Abstract
Background: Recent studies have identified empathy deficit as a core impairment and diagnostic criterion for people with autism spectrum disorders; however, the improvement of empathy focuses primarily on behavioural interventions without the target regulation. We sought to compare brain regions associated with empathy-like behaviours of fear and pain, and to explore the role of the oxytocin–oxytocin receptor system in fear empathy.
Methods: We used C57BL mice to establish 2 models of fear empathy and pain empathy. We employed immunofluorescence histochemical techniques to observe the expression of c-Fos throughout the entire brain and subsequently quantified the number of c-Fos–positive cells in different brain regions. Furthermore, we employed chemogenetic technology to selectively manipulate these neurons in Oxt-Cre−/+ mice to identify the role of oxytocin in this process.
Results: The regions activated by fear empathy were the anterior cingulate cortex, basolateral amygdala, nucleus accumbens, paraventricular nucleus (PVN), lateral habenula, and ventral and dorsal hippocampus. The regions activated by pain empathy were the anterior cingulate cortex, basolateral amygdala, nucleus accumbens, and lateral habenula. We found that increasing the activity of oxytocin neurons in the PVN region enhanced the response to fear empathy. This enhancement may be mediated through oxytocin receptors.
Limitations: This study included only male animals, which restricts the broader interpretation of the findings. Further investigations on circuit function need to be conducted.
Conclusion: The brain regions implicated in the regulation of fear and pain empathy exhibit distinctions; the activity of PVN neurons was positively correlated with empathic behaviour in mice. These findings highlight the role of the PVN oxytocin pathway in regulating fear empathy and suggest the importance of oxytocin signalling in mediating empathetic responses.
Introduction
Empathy refers to an individual’s perception and recognition of the internal emotions and feelings of others, including the ability to identify and respond to their voices, expressions, and physical sensations such as pain.1–4 It plays a crucial role in social interactions.5,6 Various components of the empathic response, which have been added layer after layer over the course of evolution, remain functionally integrated. At its core is the perception–action mechanism, which induces a similar emotional state in the observer as in the target; its most basic manifestations are imitation and emotional contagion, which are found in species with more primitive traits (e.g., rodents), as well as in humans and nonhuman primates. As the neocortex evolved, more complex patterns of empathy emerged, such as empathic concern and perspective-taking, both of which build on this core socioaffective basis while increasingly requiring emotion regulation, self–other distinction, and cognition.5,7–11 Higher-level modes of empathy depend on prefrontal functioning, but they remain fundamentally linked to the core perception–action mechanism. 2,12–15 Studies have indicated that observations of fearful emotions among others can elicit empathy-like behaviours of fear in rodents,16–20 while the nociception responses can also induce empathy-like behaviours of pain.21,22 Recent social psychological research has shown that people with autism spectrum disorder (ASD) exhibit core symptoms such as deficit in empathy, which is one of the diagnostic criteria for ASD.23–27 Furthermore, numerous studies have shown that improving empathy abilities is an important therapeutic approach for alleviating social deficits among patients with ASD.27–31 Currently, treatment approaches for improving empathy abilities primarily focus on behavioural training.32–34 Further study on neural circuits is needed to enhance understanding of this complex phenomenon.
Oxytocin is a neuropeptide composed of 9 amino acids. It is synthesized mainly within the paraventricular nucleus (PVN), supraoptic nucleus, and accessory nuclei of the hypothalamus. These regions send projections to the posterior pituitary gland, where oxytocin is secreted into the systemic circulation.35,36 Oxytocin exerts its functions by binding to its receptors.37 Oxytocin receptors are abundantly expressed in brain regions that play a role in regulating social interactions, the affective state, and adaptive behaviours, such as the amygdala, hypothalamus, and prefrontal cortex.38–42 The expression of oxytocin receptors varies across species and genders, 43,44 which may be related to behaviour patterns and emotional states.37,40,45 Furthermore, the expression level and distribution of oxytocin receptors in various brain regions are associated with different types of social behaviours.41,46–48 Studies have shown that oxytocin neurons in the PVN region are involved in the regulation of empathic behaviours.45,49 In addition, oxytocin receptors are distributed on some specific neurons in important brain regions related to social behaviour (e.g., anterior cingulate cortex [ACC], basolateral amygdala [BLA], nucleus accumbens [NAc], hippocampus).5,50–52
Despite advances in empathy research, further work is needed to explore how various forms of empathy rely on distinct brain regions and neural circuits involved in the sensory–emotional transformation process, the process by which an individual observes the behavioural state of another and elicits a behaviour that matches the emotional state.14,15,53 Thus, we sought to compare the similarities and differences in the brain regions associated with empathy-like behaviour of fear induced by electric shock and those associated with empathy-like behaviour of pain induced by complete Freund’s adjuvant. We also sought to investigate the role of the oxytocin–oxytocin receptor system in fear empathy. We explored these factors to gain a deeper understanding of the neural mechanisms underlying empathy-like behaviours of fear and pain, shedding light on the intricate nature of empathic responses.
Methods
Animals and experimental design
We purchased Oxt-Cre−/+ mice (stock no. 024234) from Jackson Laboratory in the United States and C57BL mice from the Experimental Animal Center of Lanzhou University. Animals were raised in a standardized animal room with a temperature of 22°C–25°C, a humidity of 45%–60%, and a bright–dark period of 12 hours per day. All mice were allowed to eat and drink freely, and no more than 4 mice were kept in a cage to ensure that they had sufficient space for movement. We performed the behavioural tests during the day to minimize stress stimulation to the mice.
