The intake of monosodium aspartate attenuates aggression induced by post-weaning social isolation in an ADHD rat model methods
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Methods
In brief, spontaneously Hypertensive Rats (SHR/Izm) (SLC Inc, Hamamatsu, Japan), recognized as a model for aggressive ADHD, were utilized in this study,. The rats were maintained under a 12-hour light/dark cycle, with lights on at 22:00 and off at 10:00, in a temperature-controlled environment set between 23-25 °C and an average humidity of 50 %. They were provided with chow (MFG; Oriental Yeast Co. Ltd.) and one of the following solutions ad libitum: distilled water (dH2O), 60 mM MSG (MP Biomedicals, USA, 101800), or 60 mM MSA (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) ( A). Fig. 1 The ingestion of MSA resulted in a reduction of PWSI-induced aggressive behavior, as demonstrated by the resident-intruder test. (A) This figure presents a schematic representation of the experimental protocol. The resident-intruder test was administered d...
Methods
Post-weaning social isolation (PWSI) was conducted on rats that were individually housed in cages measuring 40 × 23 × 18 cm, with minimal sawdust, from post-weaning at P25 until reaching maturity at P60, over a duration of five weeks, to induce stable aggression. Behavioral assessments were conducted during the dark phase, specifically between 13:00 and 16:00. In the resident-intruder paradigm, Wistar/ST rats were utilized as intruders and were maintained in enclosures with a 12-hour light/dark cycle, with lights activated at 8:00 and deactivated at 10:00. The intruders were provided with ad libitum access to food and water.
Behavioral tests
The Resident Intruder Test was conducted during the dark phase to evaluate aggression in SHR/Izm rats. A male Wistar/ST rat, approximately 20 g lighter than the resident, was introduced into the resident's home cage and designated as the intruder. The resident, an SHR/Izm rat, was observed for aggressive responses upon the introduction of the intruder. Behavioral recordings were performed under dim red light (<2 lx) between 13:00 and 16:00 using a video camera. The test was conducted over three consecutive days using the same resident-intruder pair to ensure consistency. Behavioral data from the second day were analyzed, as aggression levels were more stable at this time point. Aggressive behaviors were classified into three categories based on previously established criteria: anogenital sniffing (weak aggression), allo-grooming (moderate aggression), characterized by mou...
Open field test
The open field test was conducted during the dark phase prior to the resident intruder test (P60). This assessment aimed to evaluate anxiety-like behavior in a novel environment over a 10-min period. Rats were positioned at the center of a black circular arena (60 cm in diameter × 50 cm in height) during the dark phase, between 13:00 and 16:00, under red dim light (<2 lx). Anxiety-like behavior was assessed using three indicators: total distance traveled, number of entries into the center area, and duration spent in the center area over the 10-min period,. Behaviors recorded via a video camera were analyzed using the Smart software automatic tracking system (Bio Research Center Inc., Nagoya, Japan).
Immunohistochemistry of c-Fos
On the second day, the sections were rinsed in PBST containing 1 % horse serum and incubated for 120 min at room temperature (RT) with biotinylated anti-mouse IgG (Vector Laboratories, Inc., California, BA-2000, lot #ZB0622) diluted 1:200 in PBST with 1 % normal horse serum. Following PBS washes, the sections were incubated for 60 min at RT with an avidin-biotin complex (Vectastain ABC Kit, Vector Laboratories, Inc., USA, PK-4000, lot #ZK0713) diluted 9 µL/mL in PBST containing 1 % normal horse serum. Immunoreactivity was visualized on ice using 0.25 mg/mL diaminobenzidine (DAB) in PBS with 0.3 µL/mL of 30 % H₂O₂.The sections were subsequently mounted on gelatin-coated slides, dehydrated through a graded ethanol series (50-100 %), and sealed. The samples were examined using a light microscope, and...
Vagotomy
To elucidate the pathway through which MSA ingestion exerts its effects, a vagotomy was performed on male SHR/Izm rats at P24. The animals were anesthetized intraperitoneally with a mixed solution at a dosage of 2 mL/kg, comprising medetomidine (0.185 mg/mL; Fujita Pharmaceutical Co., Ltd., Tokyo, Japan), midazolam (1 mg/mL; Alfresa Holdings Co., Ltd., Tokyo, Japan), and butorphanol (1.25 mg/mL; Meiji Animal Health Co., Ltd., Tokyo, Japan). Following a median incision below the diaphragm, the liver was protected using saline-soaked gauze, and the anterior and posterior vagal trunks adjacent to the lower esophagus were severed. The organ was then repositioned appropriately, and the muscle layer and skin were sutured sequentially. Postoperatively, the rats received an intraperitoneal administration of atipamezole (0.235 mg/mL; Nippon Zenyaku Kogyo Co., Ltd...
