Nat Neurosci︱Cao Peng's lab discovers the important function of pain in the struggle for survival among species

Written by ︱ Cao Peng editor in charge ︱ Wang Sizhen Natural selection, survival of the fittest
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Struggle for existence, as the main behavior of species to adapt to the environment, has played an important role in the long process of biological evolution[1]
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Faced with the threat of natural enemies in nature, humans and animals have evolved a series of relatively conservative inter-species survival struggle behaviors
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Fanselow et al.
, a pioneer in this research field, proposed a classical theoretical model "Predatory Imminence Continuum" [2], which is used to describe the correspondence between the survival struggle between species and the "prey-natural enemy" distance
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The model points out that when the prey is far away from the natural enemy, the prey preferentially adopts defensive strategies such as standing still and fleeing; once the prey confronts the natural enemy and has nowhere to escape, the prey has to take active frontal response measures and start defensive attacks mode
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Defensive attack is the prey's last line of defense against natural enemies, and the neural mechanism of this important survival-struggle behavior is a long-standing scientific question
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 On January 3, 2022, Cao Peng's research group from the Beijing Institute of Biological Sciences published a research paper titled "Mechanically evoked defensive attack is controlled by GABAergic neurons in the anterior hypothalamic nucleus" in the prestigious journal Nature Neuroscience
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The study found that noxious mechanical stimuli are the key stimulus for triggering instinctive defensive aggressive behavior in a desperate situation where there is no escape, while gamma-aminobutyric acid in the anterior hypothalamic nucleus (AHN) can Neurons (GABAergic neurons) are the key neural groups that encode noxious mechanical stimuli
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This study not only elucidates the neural mechanism of instinctual defense against aggressive behavior, but also reveals the important function of pain sensation elicited by noxious mechanical stimuli in the struggle for survival among species
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(Extended reading: For the latest research progress of Cao Peng's research group, please refer to the "Logical Neuroscience" report: Neuron︱Cao Peng's laboratory discovered the closed-loop neural mechanism of repetitive stereotyped behavior; eLife︱Single-cell sequencing and neural circuit analysis combined to reveal brain activation Molecular Genetic Encoding Mechanisms for Instinct Behavior of Attack/Defense)
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Nociceptive mechanical stimuli are key stimuli that trigger defensive-aggressive behavior in mice First, the authors conducted a series of behavioral experiments using C57BL/6 mice to explore the key stimulus for triggering defensive-aggressive behavior
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First, the authors applied snake odor to the simulated snake to provide olfactory information of natural enemies, or attached alligator clips to apply noxious mechanical stimuli
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The simulated snakes containing the olfactory information of natural enemies did not trigger the defensive aggressive behavior of the mice, but when the alligator clips applied continuous noxious mechanical stimulation to the tails of the mice, the mice attacked the simulated snakes by biting
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This behavior persisted in the dark, suggesting that vision may not be involved in triggering this behavior
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Second, the authors attached the alligator clips to non-threatening objects (such as plastic bottle caps, wooden blocks, etc.
) and found that defensive aggressive behavior was also triggered when the alligator clips "bite" the mouse tail
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These results suggest that the object to which the alligator clip is attached is not important, but the nociceptive mechanical stimulus provided by the alligator clip.

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Third, the authors used the Cre enzyme-dependent diphtheria toxin iDTR-DT system to kill Mrgprd+ neurons in the dorsal root ganglia by intraperitoneal injection of diphtheria toxin DTX, which also significantly attenuated defensive-aggressive behavior
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Because Mrgprd+ neurons in the dorsal root ganglia are a class of sensory neurons sensitive to noxious mechanical stimuli, this experimental result reaffirms that noxious mechanical stimuli can trigger defensive-aggressive behavior through Mrgprd+ neurons
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Fourth, the authors demonstrate that this mechanical stimulus-triggered defensive-aggressive behavior is gender-neutral
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Fifth, the authors adjusted the "escapeability" in the defensive situation by changing the weight of the simulated snake and the area of ​​the experimental environment, and found that the level of "escapeability" may also be a determinant of mechanical stimuli triggering defensive-aggressive behavior
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In conclusion, the above series of experimental results suggest that noxious mechanical stimuli may be the key stimulus for triggering defensive-aggressive behavior in situations where there is no escape (Fig.
1)
.

