Method Article

Application of Optogenetics Combined with Fiber Photometry Recording in the Regulation of Neural Circuits-visceral Functions by Electroacupuncture

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DOI:

10.3791/68667

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September 19th, 2025

 ,  ,  ,  , 
1Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences, 2Northeast Asia Research Institute of Traditional Chinese Medicine, Changchun University of Chinese Medicine

Corresponding Authors: Zhu Bing <zhubing@mail.cintcm.ac.cn>, Xinyan Gao <gaoxy@mail.cintcm.ac.cn>

* These authors contributed equally

In This Article

Summary

The purpose of this experiment is to establish a novel approach for the simultaneous integration of optogenetics, intracerebral calcium signal recording, and gastric motility monitoring, offering a novel paradigm for real-time monitoring of the relationship between somatic stimulation and neural circuit-visceral function interactions, which is applied to acupuncture research.

Abstract

This protocol describes a technique for optogenetic manipulation of specific types of neurons and real-time recording of the effects of electroacupuncture at Zusanli (ST36) on nuclear calcium signals and gastric motility in mice under anesthesia in vivo. The exploration of the central mechanisms underlying the efficacy of acupuncture has been limited by technological constraints. However, the advancement of optogenetic and optical imaging technology has propelled the development of neuroscience. Fiber photometry recording, initially leveraging genetically encoded calcium indicators to visualize changes in calcium dynamics indicative of neuronal activity, stands out as a key technology for characterizing brain-behavior correlations in vivo. The application of optogenetics enables the genetic encoding of neurons, enabling their activation or inhibition in response to light stimulation. This capability facilitates the establishment of causal links between neural circuitry function and behavioral outcomes. Simultaneous fiber photometry, calcium recording, and optogenetic stimulation provide a method for real-time recording and manipulation of neuronal activity, serving as an effective approach to investigate the central mechanisms of acupuncture. Nonetheless, there remains a scarcity of studies documenting the concurrent utilization of fiber photometry calcium recording, optogenetics, and visceral function monitoring in acupuncture research. The results of this experiment show that EA-ST36 might regulate gastric motility related to PBNGlu-NTSGlu-DMVChAT circuit. Optogenetic stimulation of this circuit produces the same effect. After the superposition of the two, the changes in calcium signal and gastric motility reach their maximum. This study established a novel approach for the simultaneous integration of optogenetic, intracerebral calcium signal recording, and gastric motility monitoring, offering a novel paradigm for real-time monitoring of the relationship between somatic stimulation and neural circuit-visceral function interactions.

Introduction

In recent years, the proliferation of biomolecular fluorescent sensors has propelled the utilization of fiber photometry in molecular biology, facilitating in vivo recording1. This advancement enables the real-time monitoring of neuronal activity alterations in the brain. The integration of optogenetics allows for neuronal manipulation, offering the advantage of immediately conducting optogenetics and optical fiber recording by simply inserting the optical fiber. Combined with visceral function recording, this method offers good temporal resolution and nuclei-level targeting to observe how acupuncture modulates visceral functions via neuronal activity modifications.

Numerous studies have illustrated the distinct regulatory impact of acupuncture on gastrointestinal function2,3,4, although the precise central mechanisms remain elusive. Many ancient Chinese medical books, such as HuangDiNeiJing, also recorded that the ST36 (on the lateral side posterior to the knee joint, approximately 2 mm inferior to the caput fibula5) is often used to treat gastrointestinal diseases. Recent studies have shown that nuclei involved in gastric physiological processes (e.g., feeding behavior6, cessation of eating7, appetite suppression8) and pathological processes (e.g., gastric pain9, gastric dysfunction10) encompass the parasubthalamic nucleus (PSTn), dorsal raphe nucleus (DRN), lateral parabrachial nucleus (LPB), nucleus of the solitary tract (NTS), and dorsal motor nucleus of the vagus (DMV). Studies have shown that electroacupuncture at ST36 can increase the number of c-Fos+ cells in DMV, thereby augmenting c-Fos+ expression in the caudal subnucleus of NTS11. Additionally, it has been documented that the Paracentricular nucleus (PBN) is involved in the composition of the paracentricular nuclear complex, can project down to the medulla oblongata NTS, which plays an important role in the integration of visceral sensory information, such as gastrointestinal signals12,13. The above information suggests that acupuncture could potentially modulate gastric motility might be related to the PBNGlu-NTSGlu-DMVChAT neural circuit. Glutamatergic neurons are primarily involved in the LPB and NTS14, while cholinergic neurons are mainly involved in the DMV during gastric physiological and pathological processes15. Investigating the regulatory effects of acupuncture on neural circuits and visceral functions can provide a clearer understanding of acupuncture mechanisms and furnish theoretical backing for clinical acupuncture interventions in visceral diseases.