We randomly divided 2-month-old male C57BL mice into experimental (i.e., bystander) and control groups. Within these groups, the demonstrator produced an emotional response to specific stimulation, while the observer received this response.
To manipulate the oyxtocin neurons in the PVN, we used the chemogenetic method with Oxt-Cre−/+ mice. First, we randomly assigned male Oxt-Cre−/+ mice (the observer mice for this set of experiments) into saline and clozapine N-oxide (CNO) groups. An equal number of C57BL (demonstrator mice) were also grouped into the CNO and saline groups. The demonstrator and observer were kept in the same cage for at least 3 weeks. Second, we targeted excitability designer receptors exclusively activated by designer drugs (DREADDs) to the PVN. For chemogenetic excitability of oxytocin neurons during empathy-like behaviour of fear, we bilaterally injected the observer with 300 nL of rAAV2-Ef1a-DIO-hM3Dq (Gq)-mCherry-WPREs-pa (no. PT-0042, Shumi Technology) in PVN. Injections were aimed at the following coordinates relative to bregma: anterior–posterior −0.80 mm, medial–lateral ± 0.03 mm, and dorsal–ventral −4.8 mm. Injected observer mice were allowed to recover for 2 weeks before returning to group housing with littermates. One week after group housing with littermates, we tested mice for behaviour. Before the formal experiments, we first injected a control virus (rAAV2-Ef1a-DIO-mCherry-WPREs-pa; no. PT-0013, Shumi Technology) into Oxt-Cre−/+ mice for a pre-experiment to confirm that the virus had no effect on the normal activities of the mice and that the control virus did not react with CNO. We injected CNO (2 mg/mL; no. 28537, MedChemExpress) intraperitoneally (2 mg/kg body weight) 30 minutes before the behavioural test.
The demonstrator and observer were reared in the same cage at a 1:1 ratio for at least 4 weeks before the behavioural test.
Fear empathy model
We performed the fear test using the FCT-100M instrument (Chengdu Tai-meng). The fear box is covered with an opaque soundproof external partition box (600 × 600 × 600 mm) to block out outside noise and light, thereby reducing system errors. The fear box (230 × 230 × 300 mm) is opaque around it, with stimulus grids at the bottom and an infrared camera installed on the top to record mouse behaviour. A customized transparent partition divides the fear box into the electric shock area and the insulation area, and allows odour and sound to communicate between the 2 zones. Three days before the start of the experiment, the mice freely explored the fear box to adapt to the environment. They also received touching and grasping to reduce their stress response. To simulate the human empathy process, observers in the bystander group received unpredicted and mild electrical stimulation on day 1 of the experiment (0.4 mA for 2 s, 1-min interval, 2 shock trials). Observers in the control group freely moved in the cage without any stimulation, as shown in Figure 1A.
Establishment and evaluation of fear empathy model in C57BL mice. (A) Schematic of unconditional–conditional stimulation in fear transmission experiment for the bystander (BY) and control (Con) groups. Yellow arrows indicate that sounds and odours can perfuse normally in both regions; red lightning bolts indicate electric shock. (B) Total freezing time of the BY and Con groups during fear conditioning (p = 0.02, F3,3 = 19.75) and retrieval phase (p = 0.0002, F4,4 = 2.08). In the BY group, freezing levels on day 2 and day 3 (conditioning v. retrieval phases) were not significantly different (p = 0.3, F3,4 = 3.41). (C) Comparison of the freezing duration between the fear conditioning and habituation (baseline) phases among observers in the BY group (p = 0.03, F3,4 = 14.71). (D) Freezing duration on day 2 in the BY and Con groups (BY group: min 6 v. min 1, p = 0.005, F3,3 = 5.57; min 7 v. min 1, p = 0.009, F3,3 = 4.06; min 8 v. min 1, p = 0.02, F3,3 = 4.42; min 9 v. min 1, p = 0.1, F3,3 = 3.65. (E) Freezing duration in the BY group on day 3 (retrieval v. baseline, p = 0.01, F7,32 = 12.74). *p < 0.05, **p < 0.01, ***p < 0.001. Note: ns = not significant, SEM = standard error of the mean.
On day 2 of the experiment, the demonstrator was placed in the shock zone while the observer was placed in the insulation zone. The mice were allowed to move freely and adapt to the environment in the box, which served as a habituation period. During this period, the activity status of the observer was recorded as a baseline response. After 5 minutes of habituation, the demonstrator in the bystander group received unpredicted foot shocks (0.7 mA for 2 s; 8-s interval). This process exposed the observer to the fear emotion of the demonstrator, known as the fear conditioning period (total 4 min). The freezing time during the habituation and fear conditioning phase was recorded as an evaluation index for the observer’s reception of fear emotional information, and also as a basis for judging whether the observer empathized with the demonstrator. For the control group, the observer was similarly admitted to the insulation zone, and the demonstrator was placed in the shock zone, but, unlike the bystander group, the demonstrator in the control group did not receive shock stimuli. Otherwise, all other conditions in the test were the same as in the bystander group.
