Sex-dependent behavioral and hypothalamic receptor changes after early-life monosodium glutamate (MSG) exposure and adult social stress in Wistar rats
Juliano Ten Kathen Jung, Luiza Souza Marques, Carlos Alexandre Brambila, Fabíola Caldeira dos Santos, Cristina Wayne Nogueira
TL;DR
Early-life MSG exposure and adult social stress affect anxiety and eating behaviors differently in male and female rats.
Contribution
This study reveals sex-dependent effects of early-life MSG and adult social stress on behavior and hypothalamic receptor expression in rats.
Findings
MSG increased the Lee Index in both male and female rats at PND 60.
Social-SPS and MSG reduced food intake and induced anxiety-like behavior in a sex-dependent manner.
Hypothalamic receptor expression changes were observed and correlated with behavioral outcomes.
Abstract
Obesity is a growing public health issue, with comorbidities including metabolic syndrome and psychiatric disorders. Stress, an inherent factor in daily life, can influence mood and eating behavior and contribute to psychiatric diseases. This study investigated whether social-single prolonged stress (social-SPS) affects anxiety-like and eating patterns in male and female rats exposed to an early-life monosodium glutamate (MSG)-induced obesity model. We also evaluated changes in hypothalamic protein expression levels of leptin (Ob-R), ghrelin (GHS-R1), dopamine 1 (D1R), and dopamine 2 (D2R) receptors. Male and female Wistar rats were exposed to MSG (4 g/kg/day) from post-natal day (PND) 1 to 10, followed by social-SPS exposure at PND 60. Rats were euthanized on PND 69, and hypothalamic samples were analyzed. MSG increased the Lee Index in both sexes at PND 60. MSG-treated rats exhibited…
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TopicsBiochemical Analysis and Sensing Techniques · Regulation of Appetite and Obesity · Olfactory and Sensory Function Studies
Introduction
Obesity is a multifactorial condition that represents a serious global public health problem, associated with metabolic, cardiovascular, and psychiatric diseases (Hruby and Hu 2015). Hypothalamic obesity (HO) is a complex and rare disorder resulting from damage to the hypothalamus, leading to weight gain due to disrupted energy regulation. This condition can arise from tumors, such as craniopharyngiomas, or from surgical and radiation treatments that affect hypothalamic function. The pathophysiology of HO involves impaired satiety signaling and decreased energy expenditure, resulting in rapid weight gain (Roth and McCormack 2024). In preclinical models, neonatal administration of monosodium glutamate (MSG) has been widely used to induce neuroendocrine obesity in rodents, characterized by hypothalamic lesions, increased visceral adiposity, and alterations in feeding behavior (Rosa et al. 2016; Quines et al. 2018). Importantly, MSG-induced obesity reflects early-life hypothalamic neurotoxicity, particularly in the arcuate nucleus, leading to neuroendocrine dysfunction and adiposity redistribution, rather than simply increased food intake (Olney 1969). This model reproduces important aspects of human obesity, including leptin resistance and central inflammation, which makes it a relevant tool to investigate the neural mechanisms of metabolic dysfunction, caused by specific damage to the arcuate nucleus (ARC) of the hypothalamus, a critical region in the integration of homeostatic signals of hunger and satiety (Kayode et al. 2023).
Stress has been widely recognized as a modulating factor of energy homeostasis, directly impacting eating behavior and metabolism, especially in obese individuals (Spencer and Tilbrook 2011). Prolonged exposure to stress can exacerbate neuroendocrine changes already present in obesity, such as hypothalamic-pituitary-adrenal (HPA) axis dysfunction and leptin resistance, leading to a greater propensity for anxiety disorders and binge eating (Pecoraro et al. 2004; De Kloet et al. 2005).
The hypothalamus plays a central role in the integration of metabolic and neuroendocrine signals, coordinating behavioral and physiological responses to stress and appetite regulation (Timper and Brüning 2017). Regions such as the ARC, paraventricular nucleus (PVN), and lateral hypothalamus are particularly sensitive to the action of hormones such as leptin, ghrelin, and dopamine, which modulate energy balance and food motivation (Bouret 2022).
Chronic exposure to stress can alter the expression of Ob-R in the hypothalamus, which is also modulated by perinatal factors such as a history of maternal stress (Hosseini et al. 2024). Circulating ghrelin remains elevated in a sustained manner after stress, and its signaling in the hypothalamus contributes to increased sensitivity to aversive stimuli (Yousufzai et al. 2018). D1R and D2R are key regulators of stress and feeding behavior, integrating hypothalamic and reward pathway signals that shape energy balance and motivational responses (Jin et al. 2023; Fujioka et al. 2024). Ob-R, GHS-R1, D1R, and D2R receptors are key regulators of hypothalamic control of energy balance and stress responses. Although chronic stress is known to alter their expression, data under acute SPS conditions remain limited, particularly regarding sex-specific effects.