However, due to technical limitations, limited research has delved into the neural circuitry mechanisms of electroacupuncture in modulating visceral functions. Therefore, the present study presents simplified guidelines to introduce the detailed process of virus injection. Notably, the methodology expounded in this paper can be utilized to elucidate the circuitry mechanisms implicated in acupuncture's regulation of diverse visceral functions.

Therefore, this study aims to investigate the regulatory effects of acupuncture on gastric motility, which might be related to the PBNGlu-NTSGlu-DMVChAT neural circuit by combining optogenetics with fiber photometry for calcium imaging recording. The aim is to establish a protocol for calcium imaging and optogenetic synchronization, introducing a novel research framework for investigating visceral function and neural circuitry.

Protocol

All procedures followed the guidelines for laboratory animal care and use established by the National Institutes of Health and received approval from the Animal Care and Use Ethics Committee of the Institute of Acupuncture & Moxibustion, Chinese Academy of Medical Sciences. Adult male C57 mice weighing 25 g were employed for viral injection and optical fiber implantation. The animal experiment protocol strictly adhered to the requirements of the national standard "Guidelines for Ethical Review of Welfare of Laboratory Animals" (GB/T 35892-2018) and was approved by the Ethics Committee of the institution.

NOTE: Keep the environment clean during the process of virus injection and optical fiber implantation. Wear masks throughout the entire operation. Following the viral high-expression period (21-28 days), electroacupuncture at ST36 and optogenetics manipulations were conducted to record gastric motility. Animals were housed in the animal facility of the Institute of Acupuncture and Moxibustion, Chinese Academy of Medical Sciences, under controlled conditions (temperature: 22-24 °C, humidity: 50%-60%, 12 h light-dark cycle), with free access to food and water. Specific information regarding the reagents and equipment utilized can be found in the Table of Materials.

1. Pre-operative preparation

  1. Disinfect the surgical instrument that has undergone high-temperature sterilization, and clean the surgical area with 70% ethanol.
  2. Anesthesia: Administer an intraperitoneal injection of 1.25% tribromoethanol (0.2 mL/10 g of weight) to the mouse. Evaluate the absence of the toe pinch reflex to measure the depth of anesthesia.
  3. Shave the hair off the head and position the mice in a stereotaxic frame equipped with ear and teeth bars. Apply erythromycin ointment to the mice's eyes to prevent damage from surgical light.
  4. Soak the optical fiber ferrule (ferrule size: 1.25 mm white ferrule, fiber type: 200/0.37, fiber length: 5.5 mm, ferrule needle model: V8475) in 75% ethanol for 30 min and subsequently wash off the 70% ethanol with saline.
  5. Load the microinjector (1 µL) with paraffin (800-900 nL). Melt the glass tube and draw it into two sections. Sharpen one section at the tip using a needle grinding machine to serve as the injection needle. In order to ensure the gas tightness of the microinjector, fill the tube with paraffin and connect it to the microinjector on the tip using a glue gun.

