Negative energy balance hinders prosocial helping behavior

Abstract

The internal state of an animal, including homeostatic requirements, modulates its behavior. Negative energy balance stimulates hunger, thus promoting a range of actions aimed at obtaining food. While these survival actions are well established, the influence of the energy status on prosocial behavior remains unexplored. We developed a paradigm to assess helping behavior in which a free mouse was faced with a conspecific trapped in a restrainer. We measured the willingness of the free mouse to liberate the confined mouse under diverse metabolic conditions. Around 42% of ad libitum-fed mice exhibited a helping behavior, as evidenced by the reduction in the latencies to release the trapped cagemate. This behavior was independent of subsequent social contact reward and was associated with changes in corticosterone indicative of emotional contagion. This decision-making process was coupled with reduced blood glucose excursions and higher Adenosine triphosphate (ATP):Adenosine diphosphate (ADP) ratios in the forebrain of helper mice, suggesting that it was a highly energy-demanding process. Interestingly, chronic (food restriction and type 2 diabetes) and acute (chemogenetic activation of hunger-promoting AgRP neurons) situations mimicking organismal negative energy balance and enhanced appetite attenuated helping behavior toward a distressed conspecific. To investigate similar effects in humans, we estimated the influence of glycated hemoglobin (a surrogate of long-term glycemic control) on prosocial behavior (namely charity donation) using the Understanding Society dataset. Our results evidenced that organismal energy status markedly influences helping behavior and that hypothalamic AgRP neurons are at the interface of metabolism and prosocial behavior.

Keywords: AgRP neurons; energy status; helping behavior; hunger; hypothalamus.

Fig. 1.

Mice exhibit prosocial helping behavior toward a restrained conspecific. (A) Schematic view of the HBT consisting of three phases: housing in pairs (for 4 to 6 wk), habituation (4 consecutive days), and test. The image depicts the restraining apparatus, which is divided into two equivalent spaces where a dummy mouse and a cagemate were placed. Free mice were allowed to freely explore the arena, and these can become (a) helpers if they release the conspecific for 5 consecutive days or (b) nonhelpers if they do not. (B) Latency time to door opening when free mice were exposed to an empty restrainer (white circles) or simultaneously exposed to a trapped (blue circles) and dummy mouse (orange circles) (n = 6). Door-opening latencies are shown using the median as this variable was not normally distributed. Area under the curve (AUC) is shown as Inset. (C) Exploratory activity of nonhelper and helper mice during the HBT as measured by the number of entries into the dummy or trapped quadrants. Note that one mouse from the helper group was excluded from the study as it was identified as an outlier (using the Rout method). (D) Place preference of nonhelper and helper mice during the HBT as measured by the percentage of time spent in the dummy or trapped quadrants. (E) Social interaction time between nonhelper and helper mice with trapped mice during the HBT. Data from a second independent experiment are shown (n = 10; seven nonhelper and three helper mice). (F) Latency to door opening of helper mice when exposed to a trapped (blue circles) or dummy mouse (orange circles) in the modified HBT that prevented social contact upon the release of the conspecific (n = 5 to 8). Note the crossover experimental design. Door-opening latencies are shown using the median as this variable was not normally distributed. (G) Assessment of the anxiety-like state of helper mice by the elevated plus maze test. Time spent (%) in closed and open arms was measured after exposure to a dummy or trapped mouse. These are the same mice shown in B. (H) Plasma corticosterone increase (difference between before and after the HBT on day 5 of test) of free mice belonging to nonhelper and helper dyads. Data from a third independent experiment are shown (n = 5 to 9). (I) Correlation of plasma corticosterone increase between free and trapped mice. Data expressed as mean ± SEM or otherwise stated. Dots represent individual sample data. Statistical analysis was performed by two-way ANOVA with the Bonferroni multiple comparisons test for (B–D, F, and G), Wilcoxon test for (B Inset), unpaired t test for (E and H), and Pearson correlation test for (I). ns: not significant. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 2.

Helping behavior is highly energy demanding. (A) Blood glucose increase (difference between before and after the last session of the HBT) of nonhelper and helper mice exposed to a trapped mouse. A pool of three independent experiments is shown. (B) Concentration of diverse metabolites in the forebrain of nonhelper and helper mice after the HBT. (C) ATP:ADP ratio in the forebrain, midbrain, and hindbrain of nonhelper and helper mice after the HBT. (D) Representative immunofluorescence images showing HK staining in the mediobasal hypothalamus of nonhelper and helper mice after the HBT. 3V: third ventricle. (E) Intensity quantification of HK staining in the mediobasal hypothalamus of nonhelper and helper mice after the HBT. Dots represent brain sections from 3 to 4 mice/group. (F) Area quantification of HK staining in the mediobasal hypothalamus of nonhelper and helper mice after the HBT. Data expressed as mean ± SEM. Dots represent individual sample data. Statistical analysis was performed by an unpaired t test. *P < 0.05.