On day 3 of the experiment, the observer of the bystander or control groups was placed back into the insulation zone in the same context for 5 minutes, and the demonstrator did not participate. At this stage, we assessed the contextual observational fear memory and the duration of the freezing response, which reflected the strength of empathy-like behaviour from day 2 and the retrieval level of empathic memory. Therefore, we called this stage the retrieval period (total 5 min). All behavioural chambers were cleaned with 75% alcohol between each test. This model is based on and modified from the method of Zhou Chun-ran.54
Pain empathy model
Three days before the pain experiment, the mice were adapted to touch and grasp to reduce their stress response. Observers in both the bystander and control groups were subjected to a 24-hour social isolation period. We performed tests immediately after the end of the isolation, as shown in Figure 2A. During the experiment, the demonstrator in the bystander group was injected with 10 μL of complete Freund’s adjuvant into the left hind paw to cause localized, stable, inflammatory pain; the same volume of saline was injected into the ipsilateral paw of the demonstrator in the control group. After the injection, we placed the observer and demonstrator of each group into a clean housing cage without food or water for a 1-hour social interaction, followed by mechanical threshold testing for the observer. The above model construction is based on and modified from the method of Monique L. Smith.21
Establishment and evaluation of the pain empathy model in C57BL mice. (A) Schematic of the pain empathy model for the bystander (BY) and control (Con) groups. (B) Mechanical threshold of observers in the BY and Con groups before and after social isolation (p = 1.0, F7,7 = 1.90). (C) Mechanical threshold among observers in the BY group compared with those in the Con group (p = 0.03, F3,3 = 3.44). (D) Thermal pain threshold among observers in the BY group compared with those in the Con group (p = 0.05, F4,3 = 3.39). *p < 0.05. Note: CFA = complete Freund’s adjuvant, ns = not significant, SEM = standard error of the mean.
Figure 3 shows the experimental flow chart of fear empathy and of pain empathy.
(A) The experimental flow chart of fear empathy. (B) The experimental flow chart of pain empathy.
Measurement of mechanical sensitivity threshold
We made sure the mice were in a comfortable position, standing on all 4 legs. All the mice were first tested with 0.16 g (handle no. 3.22) Von Frey plastic fibres (Exacta). The plastic fibres were slowly and gently stimulated on the plantar surface of the left hind paw of the observer mice. We observed responses (withdrawal, shaking, or licking the paw). If there was a response, we tested lighter pressure filaments (0.07 g, handle no. 2.83), and if there was no response, we tested the next level of pressure filament (0.4 g, handle no. 3.61). We recorded the reaction pattern and the final fibre pressure. We calculated the result using the following formula: 50% g threshold = (10 [Xf + kδ])/10 000, where Xf is the value of the final filament (in log units), k is the tabular value, and δ is the mean difference between stimuli (in log units).
Measurement of thermal pain threshold
We measured the thermal pain response of mice using a hot plate apparatus. Before starting the experiment, the hot plate was adjusted to 25°C. We then placed mice in the test area for 15 minutes to acclimate.
After the acclimatization, the mice were immediately placed back in the cage. Subsequently, the temperature of the hot plate was adjusted to 55°C for testing. We exposed mice to the 55°C hot plate and removed them as soon as the injury reaction occurred (e.g., licking, flicking the back paw and jumping); the whole process takes no longer than 3 minutes. We recorded the latency as the thermal threshold of the mouse.
Immunofluorescence histochemical assay
Thirty minutes after the end of the fear or pain empathy behaviour test, we anesthetized observers (including Oxt-Cre−/+ mice) with 0.3% pentobarbital and transcardially perfused them with 4% paraformaldehyde. We performed whole-brain isolation, followed by consecutive coronal cryosectioning (Thermo Fisher Scientific) at 25-μm thickness. In accordance with the Allen Brain Atlas, we selected the following brain slices: ACC, bregma 0.74 to 0.86 mm; dorsal hippocampus, bregma −1.70 to −1.82 mm; PVN, bregma −0.82 to −0.94 mm; BLA, bregma −1.34 to −1.46 mm; LHb, bregma −1.70 to −1.82 mm; ventral hippocampus, bregma −3.08 to −3.16 mm; NAc, bregma −0.74 to −0.86 mm. We selected the brain sections according to the nucleus location. We rinsed them 3 times with 0.01 M phosphate-buffered saline (pH 7.2) at 130 rpm and then fixed them for 20 minutes in 4% paraformaldehyde at room temperature. After again washing sections 3 times with 0.01 M phosphate-buffered saline (pH 7.2) at 130 rpm, we incubated them for 1 hour in blocking solution containing 0.2% Triton X-100 and goat serum at room temperature. We then incubated sections for 12 hours with primary antibodies (rabbit anti-c-Fos, 1:2000, no. ab214672; rabbit anti-oxytocin, 1:1000, no. ab212193; anti–oxytocin receptor, 1:800, no. ab217212; Abcam). Next, we incubated sections in secondary antibody (anti-rabbit Alexa Fluor 488 immunoglobulin [Ig] G, 1:200, no. A23220, Abbkine; Alexa Fluor 594 IgG, 1:200, no. S0008, Affinity Biosciences) for 1 hour. Sections were then washed and counter-stained for 20 minutes with 4′,6-diamidino-2-phenylindole (1:2000, no. C0060, Solarbio Life Science), followed by mounting with anti-fade reagents.
