BSc Bèta-gamma
Bachelor’s Thesis in Biomedical ScienceEarly-life stress affects the reward circuitry
in a sex-dependent manner – possible
implications for reward-driven feeding?
Eva Speijer
11670649
June 2020
Supervisor
Assessor
Examiner
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Early-life stress affects the reward circuitry in a sex-dependent
manner – possible implications for reward-driven feeding?
1. Introduction
The early life environment has a considerable effect on the development of an organism. Adversities during this critical period, such as stress, malnutrition and family disruption, can increase the risk to develop metabolic disorders such as obesity later in life of adulthood (Danese & Tan, 2014; Hemmingsson, Johansson & Revnisdottir, 2014; Kopelman, 2000; Maniam & Morris, 2010). The prevalence of obesity has nearly tripled since 1975, which makes it a global health-problem (WHO, 2020). Therefore, it is important to advance knowledge of obesity and understand how risks factors such as early life stress (ELS) can influence its development.
Thus far, clinical studies have shown that having a background of ELS can increase palatable food intake (Hemmingsson, 2018; Leigh et al., 2018; Cottone et al., 2009). For example, a longitudinal study of 4800 children concluded that child maltreatment can be a predictor of obesogenic food consumption later in life (Jackson & Vaughn, 2016). Pre-clinical studies have shown similar results; an increased preference for obesogenic foods by animals that experienced ELS (Mechado et al., 2013).
Normally, there is a balance between energy intake and energy expenditure, mainly regulated by hypothalamic circuits. Peripheral metabolic hormones, such as leptin and ghrelin, interact with receptors in the hypothalamus. Hence, the central nervous system integrates signals from adipose tissue and food intake aiming to maintain stable body fat stores (Bouret, 2009; Saper et al., 2002). The primary location for the integration of these peripheral signals is the arcuate nucleus, in
which the neurons expressing
pro-opimelanocortin (POMC neurons) or
neuropeptide Y and agouti-related peptide (AgRP neurons) respond to the circulating signals (Luquet & Magnan, 2009; Bouret & Simerly, 2006). POMC neuron activation leads to loss of appetite and increased energy expenditure, whereas AgRP neuron activation leads to increased appetite and decreased energy expenditure. Besides the homeostatic regulation of energy intake and expenditure, there is also reward-driven food intake, which encompasses the stimulating and motivational properties of food (Narayanan et al., 2010; Denis et al., 2015). This is regulated by dopaminergic projections of the ventral tegmental area (VTA), which
Eva Speijer
Abstract
Early-life stress (ELS) can increase the risk to develop metabolic disorders such as obesity in adulthood. Clinical and pre-clinical studies have found that having a background of ELS can lead to altered food choice in a sex-dependent manner. It is, however, not known which neurobiological substrates underly these effects.
Normally, there is a balance between energy intake and energy expenditure, mainly regulated by hypothalamic circuits. The inhibitory neurons expressing agouti-related peptide (AgRP) are most notable for their role in regulating energy homeostasis. Besides homeostatic food intake, there is reward-driven food intake regulated by dopaminergic neurons in the ventral tegmental area (VTA), which activation is particularly stimulated by high-fat high-sugar (HFHS) foods. Recent evidence suggest AgRP plays a crucial role in the functional connection between the homeostatic system and the reward system, serving as a brake on the dopaminergic neurons in the VTA. In addition, it has been shown that there is a difference in AgRP gene expression between males and females. In this study, the effect of ELS was investigated on the neurobiological substrates of feeding (i.e. AgRP fiber and dopaminergic cell density in the VTA) in both sexes. An established chronic ELS mouse model, in the form of exposure to limited nesting and bedding material from postnatal day (p)2 to p9 in mice, was used together with exposure to a HFHS choice diet and a short stressor in adulthood.
We here present that ELS has differential effects on the AgRP fiber density and dopaminergic cell density in the VTA, depending on diet and sex of the animal. Additionally, inhibitory synapses contacting dopaminergic nuclei are influenced by diet, whereas synapses contacting the dopaminergic fibers were not affected. These alterations might contribute to different feeding behavior and energy balance, thereby leading to an increased vulnerability to develop metabolic disorders such as obesity.
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activation is particularly stimulated by high-fat high-sugar (HFHS) foods (Berridge & Robinson, 1998; Chuang et al., 2011; Narayanan et al., 2010).