Despite advances in understanding the neurobiological mechanisms that link obesity and stress, preclinical studies that comparatively evaluate the effects of these conditions in males and females are still scarce. Most research predominantly uses male rodents, which limits the understanding of sex-specific responses, especially in the context of combined models of induced obesity, such as the use of MSG, and social-SPS, a validated post-traumatic stress disorder (PTSD) model (Beery and Zucker 2011; Jung et al. 2024). This gap is particularly relevant considering that women are more vulnerable to anxiety disorders and metabolic alterations in contexts of chronic stress (Bangasser and Wicks 2017).
Given the lack of studies evaluating sex-specific responses to the combination of early-life hypothalamic disruption and adult PTSD-like stress, the present study investigated whether early-life MSG exposure modulates the behavioral and hypothalamic responses to social-SPS in male and female rats. We focused on anxiety-like behaviors, feeding patterns, and hypothalamic receptors involved in metabolic and emotional regulation.
Methods
Animals
This study used pregnant female Wistar rats obtained from the Central Animal Research Facility (250–350 g) and their newborns (male and female). The animals were kept in an animal room on a 12 h light/12 h dark cycle with the lights turned on at 7:00 am and a controlled room temperature (22 ± 3Cº). Animals were housed in a polycarbonate cage (41 cm x 34 cm x 20 cm) and had free access to filtered tap water and food (Puro Trato^®^). Food and water intake were measured every three days at the cage level and corrected per animal.
Chemicals
MSG was purchased from Labsynth S.A. All other chemicals were of analytical grade and obtained from Sigma-Merck^®^ or BioRad^®^.
Experimental procedure
Pregnant Wistar dams (n = 12) were obtained from the central animal facility. Litters were standardized to 6–8 pups per dam, and both sexes were included. Pups were randomly assigned to experimental groups, ensuring that each group contained animals from multiple litters. After maternal separation at PND21, animals were housed in groups of 2–3 per cage, depending on sex distribution within litters, and never housed alone. This arrangement was chosen to maintain stable social conditions during development and to allow the subsequent partner exchange required by the social-SPS paradigm, which demands social instability. Following completion of the social-SPS protocol, rats were housed 4–5 per cage, in accordance with standard housing density. Body weight was not used as a matching criterion. The study was conducted in two experimental cohorts, and replicability was ensured by distributing littermates across groups and maintaining identical housing and experimental conditions. Cages were considered the unit of analysis in statistical procedures.
Initially, male and female rats were divided into two groups (n = 16 each): I – Control: rats received a subcutaneous injection of saline (0.9%), and II – MSG (*n *= 16): rats received a subcutaneous injection of MSG (4 g/Kg body weight per day) during the first 10 postnatal days (Quines et al. 2018).
Rats were weaned on the 21st postnatal day. Additionally, during their development, the Lee index was recorded by measuring the body weight and nasal-anal length at the 30th and 60th postnatal days. The Lee index is a validated morphometric indicator of adiposity in rodents, allowing the detection of obesity-related changes. The Lee indices of the animals (saline and MSG groups) were calculated by – \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{\sqrt[3]{bodyweight\left(g\right)}}{nasal-anallenght\left(cm\right)}$$\end{document} . Higher Lee Index values indicate increased adiposity, consistent with obesity phenotypes (Olney 1969).
On 60th postnatal day, rats were divided into four experimental groups (n = 8 each): I: Control – (saline); II: social-SPS – (saline + social-SPS); III: MSG – (MSG); and IV: MSG + social-SPS (MSG + social-SPS). From postnatal days 21 to 60, food and water intake were recorded every three days. Cages were used as units for these measurements, and cumulative food intake was calculated by correcting consumption per animal, using the cage as the experimental unit, and summing values every three days. Water intake was expressed as mL/animal, and food intake as grams (g)/animal. In addition to cumulative measurements, food intake was also evaluated during the eating behavior test on PND 59 and PND 67, following a 12 h food deprivation, with consumption recorded over 30 min.
From postnatal days 54 to 58, animals were trained to eat from the floor in an open field apparatus to perform the eating behavior test (item 2.4.1) on days 59 (before social-SPS exposure) and 67 (after social-SPS).
On postnatal day 60, animals of groups II and IV underwent a stress protocol (item 2.3.1), a developmental stage in which rodents have already passed puberty but are not yet fully mature adults. This time point was selected to capture outcomes during a transitional period characterized by continued growth and neuroendocrine maturation. Importantly, PND 60 has also been standardized by our group in the social-single prolonged stress (social-SPS) paradigm, further supporting the relevance of this age for behavioral and molecular assessments (Jung et al. 2024). On day 68, all animals performed a spontaneous locomotor activity and the elevated plus maze test. Twenty-four hours after the last behavioral tests, the animals were weighed and then euthanized by decapitation. Adipose tissues, including brown adipose tissue (BAT), mesenteric adipose tissue (MAT), gonadal white adipose tissue (gWAT), and inguinal white adipose tissue (iWAT), were dissected, weighed, and expressed as relative weight (tissue weight [g]/body weight [g]). The hypothalamus was then collected and stored at − 80 °C for subsequent Western blot analysis (Fig. 1a).