Cell count
Each brain slice had a thickness of 25 μm, and we typically selected 4–6 brain slices per mouse in each of the bystander, control, CNO, and saline groups. For larger nuclei such as the NAc, ACC, and dorsal and ventral hippocampus, we selected 6 brain slices to ensure that all regions within the nucleus could be properly stained and observed. For smaller nuclei such as the PVN, lateral habenula, and BLA, it was sufficient to select 4 slices, with each slice separated by 2 consecutive slices, resulting in a 50-μm gap between each region. We determined fluorescence signals of whole-brain sections by fluorescence microscope (Nikon, Eclipse, Ti2-E). We demarcated the nuclei border using Image J software.
Statistical analysis
All data were expressed as means with standard errors (SEMs). We used GraphPad Prism software, version 5.0 (GraphPad) for statistical analysis. For data of c-Fos–positive cells, we compared the bystander and control groups using the Student t test. If normality assumptions were not met, we instead used the Mann–Whitney U test. For fear behavioural data, we used the Student t-test for within- and between-group comparisons, and used 2-way repeated-measures analysis of variance with Holm–Sidak post hoc tests to compare freezing times. We considered p values equal or less than 0.05 as statistically significant.
Ethics approval
All animal experiments in this study were approved by the Laboratory Animal Ethics Committee of Lanzhou University (no. 62000800000370).
Results
Evaluation of the fear and pain empathy models
For each empathy model, we included 8 observers and 8 demonstrators in each of the bystander and control groups. During the fear conditioning phase, freezing time was significantly higher among observers in the bystander group than the control group after applying electric shocks to demonstrators (Figure 1B). Furthermore, this high level in the bystander group persisted after 24 hours during the retrieval phase (Figure 1B) and notably differed from observer baseline responses (Figure 1C). As shown by the dynamic analysis of the observer freezing responses after administering the electric shock to demonstrators, freezing time per minute was significantly higher by the fifth minute compared with the first minute, with the longest time observed during the sixth minute. As the test progressed, the freezing time gradually decreased and returned to baseline level by the ninth minute (Figure 1D). This decrease may be attributed to observers consistently being in a safe zone, leading to a desensitization response. To confirm that the empathic response of the observer was triggered exclusively by the fear response of the demonstrator, we added an additional set of experiments with observers who had not experienced any stimuli tested directly with the demonstrator. We found that, unlike observers who had experienced a mild electric shock stimulus in advance, observers in the bystander group who lacked this period did not have significant differences in their freezing responses compared with the control group, which confirmed the accuracy of the model we had developed (Appendix 1, available at www.jpn.ca/lookup/doi/10.1503/jpn.230125/tab-related-content).
We did not observe any changes in the observers’ pain thresholds after acute social isolation (Figure 2B), suggesting that brief social isolation did not affect the normal pain perception of the mice. After the social interaction, the mechanical threshold of observers in the bystander group was significantly lower than the control group (Figure 2C), which also aligned with the changes in the thermal threshold test (Figure 2D). These results indicate that observers exhibited the same pain response as demonstrators, suggesting empathy-like behaviours of pain.
Brain regions involved in empathy-like behaviours of fear
To evaluate the brain regions that might be activated during empathy-like behaviours of fear or pain, we used immunofluorescence technology to investigate c-Fos–positive neurons in the whole brain. Based on the staining results, we counted the number of positive cells in the ACC, BLA, NAc, PVN, ventral hippocampus, dorsal hippocampus, and lateral habenula (Figure 4A–O). For the empathy-like behaviours of fear, the results showed a large population of c-Fos–positive cells in the ACC and BLA (Figure 4B and 4D), and higher numbers in the bystander group compared with the control group (Figure 4C and 4E). Compared with the control group, there was a significantly higher number of positive cells in the NAc (Figure 4F and 4G). In the dorsal hippocampus, the c-Fos–positive cells were primarily labelled in the granule layer of the dentate gyrus (Figure 4J). In the ventral hippocampus, they were predominantly distributed in the CA1 and CA3 regions (Figure 4L). Although the lateral habenula had the lowest number of c-Fos–positive cells of the brain regions we explored (Figure 4N), there was still a significant difference between the bystander and control groups (Figure 4O). Of the 7 brain regions, c-Fos expression was most pronounced in the PVN (Figure 4H), with a significant difference in the number of positive cells between the 2 groups (Figure 4I).
Brain regions involved in the empathy-like behaviours of fear (FE) in C57BL mice. (A) Distribution diagram of sagittal plane in each brain area. (B) Representative images of brain staining and (C) c-Fos–positive cell counts in the anterior cingulate cortex (ACC) (p = 0.0008, F3,2 = 1.71. (D) Representative images of brain staining and (E) c-Fos–positive cell counts in the basal lateral amygdala (BLA) (p = 0.009, F6,4 = 57.76). (F) Representative images of brain staining and (G) c-Fos–positive cell counts in the nucleus accumbens (NAc) (p = 0.002, F4,3 = 20.67). (H) Representative images of brain staining and (I) c-Fos–positive cell counts in the paraventricular nucleus (PVN) (p = 0.01, F3,2 = 17.40). (J) Representative images of brain staining and (K) c-Fos–positive cell counts in the dorsal hippocampus (dHPC) (p = 0.002, F3,2 = 7.62). (L) Representative images of brain staining and (M) c-Fos–positive cell counts in the ventral hippocampus (vHPC) (p = 0.002, F7,4 = 16.91). (N) Representative images of brain staining and (O) c-Fos–positive cell counts in the lateral habenula (LHb) (p = 0.01, F5,3 = 3.71). For brain images, the range of the target nucleus is within the white lines and the enlarged representative neurons are inside the white boxes. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Note: aca = anterior part of anterior commissure, BY = bystander, CeC = capsular part of central amygdaloid nucleus, CeL = lateral division of central amygdaloid nucleus, Con = control, GrDG = granular layer of the dentate gyrus, LaDL = dorsolateral part of lateral amygdaloid nucleus, LaVL = ventrolateral part of lateral amygdaloid nucleus, PoDG = polymorph layer of the dentate gyrus, Py = pyramidal cell layer of the hippocampus, SEM = standard error of the mean.