A rodent study by Denis et al. (2015) focused on understanding the functional connection of the above described homeostatic and reward-driven feeding. In this study, the authors concluded that AgRP neurons are an important drive in homeostatic feeding but become dispensable when mice are exposed to a long-term HFHS diet. Furthermore, the feeding behavior of AgRP-ablated mice was less motivated by homeostasis and more sensitive to food palatability and stress. For example, animals with AgRP-ablated neurons consumed an excess in palatable liquid after a mild stressor.
Emerging evidence supports the idea that stress, including ELS, can influence brain control of energy homeostasis (Yam et al., 2015). ELS together with a HFHS diet can have a synergic effect on weight gain and result in a higher dopamine release during palatable food consumption (Romani-Perez et al., 2016). Another study showed that impairment of AgRP neuron activity, which are inhibitory, promotes higher excitability of dopaminergic neurons in the VTA (Dietrich et al., 2012). Consequently, AgRP neurons may play a role in the set point of the hedonic neural circuitry and may be influenced by ELS. How ELS influences the homeostatic and reward feeding circuits, and the possible link between these two, remains elusive. In addition, some studies have shown that effects of ELS on metabolic vulnerability is sex-dependent (Murphy and Loria, 2017; Yam et al., 2017), but few studies have focused on the sex-specific effects of ELS on the neurobiological substrates of feeding.
We here therefore study the effect of ELS on the neurobiological substrates of feeding in both males and females. To investigate this, an established ELS mouse model (Rice et al., 2008; Yam et al., 2017) has been used in which mouse pups received abnormal, fragmented care from the dam due to limited nesting materials. In adulthood, a high-fat high sugar choice diet was introduced to investigate behavior effect of ELS and diet under basal and challenged (short stressor) circumstances. Prior results of this study show that there is a difference in food preference between males and females, males had a higher sugar intake whereas females had a higher fat intake (see Appendix A). Additionally, ELS females increased their fat intake after the short
stressor (Ruigrok et al., unpublished; see Appendix B). Hypothalamic AgRP fiber density was not changed in these animals (see Appendix C).
In the present study, we focused on the VTA. Several neural substrates were examined to investigate a potential effect of ELS and/or diet, taking possible sex differences into account. Firstly, the area fraction of AgRP fibers in the VTA was determined. Secondly, a quantification of ventral tegmental dopaminergic cells was performed. Lastly, inhibitory synapses contacting dopaminergic nuclei and fibers were quantified. These results provide new insights into the sex-specific effects of ELS and how it may contribute to the vulnerability of metabolic disorders.
2. Materials and methods
2.1 Animals and breeding
Performed by S. Ruigrok et al.
For this study, 8-weeks old female and 5-weeks old male C5B1/6J mice were obtained from Harlan Laboratories B.V. in Venray, The Netherlands. They were kept under standard 12/12 hours light/dark schedule and housing conditions (temperature 20–22 °C, 40–60% humidity, with standard chow, water ad libitum). Before breeding, the mice were allowed to acclimatize for two weeks. Afterwards, two females were put together with one male for one week to allow mating. After another week of paired housing, the pregnant females were then individually put in clean standard cages and monitored every 12 hours for the birth of pups, starting 18 days after mating. If a litter was born before 9 o’clock in the morning, the previous day was assigned as postnatal day 0 (P0).
At P9 the control (CTL) and ELS mice were transferred to new cages with standard nesting and bedding material. They were allowed to mature and were group-housed in standard cages at P21 with one or two other mice with same sex littermates. All experiments were accepted by the animal welfare committee of the University of Amsterdam and carried out in accordance with Dutch legislation and European Union directives on animal experiments.
2.2 Early life stress paradigm
Performed by S. Ruigrok et al.
The early life stress paradigm was based on limited nesting and bedding material which leads to fragmented maternal care and chronic stress in the early life period of the pups (Rice et al., 2008). This paradigm started at P2; the litters
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were culled to five-six pups (including both sexes) per dam. The dams and pups were weighted and randomly allocated to an ELS condition or control condition. The control condition consisted of standard amounts of sawdust bedding and nesting material, which includes one square piece of cotton (5 x 5 cm; Techinlab-BMI, Someren, The Netherlands). The ELS pups were placed on a fine-fauge stainless steel mesh, 1 cm underneath the mesh was a floor covered with sawdust, and on top was a reduced amount of nesting material (2.5 x 5 cm cotton). All the cages were covered with a filtertop, and the groups were left undisturbed until P9. At P9 all mice were weighted again.
