Neuron︱ Cao Peng laboratory discovers the closed-loop neural mechanism of repetitive stereotyped behaviors

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Wang Sizhen's article ︱Cheng Xinyu editor in charge ︱Wang Sizhen In daily life, people need to use various physiological repetitive stereotyped behaviors (such as washing hands and brushing teeth) to satisfy normal The needs of life
.

In mental diseases such as autism and obsessive-compulsive disorder, patients often show a series of pathological repetitive stereotypes
.

Pathological repetitive stereotypes are considered to be a window into the pathogenesis of these mental illnesses, and they are regarded by many researchers.
Attention
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When the researchers introduced genetic mutations for human autism and obsessive-compulsive disorder into the mouse genome, they were surprised to find that "diseased" mice also exhibited pathological repetitive stereotypes, which manifested as long-term repetitive stereotypes.
self comb [1-4]
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 self-comb (self-grooming) is the human and animal body to clear itself foreign objects (such as dirt) instinctive behavior has important biological significance
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Research on the neural mechanism of this type of instinctive behavior is expected to reveal the underlying logic of repeated stereotyped behaviors
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Existing studies have mostly focused on the forebrain area, including the regulatory function of the "cortex-striatum" loop on self-grooming behavior [5-7]
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However, the neural mechanism of how the brain and spinal cord coordinate and trigger repetitive stereotyped behaviors is still unclear
.

 On December 21, 2021, Cao Peng's laboratory of the Beijing Institute of Life Sciences published a research paper entitled "A brain-to-spinal sensorimotor loop for repetitive self-grooming" on the famous neuroscience journal Neuron
.

The study found that in the caudal part of the spinal trigeminal nucleus (Sp5C), neurons expressing cerebellin-2 (Cbln2) form a descending nerve pathway that projects to the spinal cord.
By innervating the motor neurons and interneurons of the cervical spinal cord, they play a decisive role in maintaining the repetitive stereotyped behavior of self-grooming
.

The researchers further proved that these neurons also receive input from the trigeminal ganglion sensory neurons and the hypothalamic paraventricular nucleus neurons.
Therefore, the author finally proposed an interesting closed-loop neural mechanism model
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 1.
Establish three behavior paradigms that induce self-grooming and a quantitative analysis method for measuring repetitive self-grooming behavior.
First, the researchers used three different external stimuli to induce repetitive self-grooming behaviors, corresponding to: corn oil dripping on the face—— Feeling irritation, subcutaneous injection of capsaicin in the face-chemical stimulation, plantar electric shock-stress stress stimulation
.

All three stimulation methods can induce repetitive self-grooming behavior on the face of mice
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In order to quantitatively analyze these behaviors, the researchers embedded micro-magnets in the forelimbs of mice and placed them in an electromagnetic field.
The electromagnetic signals generated can track repeated forelimb movements and represent repeated self-grooming behaviors
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 Then, the researchers used different volumes of corn oil to induce facial self-grooming in mice, and found that the duration of this behavior depends on the volume of corn oil
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The researchers further inactivated the specific AAV-DIO-EGFP-2A-TeNT vector by bilaterally injecting the trigeminal ganglion (TG) of TrkB-CreER, TH-2A-CreER, and Mrgprb4-TdTomato-2A-Cre mice.
Types of neurons, it is found that the inactivation of TrkB+ neurons in the trigeminal ganglion responsible for tactile stimulation can significantly reduce the duration of corn oil-induced self-grooming behavior, indicating that the TrkB+ neurons in the trigeminal ganglion responsible for touch are involved in the corn oil-induced Repetitive self-grooming behavior (Figure 1)
.