Fig. 3.

Food restriction prevents helping behavior. (A) Schematic view of the HBT on fed and food-restricted (FR) mice. (B) Latency to door opening of free mice ad libitum fed (black circles) or FR (red circles) (n = 9 to 10/group). Door-opening latencies are shown using the median as this variable was not normally distributed. Note the crossover experimental design. Area under the curve (AUC) is shown as Inset. (C) Schematic view of the modified version of the HBT in which 1% sucrose was available. (D) Sucrose intake in nonhelper and helper mice during the HBT. (E) Sucrose intake after 5-h water deprivation in nonhelper and helper mice. (F) Correlation between time in quadrant and sucrose intake in nonhelper and helper mice during the HBT. Pearson correlation indexes and P values are shown for each experimental group. Linear regression slopes from both groups were significantly different (P = 0.021). (G) Schematic view of the modified version of the HBT in which the dummy mouse was replaced by a high palatable food pellet. (H and I) Latency time to door opening of the (H) food pellet or (I) trapped mouse for ad libitum–fed (black circles) and FR (red circles) mice (n = 11/group). Door-opening latencies are shown using the median as this variable was not normally distributed. Insets represent the area under the curve (AUC). Data expressed as mean ± SEM or otherwise stated. Dots represent individual sample data. Statistical analysis was performed by two-way ANOVA with the Bonferroni multiple comparisons test for (B, H, and I), unpaired t test for (D and E), Mann–Whitney U test for (B Inset, H Inset, and I Inset), and Pearson correlation test and linear regression for (F). ns: not significant. *P < 0.05, **P < 0.01, and ****P < 0.0001.

Fig. 4.

Helping behavior is associated with the activation of oxytocin neurons in the PVN. (A and B) Representative immunofluorescence images of FOS staining in diverse brain regions from nonhelper and helper mice (A) and quantification (B). Cingulate cortex (Cg), insular cortex (Ins), lateral septum (LS), paraventricular thalamus anterior nuclei (PVA), paraventricular thalamus (PV), paraventricular nucleus of the hypothalamus (PVN), and nucleus accumbens (NAc). Orientation planes are shown (D: dorsal; V: ventral; L: lateral; M: medial). (Scale bar, 50 µm). (C and D) Representative fluorescent in situ hybridization images of oxytocin and Fos in the PVN from nonhelper and helper mice (C) and quantification (D). 3V: third ventricle; D3V: dorsal third ventricle; LV: lateral ventricle; aca: anterior cerebral artery. Data expressed as mean ± SEM. Dots represent individual sample data. Statistical analysis was performed by an unpaired t test. *P < 0.05.

Fig. 5.

Chemogenetic activation of AgRP neurons hinders helping behavior. (A) Schematic view of the experimental design. (B) Latency to door opening of free AgRP^hM3Dq mice injected with either saline (n = 9) or chemogenetic ligand CNO (n = 6) (n = 15/group considering the crossover experimental design). Door-opening latencies are shown using the median as this variable was not normally distributed. Area under the curve (AUC) is shown as Inset. (C) Exploratory activity of AgRP^hM3Dq mice injected with either saline or chemogenetic ligand CNO during the HBT, as measured by the number of entries in the dummy or trapped quadrants. Data show a random subset of mice shown in (B). (D) Place preference of AgRP^hM3Dq mice injected with either saline or chemogenetic ligand CNO during the HBT, as measured by the percentage of time spent in the dummy or trapped quadrants. Data show the same subset of mice shown in (C). Data expressed as mean ± SEM or otherwise stated. Dots represent individual sample data. Statistical analysis was performed by two-way ANOVA with the Bonferroni multiple comparisons test for (B–D) or Mann–Whitney U test (B Inset). ns: not significant. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 6.

Declined helping behavior in pathological conditions associated with negative energy balance. (A) Schematic view of the HBT in STZ-induced diabetic mice. (B) Latency time to door opening of control (saline; n = 5) and STZ-diabetic mice (n = 5). Door-opening latencies are shown using the median as this variable was not normally distributed. Inset represents the area under the curve (AUC). Data expressed as mean ± SEM. (C) Derivative (dy/dx) of HbA1c concentrations in relation to the frequency of donations in the Understanding Society dataset. Data expressed as mean ± SD. Dots represent individual sample data. Statistical analysis was performed by two-way ANOVA with the Bonferroni multiple comparisons test for (B) and Mann–Whitney U test for (B Inset). *P < 0.05, **P < 0.01, and ***P < 0.001.