2.3 Free choice high-fat high-sugar diet
Performed by S. Ruigrok et al.
One week before start of the diet (9 weeks of age), animals were housed alone to habituate. At 10 weeks of age, the mice were randomly assigned to a free choice HFHS (fcHFHS) diet or chow diet. The control chow diet consisted of ad libitum access to tap water and regular chow food (SDS, Essex, UK. 15.01 MJ/kg, where: 22 % proteins, 69 % carbohydrates and 9 % fat). The fcHFHS diet included tap water and regular chow but also a bottle of 10% sugar water (0,4kcal/mL) and pellets made of fat (beef tallow, Vandemoortele, France; 9 kcal/g). Previous studies have shown that 10% water is the most preferred concentration for C57BL/6J mice (Lewis et al., 2005; Feillet et al., 2017). Both groups were kept on their diets from P70 to P105. Every week a body weight and food intake measurements were taken from all animals to evaluate ingestion. In week 105 a short stressor (4 hours of fasting and two tail cuts) was performed, food intake was measured before and after the stressor.
2.4 Tissue preparation
Performed by S. Ruigrok et al.
Mice of 105 weeks old were anaesthetized by an IP injection of pentobarbital (EuthasolVR 120 mg/kg) and transcardial perfused with 0.9 % saline, followed by a perfusion of 4 % paraformaldehyde (PFA) in phosphate buffer (PB = 0.1M, pH = 7.4). Brains were harvested and conserved overnight in PFA/0.1M PB at 4°C. Before slicing, brains were kept in 15% sucrose for one day followed by an overnight incubation in 30 % sucrose/0.1 PB to cryoprotect the brains. Brains were sectioned in 40 μm thick coronal sections using a microtome and stored in
antifreeze solution (30% Ethylene glycol, 20% Glycerol and 50% 0.05M PBS) at -20°C until immunohistochemistry processing.
2.5 Staining
Performed by S. Ruigrok et al.
For AgRP - Tyrosine Hydroxylase (TH) - Glutamate Decarboxylase (GAD65) staining, free-floating sections were used. Slices were rinsed three times for 10 minutes in TBS solution (10% 0.05M Tris-buffer, 0.9% saline, pH=7.6). They were incubated for 1 hour in a solution consisting of 3% bovine serum albumin (BSA, immunohistochemistry grade) and 0.3% triton X-100 in TBS. Consequently, the slides were incubated with primary antibodies AgRP (Neuromics GT15023, goat-α- AgRP, 1:1000), TH (Pel-Freez P40101-150, rabbit-α-TH, 1:1000), GAD65 (Abcam 26113, mouse-α-GAD65, 1:2500), 3% BSA and 0.3% Triton in TBS during 24 hours at room temperature. After the primary antibody incubation, the slices were rinsed with TBS three times for 10 minutes. Then, an incubation of 2 hours was done in a 3% BSA and 0.3% Triton in TBS solution. The secondary antibody (Invitrogen A21206, AlexafluorTM 488, donkey-α-goat; Invitrogen A11057, AlexafluorTM 568, donkey-α-goat; Invitrogen A31571, AlexafluorTM 647, donkey- α-mouse, 1:500 in 3% BSA and 0.3% Triton in TBS) incubation was done overnight at 4°C. On the third day, the slices were rinsed with TBS three times for 10 minutes. This was followed by mounting the slices by using a DAPI solution to visualize the nuclei of the cells. Slices were kept at 4°C in the dark until imaging.
2.6 Imaging and quantification
Performed by S. Ruigrok & E. Speijer
After immunohistochemistry, four confocal images were collected per animal between bregma -2.48 mm and -3.68 mm of the VTA. This was performed by using a confocal microscope (Nikon A1) which scanned 20 µm in total, creating Z-stacks. The high-resolution digital images were taken with reference to the mouse brain stereotaxic atlas (Paxinos & Franklin, 2004). For AgRP fiber and dopaminergic (TH) cell images a 20x magnification was used, whereas a 60x magnification was used for GAD65 images.
