Nat Neurosci│Cao Peng's laboratory discovered the important function of pain in the struggle for survival between species

Editor-in-Chief | Natural selection, survival of the fittest
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As the main behavior mode for species to adapt to the environment, the struggle for survival has played an important role in the long process of biological evolution
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Faced with the threat of natural enemies in nature, humans and animals have evolved a series of relatively conservative behaviors in the struggle for survival among species
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Fanselow, a pioneer in this research field, et al.
proposed a classic theoretical model "Predatory Imminence Continuum" [1], which is used to describe the correspondence between the survival struggle behaviors of species and the distance between "prey-natural enemies"
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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 confronted with the natural enemy and there is nowhere to escape, the prey has to take active frontal countermeasures and initiate a defensive attack.
Pattern
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Defensive attack (defensive attack) is the last line of defense for prey against natural enemies, and the neural mechanism of this important survival struggle behavior is a long unresolved scientific problem
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On January 3, 2022, Cao Peng's research group from the Beijing Institute of Life Sciences published an online research paper entitled Mechanically evoked defensive attack is controlled by GABAergic neurons in the anterior hypothalamic nucleus on Nature Neuroscience
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The study found that noxious mechanical stimulus is the key stimulus (key stimulus) that triggers instinctive defense against aggressive behavior when falling into a desperate situation where you cannot escape, while the γ-aminobutyric acid energy in the anterior hypothalamic nucleus (AHN) Neurons (GABAergic neurons) are the key nerve groups that encode noxious mechanical stimuli
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This research not only clarified the neural mechanism of instinctive defense against aggressive behavior, but also revealed the important function of pain caused by noxious mechanical stimuli in the struggle for survival between species
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Noxious mechanical stimulus is the key stimulus that triggers defensive and aggressive behavior in mice.
First, the author conducted a series of behavioral experiments using C57BL/6 mice to explore the key stimulus that triggers defensive and aggressive behavior
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First, the author smeared the scent of the snake on the simulated snake to provide the olfactory information of the natural enemy, or connected the alligator clip to impose noxious mechanical stimulation
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The simulated snake containing the olfactory information of natural enemies did not trigger the defensive and aggressive behavior of the mouse, and when the alligator clip applied continuous noxious mechanical stimulation to the tail of the mouse, the mouse launched a bite and aggressive behavior on the simulated snake
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This behavior still exists in the dark, implying that vision may not be involved in the triggering of this behavior
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Second, the author connects alligator clips to non-threatening objects (such as plastic bottle caps, wooden blocks, etc.
) and found that when the alligator clip "bites" the tail of the mouse, it can also trigger defensive aggression
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These results indicate that the object connected by the alligator clip is not important, but the noxious mechanical stimulation provided by the alligator clip is important
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Third, the author used the Cre enzyme-dependent diphtheria toxin iDTR-DT system to kill Mrgprd+ neurons in the dorsal root ganglion through intraperitoneal injection of diphtheria toxin DTX, which can also significantly weaken the defensive and aggressive behavior
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Because Mrgprd+ neurons in the dorsal root ganglia are sensory neurons that are sensitive to noxious mechanical stimuli, this experimental result once again proves that noxious mechanical stimuli can trigger defensive and aggressive behaviors through Mrgprd+ neurons
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Fourth, the author proves that there is no gender difference in defensive aggression triggered by this mechanical stimulus
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Fifth, the author adjusted the "escapeability" in the defense scenario 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 stimulus triggering defensive attack behavior
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In summary, the above series of experimental results indicate that noxious mechanical stimulus may be the key stimulus to trigger defensive aggression behavior when there is no escape
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vGAT+AHN neuron is a necessary condition for triggering the defensive behavior of mice.
Then, the author looks for the central mechanism of defensive behavior
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They used fluorescence in situ hybridization (FISH) and found that the types of neurons in the AHN brain area were mainly vGAT-positive neurons
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In order to achieve inactivation of vGAT+AHN neurons, the author combined 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 area of ​​vGAT-IRES-Cre mice and the optical fiber was embedded to induce the specific expression of GtACR1 protein in vGAT+ neurons in the AHN brain area
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The author used a simulated snake connected to an alligator clip to "bite" the tail of the mouse, induced the mouse to attack the simulated snake, and then gave continuous blue light to inhibit vGAT+ AHN neurons
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Compared with mice in the control group, the attack frequency and attack duration of the mice in the experimental group were significantly reduced, suggesting that vGAT+AHN neurons are necessary for defensive behavior induced by noxious mechanical stimulation
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vGAT+ AHN neurons encode the intensity and location of mechanical stimuli.
In order to reveal the connection between vGAT+ AHN neurons and noxious mechanical stimuli, the authors used a calcium imaging optical fiber recording system and injected them into the AHN brain area of ​​vGAT-IRES-Cre mice.
AAV-DIO-GCaMP7, observed the calcium signal of vGAT+ AHN neurons under different stimuli in different scenarios
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After freely moving mice were "bitten" by the alligator clip at the tail, the calcium signal increased significantly.