Fig. 1. Scheme of the experimental protocol a. Effects of MSG exposure on Lee Index in male and female rats on PND 30 b and PND 60 c, cumulative food intake on male d and female e, cumulative water intake on male f and female g, and the mean animal weight on male h and female i. Results represent the mean ± S.E.M. of 16 rats per group. *p < .05; **p < .01; ***p < .001; ****p < .0001 when compared with saline group. Data were analyzed by two-way ANOVA b, c or two-way with repeated measures d–i followed by the Sidak’s post-test. PND means post-natal day, MSG means monosodium glutamate, SLA means spontaneous locomotor activity, EPM means elevated plus maze, social-SPS means social-single prolonged stress
Social-SPS protocol
This protocol was carried out as previously reported by our research group (Fulco et al. 2022; Jung et al. 2024). Wistar rats were exposed to three stressors: two hours of immobilization, twenty minutes of forced swimming, and then placed in a new cage with new partners. The immobilization was done with restrainers appropriate for adult Wistar rats (commonly 20–22 cm length × 6–8 cm internal diameter). The restrainer permits minimal limb movement but prevents locomotion; its ends were closed to avoid escape while allowing ventilation. The subject was then subjected to twenty minutes of forced swimming in a cylindrical tank (commonly 40 cm height × 20–25 cm inner diameter) filled with water to a depth of ~ 40 cm, thereby preventing tail support. Water temperature was maintained at 23–25 °C. After the swim, animals were gently removed, dried with soft towels, and placed for ~ 5–10 min under a heat lamp or in a warmed recovery cage until fur was no longer dripping, to avoid hypothermia, and, finally, placed in a new cage containing unfamiliar partners (social instability). Animals were housed in this new group configuration for a defined period (incubation) of seven days.
Behavioral tests
All behavioral tests (n = 8 animals/experimental group) were carried out during the daytime (7 to 12 a.m.). Stopwatches were used to measure time, and tests were evaluated by an experimenter who was unaware of the treatment groups.
Eating behavior test
To assess the rat feeding behavior, they were acclimated to the testing conditions for 30 min every morning over five consecutive days to learn how to eat the food in the new environment before the first test. Their cages were transferred to the room where the behavioral tests were conducted, where they were trained to eat food from the apparatus on the floor of the open apparatus (41 × 34 × 17 cm) (height x width x depth) in the presence of the experimenter. The behavior was assessed in two stages: the first, conducted one day before exposure to the stress protocol (PND 59), and the second, seven days after exposure to stress (PND 67), to evaluate the effect of the factors (MSG × stress). The animals were taken to the room where the behavioral tests were conducted and had a habituation time of 1 h, during which they were food-deprived. Afterward, they were placed in the apparatus without grids and without sawdust, with a light positioned above the animal and with commercial food (the same used in the animal’s diet) in a known quantity, as described (François et al. 2021). Only the day before the tests (58 and 66), the animals were subjected to a fasting period of approximately 12 h (the dark cycle of the night before the test). Aware that food deprivation for 12 h is used as an additional stressor in chronic stress protocols (Fonseca-Rodrigues et al. 2022), we considered this step important for the correct assessment of feeding behavior. Furthermore, since all the animals underwent the same deprivation, this effect was not observed as one of the study variables. The amount of food intake in 30 min was evaluated and expressed in kcal.
Spontaneous locomotor activity (SLA)
To assess the SLA of the animals, they underwent 5 min in the acrylic activity monitor (INSIGHT, Campinas, Brazil) by observing spontaneous movement (number of crossings with all four paws between the divisions of the field) on postnatal day 68. In this test, the number of crossings and elevations of the animals was assessed. The time spent in the center of the apparatus, the distance traveled, the number of crossings and rearings, the velocity, and the number of visits to the center of the apparatus were also measured (Hall 1935).
Elevated plus maze test (EPM)
Anxiety-like behavior was assessed using the EPM, a widely validated paradigm for rodents. This test was selected because it provides reliable measures of exploratory behavior and avoidance patterns, which are particularly relevant in studies involving hypothalamic and metabolic alterations. One hour after the SLA, the animals underwent the EPM test to evaluate behaviors that characterize the anxious-like phenotype. The test was performed as described by Pellow et al. (1985), in which, during 5 min, the total time spent by rats in the open and closed arms and the number of entries into the open and closed arms were recorded. An anxiety index was calculated as follows: 1 − [([Open arm time / Test duration] + [Open arms entries/Total number of entries]) / 2], and the percentage of time spent in the open arms (OAT%) and the percentage of open arms entries (OAE%) were also calculated.