2. Viral injection surgery

  1. Incise the scalp along the midline from the eyes to the back of the neck to reveal the skull. Subsequently, excise the neck muscles to expose the occipital bone. Wipe the bone surface with H2O2 for 30 s and then clean with a dry cotton swab to remove subcutaneous fascia and connective tissues (Figure 1A).
    NOTE: This step is to facilitate the covering of dental cement onto the occipital bone, making the optical fiber fixed more stably.
  2. Level the skull.
    1. X-axis leveling: Mark two points that are 3.5 mm away from the Bregema point in the coronal suture, and connect the two points to form a line. The midpoint of this line is the zero. The absolute value of the Z-axis difference on the bone surface 3.5 mm away from the origin should be ≤ 0.03 mm.
    2. Y-axis leveling: Mark two points that are 3.5mm away from the Lambda point in the lambdoid suture, set the midpoint of the two points, and connect this midpoint to the Lambda point to form a line. The midpoint of this line is another zero. The absolute value of the Z-axis difference between this zero and the zero at which the bone surface was adjusted during the X-axis should be ≤ 0.03 mm.
  3. Mark the stereotactic coordinates of the virus injection with a marker pen. For PBN: -1.1 mm Anterior-Posterior (AP,Y-axis), -5.45 mm Medial-Lateral(ML, X-axis), -3.3 mm Dorsal-Ventral (DV, Z-axis) (with bregma as the zero point); For NTS: -0.35 mm AP, 0.2 mm ML, -3.3 mm DV (with the highest point of the occipital bone as the zero point); For DMV: -0.35 mm AP, 0.0 mm (the highest point of the occipital bone), -3.5 mm DV (with the highest point of the occipital bone as the zero point) (Figure 1B). Use a high-speed microdrill (0.8 mm drill diameter) to penetrate the skull at the designated sites to expose the brain surface. Subsequently, use fine forceps to gently puncture the dura mater and remove it, apply saline to the drilled opening to maintain moisture on the brain surface.
  4. Apply Single Bond Universal adhesive to the skull surface (excluding the drilled region) and expose it to light for 30 s for curing. It is important to maintain a dry skull surface prior to applying the single bond universal adhesive to ensure strong adhesion. Additionally, the adhesive should be avoided on the muscles during application.
  5. Affix the microinjector to the mechanical arm of a stereotactic apparatus. Fill with 250 nL of virus (rAAV-CaMKIIα-CRE-WPRE-hGH polyA). Upon contact with the needle tip with the brain surface, adjust the depth (Z) to 0. Lower the needle to the target site within the parabrachial nucleus (PBN), and inject the virus into the PBN region at a rate of 35 nL/min. After injection, keep the needle in place for 10 min, then retract it at a speed of 6 mm/min. Subsequently, immerse the needle in an Eppendorf tube containing saline for cleaning (Figure 1C,D).
    NOTE: The injection doses of all the following viruses are 250 nL; DMV is 100 nL16,17.
  6. Employ the identical protocol to inject rAAV-CaMKIIα-DIO-ChrimsonR-WPREs into the nucleus tractus solitarius (NTS) and rAAV-ChAT-GCamp6s-WPRE-hGH-polyA into the dorsal motor nucleus of the vagus (DMV) (Figure 1D). All virus titers are ≥ 1 × 1012 VG/mL.

3. Optic fiber implant surgery

  1. Secure the optical fiber ferrule by the optrode holder and attach it to the mechanical arm of the stereotactic apparatus. Set the implantation coordinates corresponding to a position about 100 µm above the DMV viral injection site (Figure 1E, F).
  2. Blend 1-3 g of dental cement with 1 mL of room temperature hardened resin until uniform. Promptly administer the mixture around the optical fiber ferrule and onto the skull surface, leaving 5 mm of the upper section of the optical fiber ferrule exposed. Once the cement has solidified, release the holder screw, raise, and remove the holder (Figure 1G).
  3. Use absorbable surgical sutures (6-0) to suture the posterior occipital scalp of mice. Remove the mice from the stereotactic frame and place them in a warm environment to recuperate from anesthesia. Once the animals exhibited unrestricted movement, they were relocated to a clean and roomy cage for accommodation, with 4-5 mice housed together and allowed free access to food and water.
    NOTE: During the experiment, it was observed that a larger cage is beneficial for the normal movement of mice and is crucial for maintaining their weight. The increased space also reduces the likelihood of accidental contact with the implanted fiber, thereby minimizing potential damage. Mice weighing 25 g or more showed significant changes in intragastric pressure upon acupuncture.