The images were analyzed by using the public domain Java image processing program ImageJ (Fiji) (National Institute of Health, USA) in which the images were checked for contrast
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and brightness. All 20x magnification images were stacked and saved as tiff files. Quantification of TH cell density was conducted by manually counting TH positive cells within a defined region of interest (ROI) (62498 µm2).
The same ROI was used to determine the area fraction of AgRP fibers, for which a user-defined threshold was set described previously by Sominsky et al., 2017.
For analysis of 60x GAD65 images two quantifications were performed: GAD65 puncta contacting (1) TH fibers and (2) TH nuclei were counted. Each quantification was performed throughout two Z-stacks with an interval of one plane. For TH fibers, a ROI (303,8438 µm2) and
a threshold was used to determine the area fraction of the fibers. Within the ROI, GAD65 puncta were manually counted which contacted the fibers. Hence, a numerical density of GAD65 was determined (GAD65 puncta/µm2 fiber). For
TH nuclei, the number of terminals contacting a nucleus was counted, with an average of sixteen nuclei per animal. Total number of terminals per Z-stack were summed and used as an index for the inhibitory input onto TH positive nuclei.
2.7 Statistical analysis
Performed by E. Speijer
Statistical analysis was carried out using SPSS 20.0 (IBM software), R (version 1.1.4) and Graphpad Prism 5 (Graphpad software, Inc). All data are expressed as mean +/- SEM and were considered statistically significant when p < 0.05. If present, outliers were removed from the data. The AgRP fiber density, TH cell density and GAD65 puncta per TH fiber denisty was analyzed by using a three-way ANOVA with three fixed factors: conditions (CTL vs ELS), sex (male vs female), and diet (Chow vs fcHFHS). When a significant difference was detected, a post-hoc analysis was performed using Fisher's Least Significant Difference (LSD). For GAD65 puncta on TH nuclei a multilevel model was used with n is the number of cells, corrected for the animal the cells originated from (Field, 2013).
3. Results
3.1 AgRP fiber density decreases due to HFHS diet and ELS, and differs in males and females
To investigate the innervation of AgRP neurons in the VTA, an AgRP fiber density was determined. A synergic effect of condition, diet and sex was found to influence the AgRP fiber
density (condition * diet * sex: F1 = 11,305, P =
0.001; figure 2B). Interestingly, female ELS mice with HFHS diet showed a significant reduction in AgRP fiber density compared to female ELS mice with chow diet (post-hoc: P = 0.033; figure 1A). The same accounts for control males, in which a reduction is seen in the HFHS group compared to the chow group (post-hoc: P = 0.009). Moreover, ELS exposure in chow-fed males resulted in a lower AgRP area coverage than the control males (post-hoc: P = 0.013). This was not the case in the female chow group
(post-hoc: P = 0.411). More sex differences were
detected, such as the ELS males with chow diet which have a significantly lower density than CTL female mice with chow diet and ELS female mice with HFHS diet (post-hoc: P = 0.030; P = 0.027). On the contrary, CTL males with chow diet showed a significantly higher AgRP coverage than CTL males with HFHS diet and ELS females with HFHS diet (post-hoc: P = 0.009; P = 0.016).
3.2 Dopaminergic cell density in the VTA increases in male with chow diet after ELS-exposure
TH cell density in the VTA was determined to investigate if it is affected by ELS, HFHS diet and/or sex. We observed that ELS had an effect on the amount of TH cells per mm2 (condition: F
1
= 4.143, P = 0.045; figure 2B). In addition, a significant interaction effect of condition and diet was also observed (condition * diet: F1 = 6.461,
P = 0.012). Post-hoc analysis revealed that males on chow diet with ELS have a higher TH cell density than males (post-hoc: P = 0.15; figure 2A) and females (post-hoc: P = 0.017) on chow diet without ELS.
3.3 Diet influences number of inhibitory synapses on dopaminergic nuclei, but not on dopaminergic fibers
Next, we tested if the inhibitory innervation of the VTA was affected by ELS, diet or sex differences. There was a significant effect of diet on the number of GAD65 puncta on the TH nuclei (multilevel analysis: diet: P = 0.042; figure = 3A & B). Inhibitory synapses on TH fibers were, however, not affected by diet, nor were there significant ELS or sex-specific effects (figure 3C & D).
