Thalamic Reuniens Nucleus Glutamatergic Neurons Mediated Colorectal Visceral Pain in Mice through 5-HT2B Receptors

Background

Irritable Bowel Syndrome (IBS) is a common functional bowel disorder characterized by abdominal pain and visceral hypersensitivity. Reducing visceral hypersensitivity is crucial for effectively alleviating abdominal pain in IBS patients. However, the specific mechanisms remain unclear. Increasing evidence suggests that the thalamic Reuniens nucleus (Re) and the serotonin (5-HT) neurotransmitter system play essential roles in the development of colorectal visceral pain, but the precise mechanisms are not well understood. The neonatal maternal separation (NMD) mouse model exhibits visceral hypersensitivity, and it has been found that glutamatergic neurons in the Re region play a key role in processing colorectal visceral pain.

Paper Source

This paper was authored by Di Li, Han Du, Shu-Ting Qu, Jing-Lai Wu, Yong-Chang Li, Qi-Ya Xu, Xia Chen, Xiao-Xuan Dai, Ji-Tian Xu, Qian Wang, and Guang-Yin Xu from Suzhou University and the Chinese Academy of Sciences, among others. The paper was received on October 27, 2023, and accepted for publication by the journal Neurosci. Bull. on December 26, 2023.

Research Process Overview

1. Experimental Animals Preparation:

   • All experiments were approved by the Animal Experiment Committee of Soochow University. The mice used in the experiments were aged between 6 to 12 weeks and randomly assigned to experimental and control groups.
   • Neonatal Maternal Separation: From postnatal day 2 to 15, mice were separated from their mothers for 3 hours each day to simulate maternal separation conditions, creating the NMD model.

2. Neurophysiological Recording:

   • A single electrode was implanted in the Re region to record neuronal firing rates, aiding in assessing changes in neuronal activity under colorectal visceral pain in NMD mice.

3. Colorectal Visceral Pain Threshold Testing:

   • Using CRD (Colorectal Distension), a balloon was expanded in the colon to simulate colorectal visceral pain. The abdominal muscle electromyogram (EMG) signals of mice were recorded to assess their pain threshold.

4. Electrode Recording and Immunofluorescence:

   • Extracellular Recording: A 16-channel electrode was implanted to record neuronal firing activities in the Re region under CRD conditions.
   • Immunofluorescence: Following CRD stimulation, the expression of c-fos protein in neurons in the Re region was measured to determine the location and type of activated neurons.

5. Optogenetic Experiment:

   • Optogenetic Inhibition and Activation: Using optogenetic techniques, glutamatergic neurons in the Re region were activated or inhibited by blue and yellow light, respectively, to observe the effects on colorectal visceral pain.

6. Molecular Biology Experiment:

   • Western Blot and PCR: The expression of 5-HT2B receptors in the Re region was detected.
   • Immunofluorescence: The co-location of 5-HT2B receptors on glutamatergic neurons was explored.
   • Fiber Photometry and Probe: The release of 5-HT in the Re region during CRD was measured.

7. Local Drug Injection:

   • Local injection of 5-HT2B receptor antagonist RS-127445 was performed to observe if it could alleviate the colorectal visceral pain response in NMD mice.

Main Results

1. The Role of Reuniens Nucleus in NMD Mice Visceral Pain:

   • EMG and c-fos staining revealed that CRD-induced neuronal activation in the Re region was significantly higher in the NMD group than in the control group.
   • Single electrode recording results showed that the neuronal firing rate in the Re region of NMD mice was significantly higher than in normal mice, indicating enhanced neuronal activity in Re is related to visceral pain.

2. Increased Neuronal Excitability and Synaptic Transmission:

   • Whole-cell patch-clamp technique indicated that the neuronal excitability in the Re region of NMD mice was significantly enhanced, and synaptic transmission frequency increased, without significantly affecting synaptic current amplitude.

3. Contribution of Glutamatergic Neurons in the Re Region:

   • Immunofluorescence and optogenetic experiments confirmed the crucial role of glutamatergic neurons in the Re region in CRD-induced visceral pain.
   • Optogenetic inhibition of glutamatergic neuron activity effectively reduced the visceral pain response in NMD mice.

4. Increased 5-HT2B Receptor and 5-HT Release:

   • It was found that the expression of 5-HT2B receptors in the Re region of NMD mice increased, and the release of 5-HT significantly rose compared to the control group.
   • After local injection of the 5-HT2B receptor antagonist, the c-fos expression and visceral pain response in NMD mice significantly decreased, indicating that 5-HT2B receptors play a regulatory role in visceral pain.

Research Conclusions and Significance

Through a series of experiments, this study first revealed the significant role of glutamatergic neurons in Re in colorectal visceral pain and elucidated the important mechanism of 5-HT2B receptors in regulating visceral pain. These findings offer new therapeutic targets for IBS patients: inhibiting the 5-HT2B receptors in the Re region can alleviate visceral hypersensitivity reactions induced by early life stress and subsequently reduce abdominal pain symptoms.

The study employed various advanced technical methods, including optogenetics, patch-clamp, immunofluorescence, and local drug injection, to reveal the potential mechanisms of the Re region in colorectal visceral pain from multiple angles and levels. These results provide a scientific basis for future treatments and propose new research directions to explore the collaborative roles of Re and other brain regions in colorectal visceral pain.