Ex vivo analysis
Western Blot assay
The method was performed as previously described (Towbin et al. 1979). Total hypothalamus samples (30 µg protein/well) were homogenized in ice-cold RIPA buffer (Sigma-Merck) supplemented with protease and phosphatase inhibitors (Sigma-Merck). Homogenates were centrifuged at 14,000 g for 15 min at 4 °C, and supernatants were collected. Protein concentrations were determined using the BCA assay (Sigma-Merck). Samples were mixed with Laemmli buffer (Bio-Rad), heated at 95 °C for 5 min, and loaded onto SDS-PAGE gels with a marker protein (Bio-Rad). Proteins were separated on an SDS-polyacrylamide gel by electrophoresis. The proteins were transferred to a nitrocellulose membrane (0.45 μm, Bio-Rad) using the Transfer-Blot^®^ Turbo ™ transfer system (1.0 mA, 15 to 40 min, Bio-Rad). After blocking with a 5% bovine serum albumin (BSA) solution for 1 h, the blots were incubated overnight at 4 °C with primary antibodies. The proteins: Ob-R anti-mouse, Santa Cruz, 1:1000; GHS-R1 anti-mouse, Santa Cruz, 1:1000; D1R anti-mouse, Santa Cruz, 1:250; and D2R anti-mouse, Santa Cruz, 1:250; were marked. The protein β-actin (anti-mouse, SIGMA, 1:5000) was used as the loading control. Protein loading homogeneity was verified by Ponceau staining of membranes, which are provided in the Supplementary Material.
Statistical analysis
The data were expressed as the mean ± standard error of the mean (S.E.M). Data normality was verified using the D’Agostino and Pearson omnibus test, except for the Western blot, in which the Shapiro–Wilk test was used. Homogeneity of variance was assessed using Levene’s test. All datasets met the assumptions for parametric testing; therefore, ANOVA and t-tests were applied as described, and no data transformations or non-parametric tests were required. Comparisons among experimental groups were performed using two-way analysis of variance (ANOVA) [sex × MSG] or [social-SPS × MSG] followed by Sidak’s multiple comparisons test. Two-way repeated-measures ANOVA was carried out to analyze the food and water intake and body weight data. For repeated-measures analyses, sphericity was tested using Mauchly’s test, and when violated, Greenhouse–Geisser corrections were applied. For behavioral tests, sex was initially included as a factor, and three-way ANOVA was performed [sex × MSG × social-SPS]; however, no significant interactions were found. Subsequent analyses of behavioral and protein data were therefore conducted using two-way ANOVA. Pearson’s correlation coefficient was used to evaluate the statistical relationship between two variables. Assumptions of normality and linearity were verified prior to correlation analyses, and as all datasets met these assumptions, Pearson’s correlation was applied. Non-parametric alternatives (Spearman’s rho) were considered but not required.
Results
MSG exposure in early life enhances Lee index, increases adiposity, and reduces water consumption and body weight in male and female rats on PND 60
To assess the impact of MSG exposure on growth and metabolic parameters, we first analyzed the Lee Index, food and water intake, and body weight gain.
A two-way ANOVA of the Lee Index revealed no significant sex × MSG interaction at PND 30 (F(1, 60) = 3.93, p > .05) or PND 60 (F(1, 60) = 0.56, p > .05). At PND 60, however, significant main effects of sex (F(1, 60) = 6.27, p < .05) and MSG (F(1, 60) = 29.40, p < .0001) were observed, indicating increased adiposity in MSG-exposed rats (Figs. 1b, c).
Repeated-measures ANOVA of cumulative food intake showed a significant time × MSG interaction in males (F(11, 154) = 11.18, p < .0001), but not in females (F(11, 154) = 1.83, p > .05). Post hoc tests revealed reduced food intake in MSG-treated males from PND 24 (p < .001) to PND 57 (p < .05) (Figs. 1d, e).
Repeated-measures ANOVA of cumulative water intake indicated significant time × MSG interactions in both males (F(11, 154) = 13.71, p < .0001) and females (F(11, 154) = 43.25, p < .0001). MSG-treated rats consumed less water than controls across multiple time points (Figs. 1f, g).
Repeated-measures ANOVA of body weight revealed significant time × MSG interactions in males (F(13, 390) = 40.17, p < .0001) and females (F(13, 390) = 5.62, p < .001), with MSG-treated rats consistently weighing less than controls (Figs. 1h, i).
Two-way ANOVAs of adipose tissue weights revealed no significant sex × MSG interactions for BAT, MAT, gWAT, or iWAT. However, a significant main effect of MSG was found for MAT, gWAT, and iWAT in both sexes, and for BAT in females (Table S1).
MSG exposure and social-SPS, both individually and combined, reduce food intake on eating behavior for male and female rats
To evaluate the effects of MSG and social-SPS on eating behavior, food intake was measured before and after exposure to social-SPS.
Before social-SPS exposure, a two-way ANOVA revealed a significant sex × MSG interaction (F(1, 60) = 10.76, p < .01). Male rats showed higher food intake than females in both saline (p < .0001) and MSG-treated groups (p < .05). MSG-treated males exhibited reduced food intake than controls (p < .001), whereas MSG exposure did not alter food intake in females (Fig. 2a).