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Figure 1: AgRP fiber density decreases due to diet and early life stress exposure and is sex-dependent (A) representative confocal images (x20 magnification) indicating region of interest in the ventral tegmental area (VTA) used for analysis. In red are the agouti-related peptide (AgRP) immunoreactive fibers. (B) AgRP fiber density in the VTA in percentage in control (CTL) or early-life stress (ELS) males and females which had a chow diet or a high-fat high-sugar (HFHS) choice diet. Data are presented as mean +/- SEM; three-way ANOVA: @ interaction effect between condition x diet x sex. *LSD post-hoc test. P < 0.05. Scale bars: 100 μm.
A
A
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Dopaminergic nucleiDopaminergic nuclei Dopaminergic fibersDopaminergic fibers
(A) Confocal image (60x magnification) of a dopaminergic (TH) nucleus (green) with inhibitory pre-synapses (GAD65 puncta; white) indicated with orange arrows. (B) Confocal image (60x magnification) of TH fibers (green) with GAD65 puncta (white) indicated with orange arrows. (C) Number of GAD65 puncta on TH nuclei in the ventral tegmental area (VTA) in control (CTL) or early-life stress (ELS) males and females which were on chow diet or high-fat high-sugar (HFHS) choice diet. (D) GAD65 puncta per µm2 fiber in the VTA in CTL or ELS males and females which were on chow diet or on HFHS choice diet. Data are presented as mean +/- SEM; multi-level analysis & three-way ANOVA: $ main effect of diet. P < 0.05. Scale bars: 100 μm.
(A) Representative confocal images (x20 magnification) of TH-stained neurons in the ventral tegmental area (VTA) indicating region of interest for analysis. Upper picture is from control (CTL) chow males and lower picture is from early-life stress (ELS) chow males. (B) TH cells per mm2 in the VTA in CTL or ELS males and females which were on a chow diet or a high-fat high-sugar (HFHS) choice diet. Data are presented as mean +/- SEM; three-way ANOVA: # main effect of condition. #* interactive effect of condition x diet. *LSD post-hoc test. P < 0.05. Scale bars: 100 μm.
Figure 3: Inhibitory synapses are influenced by diet when contacting dopaminergic nuclei but not when contacting
dopaminergic fibers.
Figure 2: Dopaminergic cell density in the VTA is higher in early-life stress exposed males on chow compared to the
control males
A B
B D
A
A B
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4. Discussion
Previous research suggest ELS can increase the risk to develop obesity and also affects food choice, depending on sex (Danese & Tan, 2014; Hemmingsson et al., 2014; Kopelman, 2000; Manian & Morris, 2010). Prior results of this study showed that males had a higher sugar intake, whereas females had a higher fat intake. Additionally, ELS females increased their fat intake after a short stressor (Ruigrok et al., unpublished). In the current study, it was investigated whether ELS affects the neurobiological substrates of feeding in a sex-dependent manner. We here present differential effects of ELS on the AgRP fiber density and dopaminergic cell density in the VTA, depending on diet and sex of the animal. Additionally, inhibitory synapses contacting dopaminergic nuclei are influenced by diet, whereas synapses contacting the dopaminergic fibers were not affected.
It is suggested that ELS may alter connectivity of brain regions in the reward system (Osadchiy et al., 2019). Evidence suggest inhibitory neurons expressing AgRP play a crucial role in the functional connection between the reward system and the homeostatic system, serving as a brake on the dopaminergic neurons in the VTA (Denis et al., 2015; Dietrich et al., 2012). Despite prior research showing that AgRP fiber density in the hypothalamus was unaffected by ELS (Ruigrok et al., unpublished), we have shown here that there are differential effects on AgRP fiber density in the VTA, depending on sex and diet. Specifically, ELS was found to decrease AgRP fiber density only in males on chow. Although it is not known how (early-life) stress can alter AgRP fiber density in the VTA, there are possible indirect explanations. Studies have shown that the system of the peripheral hormone leptin, which inhibits neurons expressing AgRP (Lutter et al., 2009), is altered due to ELS exposure in both sexes (Yam et al., 2017). A study of Bouret et al., showed that leptin deficiency led to reduced density of AgRP fibers in the paraventricular nucleus (2004). There is a possibility that the altered leptin system during ELS leads to a reduction in AgRP fiber density in the VTA. Due to the fact that the reduction was only seen in males, it could be that other neurobiological systems are affected by ELS in females.