Figure 1 Induction and quantitative analysis of self-grooming instinct behavior (Source: Xie et al.
, Neuron 2021) 2.
Cbln2+ Sp5C neurons are the key neuron subtype of facial self-grooming behavior.
Next, the researchers will analyze self-grooming behavior in depth Central mechanism
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Sensory neurons in the trigeminal ganglion project to the trigeminal nerve complex in the brain
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The trigeminal nerve complex includes the principal trigeminal nucleus (Pr5) and the spinal trigeminal nucleus (Sp5)
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Among them, the caudal part of the spinal trigeminal nucleus (Sp5C) is also called the medullary dorsal horn because of its structure similar to the spinal dorsal horn
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In the research field of the spinal dorsal horn, it is known that neurons expressing Cbln2, PV, CCK, and NPY are responsible for processing mechanically stimulated information
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Therefore, the researchers injected AAV-DIO-hM4Di-mCherry virus into Sp5C of Cbln2-IRES-Cre, PV-IRES-Cre, CCK-IRES-Cre and NPY-IRES-Cre mice, and injected CNO intraperitoneally to target specific neurons.
Perform chemical genetic suppression
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They found that when Cbln2+ Sp5C neurons are inhibited, the duration of facial self-grooming behavior induced by corn oil, capsaicin, and stress can be reduced, while chemical inhibition of other neuron subtypes (PV+, CCK+, NPY+) causes The impact is not significant
.

This suggests that Cbln2+ Sp5C neurons may be an important part of the neural circuit that regulates this behavior (Figure 2)
.

Figure 2 Cbln2+ Sp5C neurons are a key neuron subtype of facial self-grooming behavior (Source: Xie et al.
, Neuron 2021) 3.
The activation of Cbln2+ Sp5C neurons can trigger forelimb movement similar to self-grooming.
Next, the researchers The Sp5C of Cbln2-IRES-Cre mice was injected with AAV-DIO-ChR2-2A-mCherry virus and the optical fiber was embedded
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They found that light-activated Cbln2+ Sp5C neurons can trigger repetitive forelimb movements that resemble facial self-grooming in mice
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After quantitative analysis, it is found that the frequency of this movement depends on the intensity and frequency of the laser
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The researchers further found that chemical genetics activation of Cbln2+ Sp5C neurons can also cause mice to repeat stereotyped facial self-grooming behavior, and the use of antidepressant fluoxetine can reduce the duration of this behavior (Figure 3)
.

Figure 3 Activating Cbln2+ Sp5C neurons triggers forelimb movement similar to self-grooming (Source: Xie et al.
, Neuron 2021) 4.
Morphological and physiological characteristics of Cbln2+ Sp5C neurons Inject the AAV-DIO-hM3Dq-mCherry vector and observe its co-localization with the marker genes of different types of neurons (such as Vglut2 and Vgat).
It is found that Cbln2+ Sp5C neurons are mainly glutamatergic neurons
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The researchers also expressed GCaMP7 in Sp5C of Cbln2-IRES-Cre mice and performed fiber-optic recordings, and found that when corn oil induced facial self-grooming behavior, the calcium signals of neurons oscillated
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After further analysis, the researchers found that the calcium signal lags behind the self-grooming behavior, so it is speculated that Cbln2+ Sp5C neurons did not initiate this behavior, but may be involved in the maintenance of this behavior
.

In addition, the researchers also tested other parts of the mouse body by adding corn oil, and used Von Frey filaments to mechanically test the ipsilateral and contralateral sides of the mouse body, and found that Cbln2+ Sp5C neurons prefer the body.
Stimulation of the same side (Figure 4)
.

Figure 4 The morphological and physiological characteristics of Cbln2+ Sp5C neurons (Source: Xie et al.
, Neuron 2021) 5.
The input and output of Cbln2+ Sp5C neurons are traced by the rabies virus.
The researchers found that Cbln2+ Sp5C neurons receive data from The input of LTMRs and TRPV1+ sensory neurons in the trigeminal ganglion also receives input from the primary somatosensory cortex (S1), paraventricular nucleus of the hypothalamus (PVH) and other brain regions
.