Fig. 2. Effect of MSG on eating behavior in male and female rats a. Effects of social-SPS and MSG on eating behavior in male b and female c rats, anxiety index in male d and female e, open arms time (%) in male f and female g rats and open arms entries (%) in male h and female i rats. Results represent the mean ± S.E.M. of 16 a or 8 b–i rats per group. *p < .05; **p < .01; ***p < .001; ****p < .0001 when compared with saline a or saline control b–i group, @p < .05 when compared with male MSG group a, #### p < .0001 when compared with male saline group a. Data were analyzed by two-way ANOVA followed by the Sidak’s post-test. MSG means monosodium glutamate, social-SPS means social-single prolonged stress
Seven days after social-SPS exposure, two-way ANOVAs revealed significant social-SPS × MSG interactions in both males (F(1, 28) = 5.69, p < .05) and females (F(1, 28) = 6.60, p < .05). Social-SPS reduced food intake in saline-treated males (p < .05) and females (p < .01). In MSG-treated rats, both MSG and social-SPS exposure reduced food intake in males (p < .0001) and females (p < .001) (Figs. 2b, c).
MSG exposure and social-SPS individually induce anxiety-like phenotype in female rats
To investigate the effects of MSG and social-SPS on anxiety-related behavior of male and female rats, we analyzed the anxiety index and open-arm parameters in the elevated plus maze.
In males, two-way ANOVA revealed no significant MSG × social-SPS interaction (F(1, 28) = 0.45, p > .05), but a main effect of social-SPS was observed (F(1, 28) = 5.56, p < .05). Anxiety index values were similar across male groups (Fig. 2d).
In females, a significant MSG × social-SPS interaction was found (F(1, 28) = 13.93, p < .001). Both MSG (p < .05) and social-SPS (p < .01) increased the anxiety index compared to controls, with no additive effect (Fig. 2e).
Analysis of OAT% revealed no significant interaction in males (F(1, 28) = 1.51, p > .05), but a significant MSG × social-SPS interaction in females (F(1, 28) = 10.96, p < .01). Social-SPS (p < .05) and MSG (p < .05) reduced OAT% in females compared to controls (Figs. 2f, g).
For OAE%, no significant interaction was observed in males (F(1, 28) = 0.09, p > .05), whereas females showed a significant MSG × social-SPS interaction (F(1, 28) = 17.03, p < .001). Female rats exposed to MSG or social-SPS spent less time in open arms compared to controls (p < .05) (Figs. 2h, i).
MSG exposure and social-SPS, both individually and combined, reduce time in the center but do not alter other locomotor parameters of male and female rats
To assess MSG and social-SPS effects on locomotor activity, we analyzed center time, crossings, rearings, distance, and speed in male and female rats in the SLA apparatus.
In males, two-way ANOVA revealed a significant MSG × social-SPS interaction for center time (F(1,28) = 15.07, p < .001). Social-SPS (p < .0001), MSG (p < .0001), and MSG + social-SPS (p < .0001) reduced center time compared to control (Table 1). Two-way ANOVAs revealed no significant MSG × social-SPS interactions for crossings, rearings, distance, or speed. However, significant main effects of MSG were observed for crossings (F(1, 28) = 47.31, p < .0001), rearings (F(1, 28) = 9.37, p < .01), distance (F(1, 28) = 12.59, p < .01), and speed (F(1, 28) = 15.58, p < .001).
In females, two-way ANOVA revealed a significant MSG × social-SPS interaction for center time (F(1, 28) = 8.48, p < .01), along with main effects of MSG (F(1, 28) = 22.72, p < .0001) and social-SPS (F(1, 28) = 11.40, p < .01). Social-SPS (p < .001), MSG (p < .0001), and MSG + social-SPS (p < .0001) reduced center time compared to controls (Table 2). No significant interactions were found for crossings, rearings, distance, or speed, but MSG exerted significant main effects on all parameters (crossings: F(1, 28) = 62.38, p < .0001; rearings: F(1, 28) = 26.82, p < .0001; distance: F(1, 28) = 58.56, p < .0001; speed: F(1, 28) = 76.95, p < .0001).
MSG exposure and social-SPS, both individually and combined, reduce Ob-R, GHS-R1, and D2R levels in the hypothalamus of male rats
To investigate the impact of MSG and social-SPS on hypothalamic receptor expression in males, we analyzed Ob-R, GHS-R1, D1R, and D2R protein levels.