4. Balloon insertion surgery

  1. At the peak of viral expression (21-28 days), fast the mice for 4-5 h. Subsequently, anesthetize the mice via intraperitoneal injection of 1.25% tribromoethanol (0.1 mL/10 g of weight). 15 min later, administer an intraperitoneal injection of 10% Ethyl carbamate solution (0.15 mL/10 g of weight). Evaluate the depth of anesthesia by confirming the absence of a toe-pinch reflex.
  2. Shave the hair of the abdominal and ST36 regions. Turn on the heating pad and set it to maintain a temperature of 37 °C, with the animal kept on the heating pad throughout the surgery to ensure a constant body temperature.
  3. Connect the latex balloon to the syringe and inject air into it to check that the latex balloon (BS4 73-2787) has no air leakage.
  4. Expose the stomach and a portion of the duodenum below the xiphoid process. Make a small incision in the duodenum approximately 0.3 cm in length, 0.5-1 cm distal to the pylorus.
  5. Insert the balloon into the stomach along the intestinal direction through the duodenal incision, with the knot positioned at the pyloric sinus.
  6. Remove the needle of the syringe, fill 5mL of pure water, discharge the air, and connect the other end of the balloon with the syringe. Pull the syringe's luer lock upward to evacuate gas from the balloon, then lower the luer lock to inject the pure water into the balloon. Connect the assembly to a three-way stopcock and a pressure transducer.

5. Synchronous recording of intragastric pressure and calcium imaging

  1. Turn on the Neurolog Digitimer Model, CED Micro1401-4, Inper Studio , Multichannel optical fiber recording system, Inper Signal software, and Spike 2 software. Set the Inper software, calcium signal recording to be triggered by Spike2 software and optogenetic stimulator, ensuring that laser stimulation and calcium signal acquisition by the Inper software align with intragastric pressure recording by Spike2 software.
  2. Open all three-way stopcocks that connect to the balloon, rotate the ZERO knob on the Neurolog digitimer Model, and observe on the Spike 2 software whether the baseline is adjusted to point 0. After equalizing the pressure inside the balloon with atmospheric pressure, inject pure water into the balloon until the intragastric pressure is displayed 15-35 cm H2O. Once both intragastric pressure and calcium signals reach a stable state, record a 60 s baseline of intragastric pressure and calcium signals in the DMV (Figure 2A).

6. Electroacupuncture at ST36 and Laser stimulation

  1. Insert an acupuncture needle (0.25 mm × 25 mm) into the left ST36 acupoint and connect it to an electroacupuncture apparatus (10 Hz, 1 mA). Administer electroacupuncture for 60 s while recording changes in intragastric pressure and calcium signals.
  2. Following stabilization of the baseline intragastric pressure and calcium signals, deliver optogenetic stimulation (635 nm, 5 ms, 5 Hz, 30 mW) through the fiber for 60 s, monitoring changes in intragastric pressure and calcium signals.
  3. Once the baseline of intragastric pressure and calcium signals stabilizes again, combine electroacupuncture at ST36 (10 Hz, 1 mA) with optogenetic stimulation (635 nm, 5 ms, 5 Hz) for 60 s, and record changes in intragastric pressure and calcium signals.

7. Histological examination of viral injection and fiber placement

  1. Following the completion of the aforementioned protocols, perfuse the mice with saline and fix with 4% paraformaldehyde via heart immediately. Harvest the mice's brains. Perform gradient dehydration using 20% and 30% sucrose solutions, then section after embedding with quick-freeze embedding agent. Compare the brain slices with the brain maps of mice to observe whether the positions of virus injection and fiber optic implantation were accurate. The precise positioning of viral injections and fiber implants upheld the validity of the intragastric pressure and calcium signal data (Figure 1H,I).