By tracing the Sp5C expressing Cre-dependent EGFP in Cbln2-IRES-Cre mice, the researchers found that these neurons traveled to the ventral posteromedial nucleus (VPM) of the thalamus, the lateral parabrachial nucleus (LPB), and the ventral horn of the spinal cord.
horn) projection
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Further experiments showed that the Cbln2+ Sp5C neurons projecting to the LPB and spinal cord were clearly separated into two groups in Sp5C
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These data suggest that Cbln2+ Sp5C neurons form a unique downward pathway leading to the cervical segment of the spinal cord for regulating the movement of the forelimbs (Figure 5)
.

Figure 5 The input and output of Cbln2+ Sp5C neurons (Source: Xie et al.
, Neuron 2021) 6.
The key role of Cbln2+ Sp5C neurons projected to the spinal cord in facial self-grooming is achieved in Cbln2-IRES-Cre mice Sp5C injection of AAV-fDIO-hM3Dq-mCherry, injection of AAV2-retro-DIO-Flp into the spinal cord, and intraperitoneal injection of CNO, the researchers found that specific chemical genetic activation of Cbln2+ neurons projecting from Sp5C to the spinal cord can cause repetitive facial self-grooming in mice Behavior
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 On the other hand, chemical genetic suppression of Cbln2+ Sp5C neurons projecting to the spinal cord can significantly reduce the duration of facial self-grooming behavior in mice caused by corn oil, capsaicin, and plantar electric shocks
.

These data indicate that the Cbln2+ Sp5C neurons projecting to the spinal cord may play a key role in the repetitive stereotyped behaviors of facial self-grooming in mice (Figure 6)
.

Figure 6 The key role of Cbln2+ Sp5C neurons projecting to the spinal cord in facial self-grooming (Source: Xie et al.
, Neuron 2021) 7.
Analysis of the characteristics of spinal cord projection Cbln2+ Sp5C neurons.
Finally, the researchers traced them with rabies virus The method traces the upstream of Cbln2+ Sp5C neurons projecting to the spinal cord
.

They found that these neurons may integrate inputs from TrkB+ neurons, TRPV1+ neurons, and PVH neurons from TG
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 In addition, they also performed fiber-optic recordings of facial self-grooming behavior on Cbln2+ Sp5C neurons projecting to the spinal cord, and observed a significant increase in calcium signals.
At the same time, these neurons also showed obvious effects on the mechanical stimulation of the ipsilateral side.
Calcium signal response
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These data indicate that the Cbln2+ Sp5C neurons projecting to the spinal cord encode mechanical stimulation of the ipsilateral side (Figure 7)
.

Figure 7 The characteristics of Cbln2+ Sp5C neurons projecting to the spinal cord (Source: Xie et al.
, Neuron 2021) Figure 8 The proposal of the "sensory-motor closed loop" model (Source: Xie et al.
, Neuron 2021) Article conclusions and Discussion, inspiration and prospects.
In summary, in the caudal part of the trigeminal nucleus spinal cord (Sp5C), neurons expressing cerebellin-2 (Cbln2) form a descending nerve pathway projecting to the spinal cord, which can affect the facial self of mice.
The repetitive and stereotyped behavior of combing is crucial
.

The research has reached three main conclusions
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First, the study revealed that the Cbln2+ Sp5C neuron in the dorsal horn of the medulla, as a unique neuron subtype, can regulate the instinctive and repetitive stereotyped behavior of facial self-grooming
.

This neuronal subtype is different from the typical neuronal subtypes (PV+, NPY+ and CCK+) that express other marker molecules
.

Second, the discovery of the spinal cord projection of Cbln2+ Sp5C neurons may provide important clues for the brain-spinal cord coordination to produce repetitive stereotyped behaviors
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Third, the researchers' data may provide new insights into mouse models of obsessive-compulsive disorder and Tourette's syndrome
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The researchers found that the activation of Sp5C neurons expressing Cbln2 triggers the repetitive stereotyped facial self-grooming behavior that is sensitive to fluoxetine treatment, suggesting that Cbln2+ Sp5C neurons may be involved in the production of excessive self-grooming behavior in a mouse model of obsessive-compulsive disorder
.