Figure 1 Noxious mechanical stimuli trigger defense-aggressive behavior in mice (Source: Xie et al.
, Nat Neurosci, 2022) vGAT+ AHN neurons are necessary for triggering defense-aggressive behavior in mice Next, the authors explored the center of defense-aggressive behavior mechanism
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Using fluorescence in situ hybridization (FISH), they found that the neuron type in the AHN brain region was mainly vGAT-positive (vGAT+) neurons
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To achieve inactivation of vGAT+ AHN neurons, the authors used a combination of the Cre-LoxP system and the inhibitory optogenetic tool GtACR1
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AAV-DIO-GtACR1-2A-EGFP was injected into the AHN brain region of vGAT-IRES-Cre mice and the optical fibers were embedded to induce the specific expression of GtACR1 protein in vGAT+ neurons in the AHN brain region
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The authors used a simulated snake attached to an alligator clip to "bite" the mouse tail, induced the mice to attack the simulated snake, and then administered continuous blue light to inhibit vGAT+ AHN neurons
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The results showed that the frequency and duration of challenge were significantly reduced in the experimental group compared with the control group, suggesting that vGAT+ AHN neurons are necessary for defensive-aggressive behavior elicited by noxious mechanical stimuli (Figure 2)
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Figure 2 vGAT+ AHN neurons are necessary to trigger defensive-aggressive behavior in mice (Source: Xie et al.
, Nat Neurosci, 2022) vGAT+ AHN neurons encode the intensity and location of mechanical stimuli.
To investigate the link between noxious mechanical stimulation, the authors used a calcium imaging fiber optic recording system, injected AAV-DIO-GCaMP7 into the AHN brain region of vGAT-IRES-Cre mice, and observed when vGAT+ AHN neurons received different stimuli in different scenarios.
calcium signal
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Free-moving mice showed a large increase in calcium signal after the tail was "bited" by alligator clips, and in other scenarios (such as free-motion or exploration), the calcium signal of vGAT+ AHN neurons was slightly increased in amplitude
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The authors also found that calcium signaling in vGAT+ AHN neurons also increased significantly when experimental mice were challenged by CD1 mice in a social setting
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These data suggest that vGAT+ AHN neurons preferentially respond to noxious mechanical stimulation
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 Next, the authors applied in vivo single-cell recording technology to deeply analyze the encoding mechanism of single vGAT+ AHN neurons for mechanical stimulation
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AAV-DIO-ChR2-mCherry was injected into the AHN brain region of vGAT-IRES-Cre mice.
After the virus was fully expressed, the head of the mouse was fixed, and a multi-channel electrode wrapped with an optical fiber was inserted vertically
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Using this "photoelectrode" technique, extracellular recordings from single vGAT+ AHN neurons can be achieved
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The authors systematically studied 15 vGAT+ AHN cells and found that they all preferred mechanical stimuli applied by alligator clips and were less responsive to snake odor stimuli
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The authors also tested experiments using Von Frey filaments and found that vGAT+ AHN neurons prefer noxious mechanical stimulation of the contralateral soma and can encode different mechanical stimulation intensities (Fig.
3)
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Figure 3 vGAT+ AHN neurons encode the intensity and location of mechanical stimulation (Source: Xie et al.
, Nat Neurosci, 2022) Monosynaptic afferents upstream of vGAT+ AHN neurons To understand the effect of vGAT+ AHN neurons on nociception Sources of information encoded by mechanical stimuli, the authors used Rabies Virus transsynaptic retrograde tracing to reconstruct the upstream projection network of vGAT+ AHN neurons
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They found that vGAT+ AHN neurons form monosynaptic connections with brain regions that process pain-related information, such as the LPB and PVT
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The authors employed chemogenetic methods to simultaneously inactivate both LPB and PVT groups of neurons and found that the defensive-aggressive behavior triggered by noxious mechanical stimuli was significantly reduced
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In addition, vGAT+ AHN neurons also receive the ventromedial and lateral phrenic nuclei of the hypothalamus, which are associated with aggressive behavior, the medial amygdala, which is associated with predator odor information, and the posterior hypothalamic nucleus, the dorsal area of ​​the anterior papillary nucleus.
, the ventral anterior motor nucleus and the subhippocampus,
etc.

The above retrograde tracer results have reconstructed the neural network related to defense attack with vGAT+ AHN as the core (Fig.
4)
.

Figure 4 Monosynaptic afferents upstream of vGAT+ AHN neurons (Source: Xie et al.
, Nat Neurosci, 2022) Photoactivation of vGAT+ AHN neurons can trigger defensive-aggressive behavior in mice Next, the authors used optogenetics means to further test whether vGAT+ AHN neurons are a sufficient condition for defensive aggressive behavior
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AAV-DIO-ChR2-mCherry was injected into the AHN brain region of vGAT-IRES-Cre mice and the fiber was embedded, and then a series of behavioral studies were performed
.