Brain regions involved in empathy-like behaviours of pain
The 7 regions selectively responded to empathy-like behaviours of pain (Figure 5A–O). A subset of neurons in the PVN, dorsal hippocampus, and ventral hippocampus were activated by empathy-like behaviours of pain (Figure 5H, 5J, and 5L), but without a significant difference in the number of c-Fos–positive cells between the bystander and control groups (Figure 5I, 5K, and 5M). This suggests that these regions did not respond significantly to empathy-like behaviours of pain. In the BLA and lateral habenula, the distributions of the c-Fos–positive cells were similar to that empathy-like behaviours of fear (Figure 5D and 5N), with a higher number of positive cells observed in the bystander group than the control group (Figure 5E and 5O). Expression of c-Fos showed the strongest difference between the bystander and control groups among all 7 brain regions in the NAc, although the absolute number of positive cells was relatively low (Figure 5F).
The brain regions involved in the empathy-like behaviours of pain (PE) in C57BL mice. (A) Distribution diagram of sagittal plane in each brain area. (B) Representative images of brain staining and (C) c-Fos–positive cell counts in the anterior cingulate cortex (ACC) (p = 0.009, F2,2 = 14.65). (D) Representative images of brain staining and (E) c-Fos–positive cell counts in the basal lateral amygdala (BLA) (p = 0.0007, F5,3 = 7.73). (F) Representative images of brain staining and (G) c-Fos–positive cell counts in the nucleus accumbens (NAc) (p = 0.0001, F3,5 = 4.23). (H) Representative images of brain staining and (I) c-Fos–positive cell counts in the paraventricular nucleus (PVN) (p = 0.07, F2,2 = 2.14). (J) Representative images of brain staining and (K) c-Fos–positive cell counts in the dorsal hippocampus (dHPC) (p = 0.1612, F2,3 = 1.98). (L) Representative images of brain staining and (M) c-Fos–positive cell counts in the ventral hippocampus (vHPC) (p = 0.2, F3,4 = 1.53. (N) Representative images of brain staining and (O) c-Fos–positive cell counts in the lateral habenula (LHb) (p = 0.01, F3,4 = 1.09). For brain images, the range of the target nucleus is within the white lines and the enlarged representative neurons are inside the white boxes. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Note: aca = anterior part of anterior commissure, BY = bystander, CeC = capsular part of central amygdaloid nucleus, CeL = lateral division of central amygdaloid nucleus, Con = control, GrDG = granular layer of the dentate gyrus, LaDL = dorsolateral part of lateral amygdaloid nucleus, LaVL = ventrolateral part of lateral amygdaloid nucleus, PoDG = polymorph layer of the dentate gyrus, Py = pyramidal cell layer of the hippocampus, SEM = standard error of the mean.
Brain regions involved in 2 types of empathy-like behaviours
In both types of empathy-like behaviours, the ACC and lateral habenula showed activation of a certain number of neurons (Figure 4B, 4N, 5B, and 5N). The ACC demonstrated a higher level of involvement in empathy-like behaviours of fear (Figure 4C), while the lateral habenula had a greater impact on pain empathy-like behaviours (Figure 5O). The NAc and BLA regions responded to both types of empathy-like behaviours, with no difference in their involvement. In addition, some neurons in the BLA, the lateral division of central amygdaloid nucleus, and other subregions of the amygdala were found to respond in both types of empathy-like behaviours (Figure 4D and 5D). Apart from that, the PVN, dorsal hippocampus, and ventral hippocampus were significantly activated in empathy-like behaviours of fear, but not pain (Figure 4H, 4J, and 4L). After comparing the brain regions activated by the 2 types of empathy-like behaviours, we found that the PVN was more responsive to empathy-like behaviours of fear and, in subsequent experiments, we found that specific activation of oxytocin neurons in the PVN region positively modulated the empathic response in mice (Figure 6).
The brain regions of empathy-like behaviours of fear and pain are different in C57BL mice. ACC = anterior cingulate cortex, BLA = basal lateral amygdala, CeC = capsular part of central amygdaloid nucleus, CeL = lateral division of central amygdaloid nucleus, CNO = clozapine N-oxide, dHPC = dorsal hippocampus, LaVL = ventrolateral part of lateral amygdaloid nucleus, LHb = lateral habenula, NAc = nucleus accumbens, OT = oxytocin, OTR = oxytocin receptor, PVN = paraventricular nucleus, vHPN = ventral hippocampus.