 Based on the above conclusions, the researchers proposed a "sensory-motor closed-loop" model of repetitive stereotyped behavior: each time the mouse combs itself, Cbln2+ Sp5C neurons are activated, which further promotes the self-grooming behavior in the next cycle, thus forming a feedback The type of closed-loop neural mechanism to maintain the occurrence of repetitive stereotyped behaviors (Figure 8)
.

 At the same time, there are still some weak links in the research
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First, the researchers only paid attention to the time of facial self-grooming, and the quantification of this behavior may be too singular
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Second, the behavior caused by optogenetic activation of Cbln2+ Sp5C neurons does not fully reproduce the facial self-grooming induced by oil
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At the same time, other neuron groups inside and outside Sp5C may also participate in this behavior.
In future research, finding other neuron groups and neural pathways involved in regulating facial self-grooming behavior will be an important direction
.

Original link: https://doi.
org/10.
1016/j.
neuron.
2021.
11.
028 Xie Zhiyong (back row, first right), Li Dapeng (front row, first left), Cheng Xinyu (front row, second left), Pei Qing (front row, left Three), Gu Huating (front row, fifth from left), Cao Peng (front row, fifth from right) (photo provided by: Cao Peng laboratory) Xie Zhiyong, Li Dapeng, Cheng Xinyu, Pei Qing and Gu Huating of Cao Peng laboratory of Beijing Institute of Biological Sciences are Co-first author of the research paper
.

Other members of the laboratory (Tao Ting, Huang Meizhu, Shang Congping, Geng Dandan, Zhao Miao, Liu Aixue) also made important contributions to the research
.

Dr.
Ma Yuanwu of the Chinese Academy of Medical Sciences, Dr.
Zhang Fan of Hebei Medical University and Dr.
Zhang Chen of Capital Medical University are the main collaborators and have made important contributions to this research
.

The research paper was strongly funded by the National Natural Science Foundation of China
.

 Cao Peng's laboratory uses neural circuit analysis tools and molecular cell biology methods to carry out basic research and transformational applications of neurodegenerative diseases
.

Now we are recruiting a number of scientific researchers to conduct in-depth research on the circuits and molecular mechanisms of neurological diseases
.

Long-term recruitment of postdoctoral and technicians, see: https://mp.
weixin.
qq.
com/s/zAmlufCr8wIRmPAfWWHvzQ selections of previous articles [1] EMBO Rep｜Kang Jiuhong's research team found that lncRNA SOX1-OT regulates human ESC neurogenesis New mechanism [2] Neuron︱ Li Yulong’s laboratory developed a new fluorescent probe to detect the spatiotemporal dynamics of extracellular ATP [3] Neurosci BullGuangyin Xu’s group revealed that GATA1 in spinal astrocytes is targeted by DNA methylation A new mechanism for combining with P2x7r to relieve visceral pain [4] Nat Rev Neurosci viewpoint article︱Probabilistic model of Alzheimer’s disease: a revision of the amyloid cascade hypothesis [5] J Neurosci︱ Wan Xiaohong’s research group reveals that the brain is not in decision-making Deterministic tasks.
General and specific neural representations [6] eLife︱ Huang Zhili’s research team found that the new nucleus of arousal in the paraventricular nucleus of the hypothalamus regulates wakefulness and drowsiness [7] Nat Commun︱ women are more likely to drink alcohol? New discovery of neural circuit to alleviate alcoholism and anxiety behaviors of men and women: paraventricular nucleus of thalamus-stria terminal nucleus [8] Nature Comm︱Song Jianren's research group discovered a new target to promote neural circuit reconstruction after spinal cord injury [9] Frontier review interpretation ︱Research progress of the basal layer in neurodegenerative diseases [10] Nat Neurosci︱The semantic representation of vision and language overlaps at the edge of the human visual cortex.
High-quality scientific research and training courses are recommended.
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