8. Data analysis

  1. Analyze calcium signal data using Inper Data Analysis software and gastric motility data by Spike2 software. Compare the differences between calcium signals or gastric motility amplitudes at the time of stimulation (60 s) and those before stimulation (60 s) to observe the regulatory effects of different stimuli on gastric motility and calcium signals. All the data shown in Figure 2 are the original data graphs.

Results

Viruses were injected into the parabrachial nucleus (PBN), nucleus tractus solitarius (NTS), and dorsal motor nucleus of the vagus (DMV) of mice, with a fiber implanted in DMV (Figure 1A-G). During the peak viral expression period, variations in intragastric pressure and calcium signals within the DMV were recorded following electroacupuncture at ST36 and optogenetic stimulation. The aim of this study is to observe the regulatory effects of electroacupuncture at ST36 and optogenetic stimulation on gastric motility, which might be related to the PBNGlu-NTSGlu-DMVChAT neural circuit.

Under baseline conditions, the peak of intragastric pressure and calcium signals showed no significant changes within the recorded 180 s (Figure 2A). Upon the application of optogenetic stimulation or electroacupuncture at ST36, there was a notable increase in both the peak of intragastric pressure and calcium signals. However, the calcium signal increased strongly with optogenetic stimulation than with electroacupuncture at ST36, while the intragastric pressure increases were weaker (Figure 2B,C). Concurrent administration of optogenetic stimulation and electroacupuncture at ST36 resulted in a substantial enhancement in both the peak of intragastric pressure and calcium signals, surpassing the effects induced by either optogenetic or electroacupuncture alone (Figure 2D). In this representative example (N = 1), optogenetic stimulation was associated with a larger calcium signal change than electroacupuncture at ST36, while the increase in intragastric pressure appeared greater during electroacupuncture (Figure 2E,F).

Glutaminergic neurons in PBN project to glutaminergic neurons in NTS and then to acetylcholinergic neurons in DMV. The virus of rAAV-CaMKIIα-DIO-ChrimsonR-WPREs is injected into NTS, causing the presynaptic neurons in NTS-DMV to carry the photosensitive protein ChrimsonR. When 635 nm light was administered through optical fibers, it activated the photosensitive protein, which increased the calcium ion influx of glutaminergic neurons in the NTS. Meanwhile, the increase in calcium signals of acetylcholinergic neurons in the DMV was recorded by optical fibers. The changes in calcium signals indicate that glutamatergic neurons in PBN project onto glutamatergic neurons in NTS, and there is a projection relationship between glutaminergic neurons in the NTS and acetylcholinergic neurons in the DMV, reflecting the formation of circuits among three brain regions and their neuronal projection relationships. At the same time, the amplitude of gastric motility was enhanced; meanwhile, the rate of calcium signals changes after EA-ST36 and photoactivation indicates that the regulatory effect of EA-ST36 on gastric motility may be related to PBNGlu-NTSGlu-DMVChAT circuit.

Optogenetic virus injection and optic-fiber implant in rodent; NTS, DMV targeting diagram.
Figure 1: Schematic diagram of viral injection procedure and virus sections. (A) Exposure of the skull. (B) Localization of PBN, NTS, and DMV brain regions. (C-D) Viral injection in PBN, NTS, and DMV brain regions. (E) Implantation of optical fiber. (F) Position of the optical fiber. (G) Fixation of the optical fiber with dental cement. (H) Histological section of viral injection (NTS). (I) Histological section of viral injection and optical fiber position sites. Please click here to view a larger version of this figure.