Oxytocin neurons in the PVN and empathy-like behaviours of fear
Based on our findings, we confirmed that PVN primarily responded to empathy-like behaviours of fear (Figure 4H). Different types of peptide neurons exist in the PVN, and we hypothesized that oxytocin neurons are more involved in empathy-like behaviours of fear. We used the mice from the fear empathy model for further investigation. Through immunofluorescence imaging, we observed the colocalization of oxytocin-positive cells and c-Fos–positive cells within the PVN (Figure 7A). Furthermore, we found that the total number of colabelled cells accounted for 46.3% of all oxytocin-positive cells and 32.3% of the total c-Fos–positive cells (Figure 7B and 7C). These results suggest that approximately 46.3% of oxytocin neurons in the PVN were activated during empathy-like behaviours of fear, meaning that around half of oxytocin neurons were associated with empathy-like behaviours of fear, which requires further research to be sure. Oxytocin is known to play a crucial role in regulating prosocial behaviour, thus we aimed to investigate whether oxytocin neurons directly regulate the fear empathy-like behaviour.
Activation of oxytocin (OT) neurons after empathy-like behaviours of fear in C57BL mice. (A) Representative images of OT (green) and c-Fos (red) expression in the paraventricular nucleus, detected by immunofluorescence, enlarged in the white box in the upper right corner. White arrowheads indicate representative neurons. (B) The ratio of neurons positive for OT to those positive for c-Fos of total c-Fos–positive neurons; the total number of c-Fos–positive neurons was 1210 and the number of neurons positive for both oxytocin and c-Fos was 391. (C) The ratio of OT-positive to c-Fos–positive neurons of total OT-positive neurons; the total number of OT-positive neurons was 844 and the number of neurons positive for both OT and c-Fos was 391.
To selectively manipulate oxytocin neurons, we used Oxt-Cre−/+ mice, which were specifically injected with Cre-dependent virus (rAAV2-Ef1a-DIO-hM3Dq(Gq)-mCherry-WPREs-pa) into the PVN so that oxytocin neurons expressed the segment of the gene carried by the drug, activated by exogenous CNO (Figure 8A). In the PVN region, nearly all neurons positive for mCherry protein were also positive for oxytocin (Figure 8C), indicating that the virus was specifically expressed in oxytocin neurons. These cells were also labelled with c-Fos (Figure 8D), demonstrating that CNO enhanced the activity of neurons expressing the hM3Dq receptor. These results confirmed the effectiveness of the CNO–hM3Dq system.
Construction of the clozapine N-oxide (CNO) hM3Dq system in oxytocin (OT) neurons of the Oxt-Cre−/+ mice, using chemogenetic technology. (A) Experimental schedules. (B) Diagram of the mechanism of the chemogenetic system. (C) Neurons positive for mCherry, co-targeted with those positive for OT or (D) those positive for c-Fos, enlarged in the white box. White arrows indicate representative neurons. AP = anterior–posterior, DV = dorsal–ventral, ML = medial–lateral.
The behavioural results revealed that the CNO-injected mice had significant enhancement of the freezing response compared with saline-treated mice on the day 2 conditioning phase (Figure 9A). In comparison with baseline levels, the increase in freezing time was more pronounced in the CNO group than in the saline group (Figure 9B), suggesting that observers in the CNO group were more sensitive to fear emotion. Time-dynamic analysis showed that both groups of observers exhibited a significant increase in freezing time during the fear conditioning period (Figure 9C); as the test progressed, the freezing time of the saline group gradually decreased, while the CNO group maintained a relatively high level (Figure 9D), indicating that upregulation of oxytocin neurons facilitated the ability to empathize with fear. On day 3, drug injections were discontinued, the freezing time of the CNO group remained at the same level as during the fear conditioning period, and the freezing level of the 2 groups in the retrieval phase was higher than their own baseline levels (Figure 9D), indicating that the extent of fear memory retrieval was not affected even without further activation of oxytocin.
Effects of activation of oxytocin neurons in the paraventricular nucleus region of Oxt-Cre−/+ mice on empathy-like behaviours of fear using clozapine N-oxide (CNO). (A) Total freezing time of the CNO and saline groups during the fear conditioning (p = 0.0001, F3,3 = 2.48) and retrieval phases (p = 0.0001, F4,4 = 1.90). In the CNO group, freezing time was not significantly different between the conditioning and retrieval periods (p = 0.06, F3,4 = 3.80). (B) Comparison of the freezing time between the fear conditioning and baseline periods among observers in the CNO (p = 0.0001, F3,4 = 1.32) and saline groups (p = 0.003, F4,3 = 4.66). (C) Freezing time on day 2 in the CNO (min 6 v. min 1, p = 0.01, F3,3 = 6.02; min 7 v. min 1, p = 0.0001, F3,3 = 1.04; min 8 v. min 1, p = 0.001, F3,3 = 1.41; min 9 v. min 1, p = 0.004, F3,3 = 3.72) and saline groups (min 6 v. min 1, p = 0.03, F3,3 = 13.56; min 7 v. min 1, p = 0.1, F3,3 = 16.13; min 8 v. min 1, p = 0.05, F3,3 = 11.29; min 9 v. min 1, p = 0.05, F3,3 = 7.77). (D) Comparison of freezing time during the retrieval (day 3) and baseline habituation periods in the CNO (p = 0.0002, F7,32 = 7.82) and saline groups (p = 0.002, F7,32 = 4.92). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Note: ns = not significant, SEM = standard error of the mean.