Two-way ANOVAs revealed significant MSG × social-SPS interactions for GHS-R1 (F(1, 16) = 7.93, p < .05), Ob-R (F(1, 16) = 22.69, p < .001), and D2R (F(1, 16) = 8.38, p < .05). Post hoc comparisons indicated that social-SPS (p < .01), MSG (p < .01), and combined MSG + social-SPS (p < .001) exposure significantly reduced hypothalamic GHS-R1 levels compared to controls (Fig. 3a). Similarly, Ob-R levels were decreased in social-SPS (p < .001), MSG (p < .0001), and MSG + social-SPS (p < .01) groups relative to controls (Fig. 3b). D2R levels were also reduced in social-SPS (p < .05), MSG (p < .01), and MSG + social-SPS (p < .01) groups compared to controls (Fig. 3c).
Fig. 3. Effects of social-SPS and MSG on the levels of GHS-R1 a, Ob-R b, D2R c, and D1R d in the hypothalamus of male rats. Results represent the mean ± S.E.M. of 5 rats per group. *p < .05; **p < .01; ***p < .001; ****p < .0001 when compared with saline control group. Data were analyzed by two-way ANOVA followed by the Sidak’s post-test. MSG means monosodium glutamate, social-SPS means social-single prolonged stress, GHS-R1 means Ghrelin receptor 1, Ob-R means leptin receptor, D2R means dopamine receptor 2, D1R means dopamine receptor 1
For D1R, no significant interaction was observed (F(1, 16) = 0.03, p > .05), but a main effect of MSG was detected (F(1, 16) = 6.69, p < .05). All groups showed similar hypothalamic D1R levels, with MSG exposure alone accounting for the reduction (Fig. 3d).
Pearson’s correlation analyses in males revealed significant positive associations between food intake and D2R levels (Fig. 4a), between GHS-R1 and D2R levels (Fig. 4b), and between Ob-R and D2R levels (Fig. 4c). In contrast, a significant negative correlation was found between food intake and anxiety index (Fig. 4d).
Fig. 4. Effects of social-SPS and MSG on the levels of GHS-R1 a, Ob-R b, D2R c, and D1R d in thehypothalamus of female rats. Results represent the mean ± S.E.M. of 5 rats per group. *p < .05; **p < .01;when compared with saline control group. Data were analyzed by two-way ANOVA followed by theSidak’s post-test. MSG means monosodium glutamate, social-SPS means social-single prolonged stress,GHS-R1 means Ghrelin receptor 1, Ob-R means leptin receptor, D2R means dopamine receptor 2, D1Rmeans dopamine receptor 1
MSG exposure and social-SPS, both individually and combined, reduce Ob-R and D1R levels in the hypothalamus of female rats
To assess the effects of MSG and social-SPS on hypothalamic receptor expression in females, we measured the protein levels of Ob-R, GHS-R1, D1R, and D2R.
Two-way ANOVAs revealed significant MSG × social-SPS interactions for Ob-R (F(1, 16) = 9.26, p < .01) and D1R (F(1, 16) = 8.78, p < .01). Post hoc analyses showed that social-SPS (p < .05), MSG (p < .01), and MSG + social-SPS (p < .05) exposure significantly reduced Ob-R levels compared to controls (Fig. 5b). Similarly, D1R levels were decreased in social-SPS (p < .01), MSG (p < .05), and MSG + social-SPS (p < .05) groups relative to controls (Fig. 5d).
Fig. 5. Pearson’s correlation test between hypothalamic levels of D2R and food intake a, D2R and GHS-R1 b, D2R and Ob-R c, and between anxiety index and food intake d in male rats. In female rats, correlation between anxiety index and food intake e, D1R levels and anxiety levels f, and D1R and food intake g. Each point represents one individual animal. GHS-R1 means Ghrelin receptor 1, Ob-R means leptin receptor, D2R means dopamine receptor 2, D1R means dopamine receptor 1
For GHS-R1 and D2R, no significant interactions were observed (GHS-R1: F(1, 16) = 0.87, p > .05; D2R: F(1, 16) = 0.26, p > .05). All groups demonstrated comparable hypothalamic levels of GHS-R1 (Fig. 5a) and D2R (Fig. 5c).
Pearson’s correlation analyses in females revealed significant negative associations between food intake and anxiety index (Fig. 4e), and between anxiety index and D1R levels (Fig. 4f). Additionally, a positive correlation was found between food intake and D1R levels (Fig. 4g).
Discussion
The results reveal a similar effect of MSG and social-SPS when evaluated independently on eating behavior in male and female rats, without evidence of synergistic or additive interactions. Notably, only female rats exhibited an anxiety-like phenotype in response to MSG or social-SPS; however, prior neonatal MSG exposure modulated the behavioral response to social-SPS, preventing the expression of this phenotype. At the molecular level, exposure to MSG and social-SPS led to a reduction in hypothalamic levels of Ob-R, GHS-R1, and D2R in male rats. The Ob-R and D1R hypothalamic levels were reduced in female rats exposed to MSG and social-SPS, both individually and in combination, indicating sex-specific differences in neuroendocrine responses.