The authors found that light activation of vGAT+ AHN neurons effectively triggered defensive aggression against simulated and real snakes in mice, but not against social peers
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Not only that, light activation of AHN vGAT+ neurons also inhibited social aggressive behavior
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Therefore, vGAT+ AHN neurons are necessary and sufficient to trigger defensive aggressive behavior in mice (Fig.
5)
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Figure 5 Photoactivation of vGAT+ AHN neurons can trigger defense-aggressive behavior in mice (Source: Xie et al.
, Nat Neurosci, 2022) The vGAT+ AHN-vlPAG pathway is a necessary and sufficient condition for triggering defense-aggressive behavior in mice for the study of vGAT+ AHN The circuit mechanism of neurons regulating defense-aggression behavior.
The authors first used the SynaptoTag anterograde tracing method, injected AAV-DIO-SynaptoTag into the AHN of vGAT-IRES-Cre mice, and found that the downstream projections of vGAT+ AHN neurons, Including the preoptic medial area MPA, the lateral phrenic nucleus LS, the medial hypothalamus ventral area VMH, the prepapillary nucleus dorsal area PMD, the central aqueductal gray matter ventrolateral area vlPAG and so on
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Both vlPAG and LS are known to be involved in attack-related behaviors, so the authors conducted a comparative study of the vGAT+ AHN-vlPAG and vGAT+ AHN-LS pathways
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By activating vGAT+ AHN neuronal fiber terminals projecting to vlPAG and LS, it was found that activation of the vGAT+ AHN-vlPAG pathway could effectively trigger defensive-aggressive behavior in mice, but not the vGAT+ AHN-LS pathway
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The above experimental results demonstrate that the defensive-aggressive behavior of mice depends on the inhibitory synaptic connection between AHN and vlPAG
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Finally, the authors validated the vGAT+AHN-vlPAG pathway using a dual approach of optogenetic inhibition and chemogenetic inhibition
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The authors found that inhibiting the axonal terminals of vGAT+ AHN neurons projecting to vlPAG by the above method significantly blocked the generation of defensive-aggressive behavior
.

These results suggest that activation of the vGAT+ AHN-vlPAG pathway is a sufficient and necessary condition for triggering defensive aggressive behavior in mice (Fig.
6)
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Figure 6 The vGAT+ AHN-vlPAG pathway is a necessary and sufficient condition for triggering defensive aggressive behavior in mice (Source: Xie et al.
, Nat Neurosci, 2022) Conclusions and discussions, inspiration and prospects Neurons are key neurons that respond to noxious mechanical stimuli and trigger defensive-aggressive behavior in mammals
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This paper not only reveals the neural circuit mechanism of defense against aggressive behavior, but also may provide new inspiration for the pathogenesis and treatment strategies of mental diseases such as anxiety and post-traumatic stress disorder
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Based on the current research, single-cell sequencing analysis of AHN will help to further reveal the neural circuits and genetic coding mechanisms that regulate mammalian defense-aggression behavior
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In addition, Professor Cao Peng's experiment will continue to conduct in-depth research on the neural circuit mechanism of instinctive behavior and the pathogenesis and treatment of nervous system diseases
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At the same time, colleagues who are interested in scientific research in this area are also welcome to join the research team of Mr.
Cao Peng
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Link to the original text: https://doi.
org/10.
1038/s41593-021-00985-4 Xie Zhiyong (the first from the right in the back row), Gu Huating (the fifth from the left in the front row), Huang Meizhu (the second from the left in the front row), and researcher Cao Peng (the front row).
Fifth from the right) (Photo provided by: Cao Peng's laboratory) Xie Zhiyong, Gu Huating, and Huang Meizhu of the Cao Peng Laboratory of the Beijing Institute of Life Sciences are the co-first authors of the paper, and researcher Cao Peng is the corresponding author of the paper
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Other members of the laboratory (Cheng Xinyu, Shang Congping, Tao Ting, Li Dapeng) also made important contributions to this research
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Zhang Zhibin's laboratory of the Institute of Zoology, Chinese Academy of Sciences, Zhang Fan's laboratory of Hebei Medical University, Tang Zongxiang's laboratory of Nanjing University of Traditional Chinese Medicine, and Zhancheng's laboratory of Beijing Institute of Life Sciences also participated in this project and made important contributions
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The research paper was heavily funded by the Beijing Institute of Life Sciences and the National Natural Science Foundation of China
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Darwin, C.
On the Origin of Species, Chapter III: Struggle for existence.
71-90 (1859) 2.
Fanselow, MS & Lester, LS