Diet did have an effect in both males and females on AgRP fiber density in the VTA. More
specifically, HFHS diet led to a reduction in AgRP fiber density in control males and ELS females. Alterations in AgRP fiber density in the VTA due to diet have not been shown before, however, it has been determined that HFHS diet can result in altered neuropeptide expression in the hypothalamus (POMC, neuropeptide Y and AgRP) (La Fleur et al., 2010; Ziotopoulou et al., 2000). Although another study showed that the neuropeptide expression of POMC and AgRP neurons were unaffected by a high fat diet, they did find that the formation of POMC and AgRP projections in the hypothalamus was severely impaired (Vogt et al., 2014). This confirms the idea that diet can influence neurons expressing AgRP. Importantly, due to a HFHS diet, adipose tissue increases which can lead to altered metabolic hormone levels. These altered levels, in turn, can also contribute to changes in neurobiological substrates of feeding (Bouret et al., 2004). Further research needs to be done into the mechanisms behind the alterations in AgRP fiber density in the VTA due to diet.
While an effect of diet on AgRP occurred in both sexes, many other differences in density were sex-dependent. As seen in the results, ELS males on chow had a lower density than CTL females on chow and ELS females on HFHS diet. CTL males on chow had, on the contrary, a higher AgRP coverage than ELS females on HFHS diet. The probable sex-specific regulation of the AgRP fiber density corresponds to the finding that there is a difference in AgRP gene expression in males and females (Ruigrok et al., unpublished). In addition, sex-specific regulation is seen more often in the feeding circuitry (Dearden et al., 2018; Nohora et al., 2011), which probably contributes to the behavioral differences in food intake and differences in vulnerabilities for obesity. It remains difficult to determine what causes the sex-specific effects of ELS, but it may be due to the sexual organization of the brain that already takes place in the early-life period (Cosgrove et al., 2007). Therefore, the brain could already respond differently to early-life adversities in males and females due to sexual dimorphism. Clearly, this still requires further investigation.
To further explore brain anomalies associated with altered food intake, we examined the ventral tegmental dopaminergic neurons. These neurons play an import role in reward-driven food intake, and they are particularly stimulated by palatable foods (Berridge &
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Robinson, 1998; Chuang et al., 2011; Narayanan et al., 2010). Studies have shown that chronic exposure to stress or chronic consumption of palatable foods can induce alterations in the dopaminergic neurons of the reward system, although specific effects differ across studies (Romani-Perez et al., 2016; Carlin et al., 2016; Chuang et al 2011; Anstrom et al., 2009; Cao et al., 2010). Our results also show an effect of both ELS and diet on the reward system, in particular on the dopaminergic cell density in the VTA.
Moreover, chow males with a background of ELS showed an increase in dopaminergic cell density compared to the control males. No other studies have looked at the effect of ELS on the number of dopaminergic cells in the VTA before, but there are several studies that focused on the activity of those cells. They show that there is an increase of activity in VTA dopamine neurons after stress (Anstrom et al., 2009; Cao et al., 2010; Deutch et al., 1985), which may contribute to stress-induced feeding abnormalities. The exact mechanism behind altered activity or density is unknown, however Chuang et al., have proposed a possible role of metabolic hormones (2011). They show that stress can lead to elevations in levels of ghrelin, which, in turn, interacts with dopaminergic cells (likely to include the VTA). Ghrelin induces changes on these neurons, possibly leading to food-reward behavior and increased intake of highly palatable foods. Additionally, the metabolic hormone leptin also influences dopamine neurons, and the leptin system can, as mentioned before, be disrupted by ELS (Hommel et al., 2006; Fulton et al., 2006; Yam et al., 2017).
Notably, only ELS males on chow showed an increase in cell density whereas males on HFHS and females did not show any significant difference. These results, together with the other results of the AgRP fiber density in chow males, are interesting when compared to the behavior observations. Firstly, we saw no effect of ELS on food intake and TH/AgRP density in males on HFHS diet, though we did see ELS effects on TH/AgRP density in chow males. It could be that the HFHS diet overrules or normalizes the effects of ELS. Secondly, males had no differential food intake after ELS in both basal and challenged circumstances, whereas ELS females did take in higher fat amounts after a short stressor. Therefore, it might be that other neurobiological substrates of feeding are affected between sexes. A reason for this could be that both circulating levels and the sensitivity to metabolic hormones
(i.e. leptin) varies vastly between males and females, which can potentially mediate some of the sex differences in response to ELS (Clegg et al., 2003).