Early exposure to MSG resulted in significant changes in rat metabolism, as reflected by an increase in the Lee index at PND 60. The increase in adiposity, accompanied by a reduction in body weight, suggests a possible redistribution of body mass, favoring adiposity over total body mass, a phenotype consistently described in experimental models of neonatal MSG exposure (Olney 1969; Quines et al. 2018). The absence of changes at PND 30 indicates that the MSG effects are progressive and may depend on changes in the neuroendocrine axis that manifest with maturation. The reduction in water intake observed in male and female rats treated with MSG further supports hypothalamic involvement, given the established role of these nuclei in thirst control and osmoregulation (Caputo and Scallet 1995).
The hypothalamus, especially the ARC and PVN nuclei, integrates hormonal and environmental signals to regulate both energy homeostasis and the stress responses. Neonatal MSG exposure has been shown to disrupt leptin and ghrelin signaling pathways, which, in addition to controlling appetite, also modulate anxiety-related behaviors via receptors expressed in pro-opiomelanocortin neurons (POMC) and agouti-related peptide neurons (AgRP), as well as in other hypothalamic regions (Park and Ahima 2015; López 2018). Although our findings are consistent with functional outcomes previously associated with MSG exposure, we did not directly assess hypothalamic integrity; therefore, hypothalamic alterations should be interpreted as mechanistic inferences based on prior literature, rather than demonstrated effects in the present study. Notably, female rats maintained normal food intake despite increased adiposity, suggesting a role for sex hormones in modulating hypothalamic sensitivity to metabolic cues. Estrogen interacts with leptin and insulin pathways in the hypothalamus, influencing both energy homeostasis and stress responsiveness, which may underlie the heightened anxiety phenotype observed in females under stress (Clegg et al. 2006; Mahboobifard et al. 2022).
In both sexes, exposure to MSG and social-SPS resulted in a significant reduction in hypothalamic Ob-R levels, suggesting a disruption of the leptin pathway, consistent with impaired hypothalamic sensitivity to metabolic cues (Timper and Brüning 2017; Pan and Myers 2018). In male rats, the reduction in GHS-R1 and D2R levels suggests a coordinated disruption of receptor signaling, pointing to alterations in a shared regulatory pathway (Zigman et al. 2016; Andermann and Lowell 2017). Importantly, the positive correlations between food intake and D2R, as well as between D2R and GHS-R1, provide direct experimental support for the existence of a hypothalamic ghrelin–dopamine functional axis involved in the regulation of food motivation and stress-related behaviors (Stuber and Wise 2016).
In male rats, both neonatal MSG administration and adult social-SPS exposure reduced time spent in the center of the SLA, suggesting increased avoidance of open and potentially threatening environments (Seibenhener and Wooten 2015). This behavioral change suggests heightened emotional reactivity rather than a fully expressed anxiety-like phenotype, as no alterations were observed in elevated plus maze parameters, indicating preserved risk-assessment behavior in this task. Accordingly, despite evidence that social stress exacerbates anxiety-like behavior in rodents (Fulco et al. 2022), male rats subjected to the present protocol did not exhibit a consistent anxiety-like phenotype across behavioral paradigms, supporting the interpretation of a context-dependent or subthreshold emotional response.
Social-SPS in the context of neonatal MSG exposure prevented the expression of anxiety-like behavior in female rats. The susceptibility of female rats to an anxiety-like phenotype observed in the elevated plus maze following exposure to MSG or social-SPS as isolated factors suggests a critical interaction between hypothalamic and limbic circuits, potentially modulated by hormonal influences (Kundakovic and Rocks 2022). Instead, this behavioral profile may reflect a context-dependent modulation of stress responsiveness, potentially involving dopaminergic receptor function. This interpretation is supported by the reduction in hypothalamic D1R levels observed in female rats exposed to MSG and social-SPS, which may contribute to the expression of anxiety-like behavior, given the established role of D1 receptors in emotional regulation and stress adaptation. Activation of D1 receptors within limbic–hypothalamic circuits has been associated with anxiolytic effects, whereas their downregulation is linked to heightened emotional reactivity and reduced motivation to feed (Cabib and Puglisi-Allegra 2012; Zhang et al. 2020). Importantly, the observed correlations between hypothalamic D1R levels, food intake, and anxiety-related measures provide support for a functional involvement of dopaminergic signaling in the sex-specific behavioral outcomes induced by early-life metabolic disruption and social stress. Sex hormones are known modulators of dopaminergic tone and receptor expression across brain regions (Inagaki et al. 2010), and fluctuations in estrogen during development can influence dopamine synthesis and D1R sensitivity (Paolo 1994). Although circulating hormone levels were not measured in the present study, developmental differences in estrogen signaling may contribute to the increased vulnerability of dopaminergic receptor function observed in females, a hypothesis supported by experimental studies demonstrating hormone-dependent modulation of dopaminergic systems during critical periods of brain maturation (Purves-Tyson et al. 2012; Cao et al. 2018).
