These authors contributed equally to this study as co-first authors.
Suicidality can be a serious feature of psychiatric symptoms in encephalitis. Investigating the psychiatric behavior associated with suicidality in animal models of encephalitis is important; thus, determining whether normal laboratory animals are aware of death is necessary.
To examine the behavioral and brain activity changes associated with death of conspecifics, laboratory mice were exposed to a cadaveric mouse or an anesthetized mouse. Behavioral tasks associated with anxiety and locomotion were conducted after repeated exposure. Neural activity in the medial prefrontal cortex during the cadaver exploration was investigated using electroencephalographic recordings.
During repeated exposure, mice in the cadaver group showed a gradual decrease in time exploring the cadaver, which was not observed in mice in the anesthesia group. The cadaver group also exhibited increased levels of anxiety in the light/dark transition and elevated plus maze tasks and displayed increased locomotor activity in the open field test. In an electrophysiological study, different brain oscillations were observed when mice were exposed to a cadaveric mouse and an anesthetized mouse. Enhanced delta-band activity and reduced theta- and alpha-band activities were observed during cadaver exploration.
The present study results showed that experiences involving dead conspecifics strongly affect mouse behavior and brain activity. These findings may be helpful in treating patients with psychiatric symptoms and aid in understanding the concept of death recognition/awareness in laboratory animals.
Encephalitis can often be accompanied by emotional and psychological problems, such as anxiety, depression, and mood swings. In antibody-mediated autoimmune encephalitis, such as anti-
Many nonhuman mammalian species show unique responses to dead conspecifics, such as remaining next to the deceased conspecific, vocalizations, grooming, licking, or carrying the body [
In the wild, animals may occasionally observe dying conspecifics or may encounter dead conspecifics due to events such as aging, disease, or enemy attack. This experience may be traumatic as the animals come to recognize or become aware of death. However, animals that live in a laboratory cannot have such observational experiences involving dead bodies of their conspecifics owing to animal welfare practices and the control of experiments in accordance with the guidelines of the Institutional Animal Care and Use Committee [
Mice are commonly used experimental animals. They are highly social and even display empathy-like behaviors toward each other [
Male C57BL/6 mice were used in the present study. Animals were maintained with free access to food and water under a 12-hour light/dark cycle. All experiments were approved by the Institutional Animal Care and Use Committee in Seoul National University Hospital (SNUH-IACUC, No. 14-0210-S1A1) and animals were maintained in the facility accredited AAALAC International (#001169) in accordance with Guide for the Care and Use of Laboratory Animals 8th edition (National Research Council 2011). All efforts were made to minimize suffering. The overall study consisted of two major experiments shown in
The experiment was performed as previously described [
For assessment of anxiety, elevated plus maze and light/dark transition tasks were performed as previously described [
For the light/dark transition task, a light/dark box (30 × 45 × 27 cm) made of plastic comprised of a dark compartment (one third of the total area) and light compartment with a hole in the middle. The light compartment was illuminated at 400 lux. The elapsed time to entry (all four paws) into the light compartment (latency) and the amount of time (duration) spent in each compartment were measured over a 5-minute period by video monitoring.
To assess locomotor activity, the open field test was performed as previously described [
Electroencephalographic (EEG) surgery and recording
All data are presented as means ± standard error of the mean. Analysis of variance (ANOVA) was used to conduct multiple comparisons of means. Student t-test was performed to determine statistical differences between two means. A p-value < 0.05 was considered statistically significant.
To determine how laboratory mice are affected by a dead conspecific, mice were first allowed to explore a dead conspecific (cadaver). The social investigative activity was assessed as duration of contact with the target (a cadaver or an anesthetized mouse). On the first day, the cadaver (exposed to a cadaveric conspecific) (n = 17; 147.88 ± 15.65 seconds over 5 minutes,
After the 3-day exposure experiment, the cadaver and anesthesia groups were subjected to elevated plus maze, light/dark transition, and open field tests to assess anxiety and locomotion. In the elevated plus maze task (
In the light/dark transition task (
To assess locomotor activity, an open field test was performed. Mice in the cadaver group traveled a greater distance (n = 15, 3,758.78 ± 106.85 cm) than mice in the control group (n = 17, 3,323.42 ± 95.95 cm, p < 0.01, Student t-test). However, mice in the anesthesia group (n = 13, 3,395.06 ± 90.06 cm) traveled a distance similar to mice in the control group.
Taken together, these results demonstrated that mice exposed to a cadaveric conspecific had increased levels of anxiety and locomotor activity compared with naïve control mice or mice exposed to an anesthetized conspecific.