Oxytocin receptor and empathy-like behaviours of fear
To determine the specific modulation of fear empathy by oxytocin, we performed immunofluorescence colocalization staining for oxytocin receptor, as well as c-Fos, in brain regions activated by empathy-like behaviours of fear. We detected oxytocin receptor in the ACC, BLA, dorsal hippocampus, NAc, and PVN (Appendix 2, available at www.jpn.ca/lookup/doi/10.1503/jpn.230125/tab-related-content). A portion of neurons activated by empathy-like behaviour of fear (c-Fos–positive cells) in these regions were labelled with oxytocin receptor (Figure 10A, 10C, 10E, 10G, and 10I). After counting cells positive for both oxytocin receptor and c-Fos in these 5 regions, almost all neurons in the PVN region activated in empathy-like behaviours of fear were oxytocin receptor–positive neurons. Colabelled cells accounted for 70.4% of c-Fos–positive cells in the NAc and 89.7% in the dorsal hippocampus, while the data for the ACC and BLA were 81.4% and 90.3%, respectively. We hypothesize that these 4 brain regions may be differentially modulated by oxytocin neurons during fear empathy in the PVN (Figure 10B, 10D, 10F, 10H, and 10J).
Activation of oxytocin receptor (OTR) after empathy-like behaviours of fear in C57BL mice. (A) Representative images of OTR and c-Fos expression and (B) proportion of c-Fos and OTR positivity in the paraventricular nucleus (PVN). (C) Representative images of OTR and c-Fos expression and (D) proportion of c-Fos and OTR positivity in the nucleus accumbens (NAc). (E) Representative images of OTR and c-Fos expression and (F) proportion of c-Fos and OTR positivity in the dorsal hippocampus (dHPC). (G) Representative images of OTR and c-Fos expression and (H) proportion of c-Fos and OTR positivity in the anterior cingulate cortex (ACC). (I) Representative images of OTR and c-Fos expression and (J) proportion of c-Fos and OTR positivity in the basal lateral amygdala (BLA). Positivity detected by immunofluoresence, with OTR expression shown in green and c-Fos expression shown in red. White arrows indicate representative neurons. A total of 6 brain slices were selected for counting statistics for the NAc, ACC, and dHPC, while a total of 4 brain slices were selected for counting for the PVN and BLA. In the PVN, NAc, dHPC, BLA, and ACC, the total number of c-Fos–positive cells was 312, 27, 58, 31, and 177, respectively. Note: GrDG = granular layer of the dentate gyrus, PoDG = polymorph layer of the dentate gyrus.
Discussion
Empathy can be categorized into cognitive empathy (consolation, perspective-taking, and targeted helping) and affective empathy (motor mimicry and emotional contagion). The regulation of cognitive empathy requires a more developed cortex, and there is no strong evidence to prove that rodents can produce cognitive empathy. However, as the core of empathic behaviour, affective contagion exists widely among species.1 In rodents, affective empathy is primarily characterized by the perception–action mechanism.7,13,15,16,55 The demonstrator’s fear or pain response serves as an emotional stimulus for the observer, eliciting a similar emotional and behavioural reaction known as emotional empathy.2,5,11,56 In this study, we investigated the similarities and distinctions in the brain regions implicated in 2 forms of emotional empathy. Our findings showed that, when the demonstrator exhibited a fear response to electric shock, the observer showed an emotional response, specifically freezing behaviour, which differed significantly from the control group of mice that did not receive fear stimuli. These behavioural results indicate that empathy-like behaviour in rodents cannot be simply defined as a behavioural imitation. Furthermore, even in the absence of a demonstrator, the observer still exhibited a response when reexposed to the previously empathized context after 24 hours. Although the level of freezing response was not identical to the empathy-like period (conditioning stimulus period), it was significantly different from the control group of mice that did not display empathy-like behaviours. These findings suggest that the generation of empathy information and the retrieval of empathy memory were directly influenced by the empathy environment.
In addition, we further investigated the functional regions of the brain that responded to these 2 types of empathic behaviours and observed differences in their activation levels during empathy-like behaviours. It is important to note that there are significant mechanistic differences between the 2 types of empathy-like behaviours. The dorsal hippocampus and ventral hippocampus, responsible for memory and learning, demonstrated lower levels of activation in empathy-like behaviours of pain but exhibited relatively higher responses in empathy-like behaviours of fear. In both empathic behaviours, neurons in the PVN were highly activated, but we found no statistically significant difference in empathy-like behaviours of pain. These findings suggest that the PVN region is very important for empathy-like behaviours of fear, but has no role in the occurrence of empathy-like behaviours of pain in the current experiment. Previous studies have shown that the amygdala plays an important role in the generation and extinction of fear memories.57–61 Our study also found that the BLA exhibited highly active responses during empathy-like behaviours of fear. Other brain regions such as the ACC, NAc, and lateral habenula were found to be significantly activated to varying degrees in both types of empathy-like behaviours. We speculate that they are all related to fear empathy to varying degrees. Interestingly, there was no difference in the number of c-Fos–positive neurons in the NAc between the 2 types of empathy-like behaviours. However, the lateral habenula exhibited a higher response to empathy-like behaviours of pain, while the ACC showed a stronger exhibition in empathy-like behaviours of fear. However, given the different characteristics of fear and pain, they will show different nuclear responses under each empathic behaviour. Specifically, the PVN, ventral hippocampus, and dorsal hippocampus were specific brain regions that exhibited empathy-like behaviours of fear. The reason for this difference is that empathy-like behaviours of pain and fear not only share similarities but also have their own characteristics. Empathy-like behaviour of fear is characterized by short durations, whereas that of pain is often accompanied by longer durations. Empathy-like behaviour of fear is usually accompanied by stressful stimuli, and thus activation of the PVN region is evident, in which the amygdaloid–hippocampus circuit may play an important role.62 Empathy-like behaviour of pain is more physiologically manifested, aversion-like reflexes are more pronounced, and, thus, the amygdala–cingulate circuit plays an important role.63–65 In particular, the transmission of pain emotion is relatively weak, compared with the transmission of fear emotion, and requires more conditioning to activate the response.