Estrogen can upregulate D1R expression via estrogen receptor α (ERα) mediated enhancement of cAMP response element-binding protein (CREB) phosphorylation and promote binding in dopaminergic neurons, particularly within the ARC and preoptic areas, which are sexually dimorphic (Almey et al. 2015). Beyond dopaminergic regulation, sex hormones also interact with metabolic signaling pathways. Estradiol enhances hypothalamic leptin and insulin sensitivity by promoting Ob-R and insulin receptor signaling in POMC neurons, thereby facilitating satiety and stress resilience (Bendis et al. 2024). These mechanisms may underlie the observed sex-specific vulnerability of D1R and D2R to early metabolic disruption and psychosocial stress in our model.
The neuroendocrine and neurotransmitter changes reported during late adolescence, including around PND 60, warrant caution when interpreting findings in light of adult literature. Evidence suggests that differences in behavior and physiological responses between juvenile and adult animals may be influenced by ongoing maturation processes, as the balance between excitation and inhibition mediated by neurotransmitter systems continues to be refined during this period (Kilb 2012; Kang et al. 2018). Therefore, comparisons with adult literature should be interpreted with caution, as age-dependent differences in neuroendocrine and neurotransmitter signaling may influence both behavioral and molecular outcomes.
Some methodological considerations should be taken into account when interpreting the present findings. The absence of a true neonatal control group represents an important constraint, as neonatal injections and handling alone are known to influence later behavior; therefore, the possibility that these procedures contributed to the observed outcomes cannot be excluded. Future studies will incorporate non-injected neonatal controls to better isolate the specific effects of MSG exposure. Similarly, food intake was measured every three days in group-housed animals, which provides reliable cumulative intake values but limits temporal resolution and may mask short-term fluctuations in feeding behavior. Additionally, the inclusion of complementary behavioral paradigms, such as depression-related tests, could have provided a broader characterization of emotional outcomes. From a technical standpoint, Ob-R, GHS-R1, D1R, and D2R are low-abundance, membrane-associated receptors that are challenging to detect reliably in total hypothalamic lysates using standard RIPA extraction and denaturing conditions. Although normalization to β-actin may not fully correct for variability in membrane protein recovery, Ponceau staining confirmed homogeneous protein loading across samples. Accordingly, the Western blot results should be interpreted as relative comparisons among groups rather than as absolute quantifications. Taken together, the molecular data suggest that the observed effects likely reflect functional modulation of hypothalamic signaling rather than structural changes to the established ARC lesion, a hypothesis that should be addressed in future histological and circuit-level studies.
Another limitation is that molecular analyses were performed on the total hypothalamus, without discrimination among specific nuclei such as the ARC, PVN, and lateral hypothalamus, which play distinct roles in the regulation of feeding and emotional behavior and display different expression profiles of Ob-R, GHS-R1, D1R and D2R (Gao and Sun 2015). A further limitation is the absence of direct measurements of hormonal and neurotransmitter concentrations at PND 60. Given that this developmental stage marks a transition from puberty to full adulthood, age-related fluctuations in metabolic hormones and neurotransmitter systems may contribute to the observed behavioral and molecular outcomes. Therefore, comparisons with fully adult literature should be interpreted with caution. Future studies incorporating direct hormonal and neurotransmitter measurements at this age will be essential for refining mechanistic interpretations. The lack of HPA-axis measurements, including corticosterone or ACTH levels, further restricts interpretation of the stress-related findings.
Previous studies have shown that neonatal MSG exposure can alter estrous cycle dynamics in female rodents (Mondal et al. 2018). However, as the present study focused primarily on anxiety-like and feeding behaviors, reproductive status or fertility markers were not evaluated. Furthermore, the phases of the estrous cycle were not controlled during behavioral and molecular assessments in females, which may have introduced variability given the modulatory role of sex steroids on hypothalamic circuits and stress responses (Ter Horst et al. 2012; Oyola and Handa 2017). Finally, although the social-SPS model captures relevant aspects of psychosocial adversity, it represents only one dimension of stress exposure, and caution is warranted when generalizing these findings to other forms of early-life or adult stress (McEwen and Akil 2020).
This study demonstrates that neonatal MSG exposure and adult social-SPS may affect feeding behavior and anxiety-like phenotypes in rats, possibly associated with alterations in hypothalamic membrane-linked proteins. Behavioral alterations were accompanied by modulations in the hypothalamic levels of receptors related to the regulation of energy and emotional homeostasis, such as Ob-R, GHS-R1, D1R, and D2R. Importantly, these molecular changes directly illustrate how metabolic and emotional signals converge within hypothalamic circuits, reinforcing the integrative role of these receptor systems in shaping both feeding behavior and stress-related responses.
Supplementary Information
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Supplementary Material 1
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- 1Kang S, Cox CL, Gulley JM (2018) High frequency stimulation-induced plasticity in the prelimbic cortex of rats emerges during adolescent development and is associated with an increase in dopamine receptor function. 10.31219/OSF.IO/54EB 310.1016/j.neuropharm.2018.08.037PMC 620743530165079 · doi ↗ · pubmed ↗