The mPFC has an important role in social cognitive behavior, including novelty detection and fear recognition [
In the wild, animals may frequently encounter dead bodies and observe the death of their family members. These experiences, together with education from family or social groups, may help animals understand or become aware of death and may cause them to exhibit specific behavioral responses to the dead (or to react differently to dead bodies), such as remaining near or carrying the deceased conspecific, as well as vocalizations, grooming, or licking [
Death-related behavior in primates has been reported in several studies [
In the present study, the effects of a dead body on a laboratory mouse that has never previously encountered a dead conspecific and likely has not received any education regarding death from its family (parents or cagemates) were investigated. Exposure to a cadaveric conspecific rendered mice more anxious than exposure to an anesthetized conspecific. In the social investigation experiment, differences were not observed in contact time between the cadaver and anesthesia groups on the first day; however, differences were observed on the second and third days. If mice had an innate fear of dead bodies, mice exposed to a cadaveric conspecific should have exhibited reduced contact time compared with mice exposed to an anesthetized conspecific on the first day. The presence or smell of a predator provokes immediate innate fear in rodents such as rats and mice. Mice avoid the location of the predator, even when they have never been exposed to it and have no prior learning experience [
Although difference was not observed in the contact duration between the two groups on the first day, mice might have suspected a problem with the cadaver and acquired some information regarding the cadaver during the first investigation; they exhibited different neural activities in the mPFC when investigating a cadaveric conspecific or an anesthetized conspecific in the electrophysiological experiment. The mice may have learned to fear or experienced anxiety from repeated exposure to cadaveric conspecifics on the second and third days. However, the mice may simply have lost interest more quickly in a body that was dead compared with an alive but anesthetized body. In addition, whether the fear of a cadaveric conspecific in this study included fear of mortality could not be determined [
In the present study, several experiments were performed to determine how exposure to a cadaveric conspecific affects laboratory mice and what relevance it has. Notably, exposure to the cadaveric conspecific rendered laboratory mice fearful or anxious, and changed their brain activities. Encountering the dead body of a conspecific may be a traumatic experience for laboratory mice and they may acquire fear of a cadaver without experiencing an innate fear of death. Finally, the results indicated that laboratory mice can learn about death through fear or anxiety induced by exposure to a cadaveric conspecific. Although whether laboratory mice learned or became aware of death through investigation of a cadaver conspecific is unknown, experiments using animal cadaveric conspecifics may provide information and facilitate further studies of brain disorders such as suicidality and psychosocial behaviors.
Daejong Jeon has been an associate editor of
Conceptualization, Methodology: Jeon D, Lee SK, Chu K; Investigation, Data curation, Formal analysis: Jeon D, Kim S; Resources: Lee SK, Chu K; Funding acquisition: Jeon D, Lee SK, Chu K; Writing–original draft: Jeon D; Writing–review and editing: all authors.
We would like to thank Ah reum Yang (Laboratory for Neurotherapeutics, Department of Neurology, Biomedical Research Institute, Seoul National University Hospital, Seoul, Korea) for her help in the behavioral tests. This work was supported by Korea Health 21 R&D grants (HI12C0035) funded by Ministry of Health and Welfare and supported by a grant from Advanced Neural Technologies (0620182930).
(A) Simple schematic for the experiments: 1) Behavioral tasks after the social investigation of a cadaveric mouse target or an anesthetized mouse target for 3 days; 2) electroencephalographic (EEG) recordings during the social investigation. (B, C) In the 3-day social investigation of a cadaveric mouse target or an anesthetized mouse target, the contact duration was assessed for the first 5 minutes (B) and 10 minutes (C) on each day. The duration of contact in the cadaver group, but not in the anesthesia group, continued to decrease for 3 days, and the cadaver group showed reduced duration of contact compared with the anesthesia group.
EPM, elevated plus maze; LD, light/dark transition; OP, open field.
Comparison between the two groups: *p < 0.05, **p < 0.01; Student t-test. Comparison within each group: ##p < 0.01; one-way analysis of variance.
(A) Elevated plus maze task: the cadaver and anesthesia groups spent less and more time in the open and closed arms, respectively, than the control group. There was no difference between the cadaver and the anesthesia groups. Comparison among groups: ##p < 0.01; one-way analysis of variance. (B) Light/dark transition task: the cadaver group, but not the anesthesia group, showed a longer latency to enter the light compartment, spent less time in the light compartment, and spent more time in the dark compartment than the control group. There was no difference between the control and the anesthesia groups. Comparison with control group: *p < 0.05; Student t-test. (C) Open field test: the cadaver group, but not the anesthesia group, exhibited an increase in total distance traveled in the open field box compared with the control group. There was no difference between the control and anesthesia groups. Comparison with control or anesthesia group: **p < 0.01; Student t-test.
(A) Sample traces of electroencephalographic (EEG) signals from mouse medial prefrontal cortex neurons during investigations of the cadaveric and anesthetized mouse targets. (B, C) Power spectra analyses of two epochs of EEG signal: power spectra analyses revealed that mice showed increased delta-band activity and reduced theta- and alpha-band activities when they explored a cadaveric mouse target than when they explored an anesthetized mouse target.
Comparison between the two targets in each band: *p < 0.05, **p < 0.01; Student t-test.