Previous studies have demonstrated that oxytocin neurons directly regulate fear stimulation.66,67 In our study, we also observed activation of oxytocin neurons in empathy-like behaviours of fear, while their effect on empathy-like behaviours of pain was not significant. This may be attributed to the analgesic effects of oxytocin.39 Further research has indicated that enhancing the activity of oxytocin neurons leads to a corresponding increase in the empathy-like abilities of the observer. When investigating the relationship between oxytocin neurons and brain regions associated with fear empathy — such as the ACC, NAc, BLA, dorsal hippocampus, and PVN — we found that oxytocin receptors were expressed on neurons activated during empathy-like behaviours of fear. We hypothesize that oxytocin neurons may influence empathy-like behaviours of fear by modulating these brain regions to varying degrees. These results suggest that at least some neurons depended on the regulation of oxytocin neurons. However, in other nuclei, such as the lateral habenula and ventral hippocampus, although neurons expressed c-Fos, the neurons did not colabel oxytocin receptor, which indicates that these regions were indeed activated in fear empathy-like behaviours, but their activation did not depend on the regulation of oxytocin neurons. However, to further investigate whether this effect is mediated by oxytocin projections to other emotion-regulatory brain regions, it is necessary to examine if there is an increase in activity in these oxytocin-projecting regions. Previous studies have shown that oxytocin acts on the oxytocin receptor system,68 which is widely present in numerous brain regions. Studies have shown that fibres from oxytocin neurons can project to a wide range of brain regions and release oxytocin from axon terminals by local diffusion to influence activity in those brain regions, including the aforementioned brain regions involved in empathy-like behaviours.5,6
Empathy deficit is common in patients with ASD, so improving individual empathy ability could help improve the social deficit and alleviate related symptoms for these patients. However, traditional treatment programs are mostly to correct the behaviours of patients at the macro level. Peripheral injection or nasal inhalation of oxytocin is effective among some patients with ASD, and the mechanism may be related to the involvement of oxytocin in the regulation of empathic behaviours. The results of this study may provide new ideas for the treatment of ASD symptoms by intervening in the micro-circuits of empathic behaviours.
Limitations
We used male animals for all tests, which limits a broader interpretation of the findings. Given the small sample size, the statistical results of c-Fos–positive cells do not indicate whether the PVN region plays a role in pain empathy behaviours, which needs to be further verified by increasing the sample size in subsequent experiments and by manipulating the activity and behavioural tests of oxytocin neurons. In addition, although the positive effects of oxytocin neurons on empathy-like behaviours of fear have been explored using chemogenetic techniques, we did not conduct further complementary studies on the role of empathy-like behaviours of pain. The specific molecular mechanisms of these 2 empathy-like behaviours remain to be further investigated. One approach could be to investigate the projection circuitry of oxytocin neurons in the PVN region. By studying the projection pathways of oxytocin neurons, we could explore the specific activities in the targeted brain regions and elucidate the mechanisms following the empathy behaviour paradigms. In demonstrating the positive effect of oxytocin neurons on empathy-like behaviours of fear, the results of immunofluorescence staining of the oxytocin receptor should be replaced by reverse validation using oxytocin receptor–knockout mice as a control group to more rigorously illustrate the correspondence between the oxytocin receptor and c-Fos.
Conclusion
We found that the brain regions involved in empathy-like behaviours of fear and pain differed. The oxytocin neurons in the PVN positively regulated empathy-like behaviours, and upregulating the activity of oxytocin neurons could enhance empathy-like responses. The oxytocin neurons in the PVN region seem to regulate empathy-like behaviours by acting on the oxytocin receptor in the empathy-participating brain regions.
Footnotes
Competing interests: None declared.
Contributors: Yu-Hong Jing contributed to the conception and design of the work. All of the authors contributed to data acquisition, analysis, and interpretation. Lu Zhang drafted the manuscript. All of the authors revised it critically for important intellectual content, gave final approval of the version to be published, and agreed to be accountable for all aspects of the work.
Funding: This work is partly supported by National Natural Science Foundation of China (no. 81870949) and Natural Science Foundation of Gansu Province (no. 20JR10RA601 and no. 21JR7RA452).
- Received August 30, 2023.
- Revision received February 6, 2024.
- Revision received March 12, 2024.
- Accepted March 22, 2024.
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