In other scenarios (such as free movement or exploration), the calcium signal of vGAT+ AHN neurons increased slightly
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The author also found that when the experimental mice were attacked by CD1 mice in a social setting, the calcium signal of vGAT+ AHN neurons also increased significantly
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These data indicate that vGAT+ AHN neurons preferentially respond to nociceptive mechanical stimuli
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Next, the author applied the in vivo single-cell recording technology to deeply analyze the coding mechanism of a single vGAT+AHN neuron to mechanical stimulation
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AAV-DIO-ChR2-mCherry was injected into the AHN brain area of ​​vGAT-IRES-Cre mice.
After the virus was fully expressed, the head of the mouse was fixed, and the multi-channel electrode that also wrapped the optical fiber was inserted vertically
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Using this "photoelectrode" technology, extracellular recording of a single vGAT+AHN neuron can be achieved
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The author conducted a systematic study on 15 vGAT+ AHN cells and found that they all prefer the mechanical stimulation exerted by the alligator clip, but have a weaker response to the scent stimulation of the snake
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The author also used Von Frey filaments for test experiments and found that vGAT+AHN neurons prefer nociceptive mechanical stimulation of the contralateral body and can encode different mechanical stimulation intensities
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Single synaptic neural afferent upstream of vGAT+ AHN neurons In order to understand the source of information that vGAT+ AHN neurons encode nociceptive mechanical stimuli, the author used the Rabies Virus trans-synaptic reverse tracing method, the upstream projection network of vGAT+ AHN neurons Rebuilt
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They found that vGAT+AHN neurons form a single synaptic connection with brain regions that process pain-related information, such as LPB and PVT
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The authors used chemical genetics to simultaneously inactivate two groups of neurons, LPB and PVT, and found that the defensive and aggressive behavior triggered by noxious mechanical stimuli was significantly reduced
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In addition, vGAT+ AHN neurons also receive the ventromedial hypothalamic nucleus and lateral diaphragmatic nucleus related to aggressive behavior, the medial amygdala related to predator odor information, and the posterior hypothalamic nucleus and the dorsal area of ​​the prepapillary nucleus , Ventral anterior motor nucleus, hippocampus and so on
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The above retrograde tracing results reconstructed the neural network related to defense attacks with vGAT+AHN as the core
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The light activation of vGAT+ AHN neurons can trigger defensive and aggressive behavior in mice.
Next, the author uses optogenetics to further test whether vGAT+ AHN neurons are sufficient conditions for defensive and aggressive behavior
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AAV-DIO-ChR2-mCherry was injected into the AHN brain area of ​​vGAT-IRES-Cre mice and the optical fiber was embedded, and then a series of behavioral studies were carried out
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The author found that light-activated vGAT+ AHN neurons can effectively trigger the defensive attacks of mice against simulated snakes and real snakes, but do not launch attacks on social companions
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Not only that, light-activated AHN vGAT+ neurons can also inhibit social aggressive behavior
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Therefore, vGAT+AHN neurons are necessary and sufficient to trigger defensive aggressive behavior in mice
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The vGAT+ AHN-vlPAG pathway is a necessary and sufficient condition to trigger the defensive behavior of mice.
To study the loop mechanism of vGAT+ AHN neurons regulating defensive behavior, the author first used the SynaptoTag anterograde tracing method in vGAT-IRES-Cre mice.
AAV-DIO-SynaptoTag was injected into the AHN, and the downstream projections of vGAT+ AHN neurons were found, including the anteromedial area MPA, the lateral diaphragmatic nucleus LS, the medial hypothalamic ventral area VMH, the dorsal area of ​​the papillary nucleus PMD, and the central guide The ventrolateral area of ​​gray matter around the water pipe vlPAG and so on
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It is known that vlPAG and LS are both involved in attack-related behaviors, so the author conducted a comparative study on the vGAT+AHN-vlPAG and vGAT+AHN-LS pathways
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By activating the vGAT+AHN neuron fiber ends projected to vlPAG and LS, it is found that the activation of vGAT+AHN-vlPAG pathway can effectively trigger the defensive and aggressive behavior of mice, but the vGAT+AHN-LS pathway cannot
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The above experimental results prove that the defensive and aggressive behavior of mice depends on the inhibitory synaptic connection between AHN and vlPAG
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Finally, the author used the dual means of optogenetic inhibition and chemical genetic inhibition to verify the vGAT+AHN-vlPAG pathway
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The authors found that inhibiting the axon terminals of vGAT+AHN neurons projected to vlPAG by the above methods can significantly block the defensive and aggressive behavior
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These results indicate that the activation of the vGAT+AHN-vlPAG pathway is a necessary and sufficient condition for triggering the defensive behavior of mice.

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Xie Zhiyong, Gu Huating, and Huang Meizhu of Cao Peng Laboratory of Beijing Institute of Life Sciences are the co-first authors of the research paper
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Other members of the laboratory (Cheng Xinyu, Shang Congping, Tao Ting, Li Dapeng) also made important contributions to the research
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The Zhang Zhibin laboratory of the Institute of Zoology of the Chinese Academy of Sciences, the Zhang Fan laboratory of Hebei Medical University, the Tang Zongxiang laboratory of Nanjing University of Traditional Chinese Medicine, and the Zhancheng Laboratory of Beijing Institute of Biological Sciences also participated in this project and made important contributions
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Original link: https:// Platemaker: 11 References 1.
Fanselow, MS & Lester, LS A functional behavioristic approach to aversively motivated behavior: Predatory imminence as a determinant of the topography of defensive behavior.
Evolution and Learning, 185-211 (1988).
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