Next to the investigation of differential dopaminergic cell density in the VTA, it was also examined whether the inhibitory innervation of the cells is affected. Although results must be taken with caution due to low sample size, no effect was found on the inhibitory synapses contacting dopaminergic fibers. However, a diet effect was found on synapses contacting dopaminergic nuclei. Prior rodent studies have found that chronic consumption of palatable foods can indeed induce adaptations in the dopamine system. The majority of these studies concluded a decreased effect on the dopamine activity or release (York et al., 2010; Carlin et al., 2016; Romani-Perez et al., 2016). The authors suggested that this could lead to higher palatable food intake to attain the necessary dopamine reward. It is, however, not clear whether the alteration in inhibitory input seen in our study leads to decreased dopamine activity. Further research needs to be done to explore this.
It is interesting to note that, whilst it is known that the studied circuitry of inhibitory AgRP and dopaminergic neurons influences each other (Denis et al., 2015; Dietrich et al., 2012), it is unknown to what extend and in which manner. We have seen that all parameters are influenced by diet and both AgRP fiber density and dopaminergic cell density in chow male are influenced by ELS. It is, however, difficult to determine if these results are connected. Further research could focus on the specific connection and dependency between AgRP fibers, inhibitory input and dopaminergic neurons by, for example, a knockout study. Additionally, the studied parameters showed a sex-dependent effect, which was also seen in food intake. However, it is unclear if these results are connected, and this still requires further investigation.
Although the mechanism underlying these neurobiological alterations and sex-differences require further elucidation, we showed that early-life stress influences the neurobiological substrates of hedonic feeding in a diet- and sex-dependent manner. These alterations might contribute to different feeding behavior and energy balance, thereby leading to an increased vulnerability to develop metabolic disorders such as obesity.
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6. Appendix
Appendix A
Differences in food intake between males and females
The behavioral observations of this study show that females have a higher fat intake compared to males (figure 1A). Males, on the other hand, took in a higher sugar amount compared to females (figure 1B). No early-life stress effect was detected under basal circumstances.
Figure 1: Female take in higher fat amounts, whether male take in higher sugar amounts
(A) Kilocalories due to fat intake per week in males (blue lines) and females (pink lines) with a background of early-life stress (ES) or control (CTL) (B) Kilocalories due to sugar intake per week in males (blue lines) and females (pink lines) with a background of early-life stress (ES) or control (CTL). Data are represented as mean +/- SEM; three-way ANOVA: @ week effect. $ sex effect. P < 0.05. Retrieved from Ruigrok et al., unpublished.
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Appendix B
Early-life stress females increase fat-intake after a short stressor
In the behavioral research after a short stressor, a sex effect and synergic effect of sex and condition was found in fat intake (image 2A). Post-hoc revealed that ELS females took in a higher amount of fat compared to control females and males. There was no difference seen in sugar intake (image 2B).
Figure 2: Early-life stress females show a higher fat intake after a short stressor compared to control females and males (A)Kilocalories due to fat intake per 24 hours in males (M) and females (F) with a background of early-life stress (ES) or control (CTL) (B) Kilocalories due to sugar intake per 24 hours in males and females with a background of ES or CTL. Data are represented as mean +/- SEM; three-way ANOVA: $ sex effect. $* sex*condition effect. *LSD post-hoc test. P < 0.05. Retrieved from Ruigrok et al., unpublished.
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Appendix C
No effect of ELS, diet or sex on AgRP fiber density in the hypothalamus
Figure 3: Hypothalamic AgRP not different in male and female, nor effected by ELS or diet
AgRP fiber density in the hypothalamus in percentage in control (CTL) or early-life stress (ES) males (M) and females (F) which had or a chow diet or a high-fat high-sugar (HFHS) choice diet. Data are represented as mean +/- SEM; three-way ANOVA. P < 0.05. Retrieved from Ruigrok et al., unpublished.