Statistical analysis
Statistical analyses were executed using the EZR software, with a threshold for statistical significance set at p ≤ 0.05. The data are reported as mean ± standard error of the mean (SEM). In the resident intruder test, the frequency and duration metrics were examined using one-way ANOVA, followed by Tukey's post hoc test. Latency was assessed through non-parametric analysis, followed by the Steel test, due to the endpoint being established at 10 min. In the vagotomy experiment, aggression-related data, excluding latency, were subjected to one-way ANOVA, with Dunnett's post hoc test applied subsequently. Latency was analyzed independently using non-parametric methods, followed by the Steel test. The open field test and c-Fos immunohistochemistry (IHC) data were both analyzed using one-way ANOVA, followed by Tukey's post hoc test.
The reduction in aggression resulting from MSA ingestion is facilitated by the vagus nerve
To evaluate the role of the vagus nerve in mediating the aggression-suppressing effects of MSA ingestion, aggressive behaviors were compared between sham-operated (sham) and vagotomized (vagotomy) groups following a five-week period of individual housing and MSA ingestion ( A). Fig. 5 The role of the vagus nerve in mitigating aggression following MSA consumption. (A) Schematic illustration of the experimental protocol. (B-C) Vagotomy effectively counteracted the decrease in both the frequency and duration of aggressive grooming and attack behaviors that were induced by MSA ingestion, thereby restoring aggression levels to those comparable to the dH2O ingestion group. (D) The latency of attack was markedly reduced in the MSA + vagotomy group. (E) The expression of c-Fos in the iNTS was markedly diminished in the MSA + vagotomy group compared to the MSA + sham group. (...
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Evidence quotes (8)
In brief, spontaneously Hypertensive Rats (SHR/Izm) (SLC Inc, Hamamatsu, Japan), recognized as a model for aggressive ADHD, were utilized in this study,. The rats were maintained under a 12-hour light/dark cycle, with lights on at 22:00 and off at 10:00, in a temperature-controlled environment set between 23-25 °C and an average humidity of 50 %. They were provided with chow (MFG; Oriental Yeast Co. Ltd.) and one of the following solutions ad libitum: distilled water (dH2O), 60 mM MSG (MP Biomedicals, USA, 101800), or 60 mM MSA (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) ( A). Fig. 1 The ingestion of MSA resulted in a reduction of PWSI-induced aggressive behavior, as demonstrated by the resident-intruder test. (A) This figure presents a schematic representation of the experimental protocol. The resident-intruder test was administered during the subjective night following a five-week housing period. (B, C) MSA ingestion led to a decrease in both the frequency and duration of attack behavior. (D) No significant difference was observed in the latency of aggressive behavior between the MSA and control groups. Data are presented as th...
Post-weaning social isolation (PWSI) was conducted on rats that were individually housed in cages measuring 40 × 23 × 18 cm, with minimal sawdust, from post-weaning at P25 until reaching maturity at P60, over a duration of five weeks, to induce stable aggression. Behavioral assessments were conducted during the dark phase, specifically between 13:00 and 16:00. In the resident-intruder paradigm, Wistar/ST rats were utilized as intruders and were maintained in enclosures with a 12-hour light/dark cycle, with lights activated at 8:00 and deactivated at 10:00. The intruders were provided with ad libitum access to food and water.
The Resident Intruder Test was conducted during the dark phase to evaluate aggression in SHR/Izm rats. A male Wistar/ST rat, approximately 20 g lighter than the resident, was introduced into the resident's home cage and designated as the intruder. The resident, an SHR/Izm rat, was observed for aggressive responses upon the introduction of the intruder. Behavioral recordings were performed under dim red light (<2 lx) between 13:00 and 16:00 using a video camera. The test was conducted over three consecutive days using the same resident-intruder pair to ensure consistency. Behavioral data from the second day were analyzed, as aggression levels were more stable at this time point. Aggressive behaviors were classified into three categories based on previously established criteria: anogenital sniffing (weak aggression), allo-grooming (moderate aggression), characterized by mounting and pouncing, and attack (strong aggression), characterized by biting and keeping down. Each aggression category was analyzed for frequency, duration, and latency over a 10-minute observation period. Behavioral assessments were manually conducted using the Smart software (Bio Research Center...
