggers sustained increase in food intake.\u003c/em\u003e Nat Commun, 2016. \u003cstrong\u003e7\u003c/strong\u003e: p. 10268.\u003c/li\u003e\n\u003cli\u003eCai, J., et al., \u003cem\u003eAgRP neurons are not indispensable for body weight maintenance in adult mice.\u003c/em\u003e Cell Rep, 2023. \u003cstrong\u003e42\u003c/strong\u003e(7): p. 112789.\u003c/li\u003e\n\u003cli\u003eBeutler, L.R., et al., \u003cem\u003eObesity causes selective and long-lasting desensitization of AgRP neurons to dietary fat.\u003c/em\u003e Elife, 2020. \u003cstrong\u003e9\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eBriggs, D.I., et al., \u003cem\u003eDiet-induced obesity causes ghrelin resistance in arcuate NPY/AgRP neurons.\u003c/em\u003e Endocrinology, 2010. \u003cstrong\u003e151\u003c/strong\u003e(10): p. 4745-55.\u003c/li\u003e\n\u003cli\u003eEnriori, P.J., et al., \u003cem\u003eDiet-induced obesity causes severe but reversible leptin resistance in arcuate melanocortin neurons.\u003c/em\u003e Cell Metab, 2007. \u003cstrong\u003e5\u003c/strong\u003e(3): p. 181-94.\u003c/li\u003e\n\u003cli\u003eWei, W., et al., \u003cem\u003eDiet composition, not calorie intake, rapidly alters intrinsic excitability of hypothalamic AgRP/NPY neurons in mice.\u003c/em\u003e Sci Rep, 2015. \u003cstrong\u003e5\u003c/strong\u003e: p. 16810.\u003c/li\u003e\n\u003cli\u003eOlofsson, L.E., et al., \u003cem\u003eModulation of AgRP-neuronal function by SOCS3 as an initiating event in diet-induced hypothalamic leptin resistance.\u003c/em\u003e Proc Natl Acad Sci U S A, 2013. \u003cstrong\u003e110\u003c/strong\u003e(8): p. 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A prime environmental regulator of neuronal activity is diet, and previous work has highlighted changes in hypothalamic connections in response to diets high in dietary fat and elevated sucrose. We sought to determine if the change in hypothalamic neuronal connections was driven primarily by an elevation in dietary fat alone. Analysis was performed in both male and female animals. We measured Agouti-related peptide (AgRP) neuropeptide and Synaptophysin markers in the paraventricular nucleus of the hypothalamus (PVH) in response to an acute 48h high fat diet challenge. Using two image analysis methods described in previous studies, an effect of a high fat diet on AgRP neuronal projections in the PVH of male or female mice was not identified. These results suggest that it may not be dietary fat alone that is responsible for the previously published alterations in hypothalamic connections Future work should focus on deciphering the role of individual macronutrients on neuroanatomical and functional changes.\u003c/p\u003e","manuscriptTitle":"Elevated dietary fat alone is not sufficient to decrease AgRP projections in the paraventricular nucleus of the hypothalamus in mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-14 17:44:19","doi":"10.21203/rs.3.rs-4358544/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-05-20T06:56:29+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-19T03:21:44+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-17T21:12:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"138853530022532067281431618161117692655","date":"2024-05-08T17:33:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"183327342620573291108392193548086591799","date":"2024-05-08T16:31:11+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-07T05:54:12+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-07T05:41:33+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-05-06T05:24:32+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-06T05:22:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-05-02T10:29:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6733b618-a4e3-4b1e-b9d3-fd4f3c6471bc","owner":[],"postedDate":"May 14th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-02T16:05:07+00:00","versionOfRecord":{"articleIdentity":"rs-4358544","link":"https://doi.org/10.1038/s41598-024-70870-0","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-08-29 15:58:15","publishedOnDateReadable":"August 29th, 2024"},"versionCreatedAt":"2024-05-14 17:44:19","video":"","vorDoi":"10.1038/s41598-024-70870-0","vorDoiUrl":"https://doi.org/10.1038/s41598-024-70870-0","workflowStages":[]},"version":"v1","identity":"rs-4358544","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4358544","identity":"rs-4358544","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}