The open field test was conducted during the dark phase prior to the resident intruder test (P60). This assessment aimed to evaluate anxiety-like behavior in a novel environment over a 10-min period. Rats were positioned at the center of a black circular arena (60 cm in diameter × 50 cm in height) during the dark phase, between 13:00 and 16:00, under red dim light (<2 lx). Anxiety-like behavior was assessed using three indicators: total distance traveled, number of entries into the center area, and duration spent in the center area over the 10-min period,. Behaviors recorded via a video camera were analyzed using the Smart software automatic tracking system (Bio Research Center Inc., Nagoya, Japan).
On the second day, the sections were rinsed in PBST containing 1 % horse serum and incubated for 120 min at room temperature (RT) with biotinylated anti-mouse IgG (Vector Laboratories, Inc., California, BA-2000, lot #ZB0622) diluted 1:200 in PBST with 1 % normal horse serum. Following PBS washes, the sections were incubated for 60 min at RT with an avidin-biotin complex (Vectastain ABC Kit, Vector Laboratories, Inc., USA, PK-4000, lot #ZK0713) diluted 9 µL/mL in PBST containing 1 % normal horse serum. Immunoreactivity was visualized on ice using 0.25 mg/mL diaminobenzidine (DAB) in PBS with 0.3 µL/mL of 30 % H₂O₂.The sections were subsequently mounted on gelatin-coated slides, dehydrated through a graded ethanol series (50-100 %), and sealed. The samples were examined using a light microscope, and images were captured in accordance with the brain atlas. c-Fos-positive cells (c-Fos+ cells) were manually quantified utilizing ImageJ software.
To elucidate the pathway through which MSA ingestion exerts its effects, a vagotomy was performed on male SHR/Izm rats at P24. The animals were anesthetized intraperitoneally with a mixed solution at a dosage of 2 mL/kg, comprising medetomidine (0.185 mg/mL; Fujita Pharmaceutical Co., Ltd., Tokyo, Japan), midazolam (1 mg/mL; Alfresa Holdings Co., Ltd., Tokyo, Japan), and butorphanol (1.25 mg/mL; Meiji Animal Health Co., Ltd., Tokyo, Japan). Following a median incision below the diaphragm, the liver was protected using saline-soaked gauze, and the anterior and posterior vagal trunks adjacent to the lower esophagus were severed. The organ was then repositioned appropriately, and the muscle layer and skin were sutured sequentially. Postoperatively, the rats received an intraperitoneal administration of atipamezole (0.235 mg/mL; Nippon Zenyaku Kogyo Co., Ltd., Fukushima, Japan) at a dosage of 1 mL/kg. The day following surgery, the rats were individually housed and provided with MSA via a drinking bottle for a duration of five weeks until the commencement of behavioral tests.
Statistical analyses were executed using the EZR software, with a threshold for statistical significance set at p ≤ 0.05. The data are reported as mean ± standard error of the mean (SEM). In the resident intruder test, the frequency and duration metrics were examined using one-way ANOVA, followed by Tukey's post hoc test. Latency was assessed through non-parametric analysis, followed by the Steel test, due to the endpoint being established at 10 min. In the vagotomy experiment, aggression-related data, excluding latency, were subjected to one-way ANOVA, with Dunnett's post hoc test applied subsequently. Latency was analyzed independently using non-parametric methods, followed by the Steel test. The open field test and c-Fos immunohistochemistry (IHC) data were both analyzed using one-way ANOVA, followed by Tukey's post hoc test.
To evaluate the role of the vagus nerve in mediating the aggression-suppressing effects of MSA ingestion, aggressive behaviors were compared between sham-operated (sham) and vagotomized (vagotomy) groups following a five-week period of individual housing and MSA ingestion ( A). Fig. 5 The role of the vagus nerve in mitigating aggression following MSA consumption. (A) Schematic illustration of the experimental protocol. (B-C) Vagotomy effectively counteracted the decrease in both the frequency and duration of aggressive grooming and attack behaviors that were induced by MSA ingestion, thereby restoring aggression levels to those comparable to the dH2O ingestion group. (D) The latency of attack was markedly reduced in the MSA + vagotomy group. (E) The expression of c-Fos in the iNTS was markedly diminished in the MSA + vagotomy group compared to the MSA + sham group. (F) the MSA-induced reduction in c-Fos-positive neurons were blocked by vagotomy, showing a significance increasing in MSA + vagotomy group compared with MSA + sham group. Data are expressed as mean ± SE (n = 9-11 for the behavioral experiment, n =...
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"headline": "The intake of monosodium aspartate attenuates aggression induced by post-weaning social isolation in an ADHD rat model",
"datePublished": "2026",
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