Fiber photometry, gastric motility, photoactivity recordings in mice; diagrams, charts, research data.
Figure 2: Schematic diagram of electroacupuncture combined with optogenetic stimulation. (A) Balloon pressurized to 35 cm H2O, recording 180 s baseline of calcium signals and intragastric pressure. (B-C) Electroacupuncture at ST36 or laser stimulation, recording calcium signals and intragastric pressure (60 s baseline, 60 s stimulation, and 60 s after stimulation). (D) Electroacupuncture at ST36 combined with laser stimulation, recording of calcium signals and intragastric pressure (60 s baseline, 60 s laser stimulation+ Electroacupuncture at ST36, and 60 s after stimulation). (E-F) Peak of intragastric motility and rate of calcium signal change during Base, EA-ST36, Red Light (RL), RL+EA-ST36, (N = 1). Please click here to view a larger version of this figure.

Discussion

The central scientific inquiry of acupuncture pertains to the regulation of target organs through acupoint stimulation of autonomic nerves. The findings suggested by Q. Ma suggested that acupuncture's influence on remote organ function modulation arises from somatic autonomic reflexes that activate sympathetic and/or parasympathetic pathways, especially those related to gastrointestinal diseases and systemic inflammation18. Nonetheless, extant research has predominantly centered on peripheral nerve investigations or isolated brain regions19,20. Individual neurons work together in neural circuits with specific projection relationships to process information21. These neural circuits are pivotal in elucidating the mechanisms underlying acupuncture's regulation of target organs. However, solely recording neuronal activation in nuclei in response to acupuncture or evaluating acupuncture's effects on visceral functions is insufficient to directly establish the link between acupoint stimulation, autonomic nerve modulation, and target organ regulation18.

In fiber photometry, the activity level of neuronal populations is measured by activating photosensitive proteins with light and recording the changes in fluorescence intensity of calcium fluorescent proteins14. Calcium imaging, using genetically encoded calcium indicators to measure Ca2+ dependent fluorescence, represents a standard method for tracking neuronal activity in specific neurons and neural networks12. The combination of fiber photometry and optogenetics innovatively integrates recording and manipulation of neuronal activity, a methodology that has found extensive application in numerous neuroscience investigations22,23,24. While the integration of optogenetics with fiber photometry calcium imaging has been deemed to be a useful technology in neuroscience studies, its application in elucidating neuromodulatory mechanisms underlying acupuncture remains challenging,such as the lack of operations for synchronously recording target organs, etc. In the present study, we have provided simplified guidelines for the virus injection method and set up a test method for calcium imaging, optogenetics, and simultaneous recording of gastric motility. This methodology provides a powerful approach for elucidating the neurophysiological mechanisms linking somatic afferent stimulation to visceral organ regulation via neural circuitry pathways. Even though we only built such a method, the experiment is reproducible, and the data statistics could be counted in future research.

Electrophysiology serves to capture neuronal activity through the detection of alterations in electrical discharges, providing a superior temporal and spatial resolution. This technique stands as a fundamental tool in neuroscience; however, conventional approaches face constraints in monitoring distinct neuron subtypes. Fiber photometry, despite its reduced spatial resolution compared to calcium imaging, resulting from photon amalgamation through a single channel10, can target specific neurons when combined with such techniques. Notably, fiber photometry presents advantages of cost efficiency, minimal invasiveness, and wide-ranging recording capabilities, but a disadvantage is that it lacks single-neuron spatial resolution. Data analysis proves to be uncomplicated and exhibits enduring stability with minimal constraints on animal mobility. While GRIN lens microscopes can capture the discharges between individual neurons and have stronger persuasiveness in the connections between single-neuron, they entail heightened complexity, costs, and surgical invasiveness25. Conversely, fiber photometry emerges as a more accessible and sensitive alternative for fluorescent surveillance of neuronal activity, finding extensive application in neuroscience and acupuncture research. During the experiment, it was observed that since this experiment involves optogenetic operations, the accuracy requirements for virus injection localization and fiber optic implantation localization are relatively high. The optical fiber patch cord is prone to photobleaching, it is necessary to wait for the calcium signal baseline to be stable before conducting the experiment. During the data acquisition process, the Inper software can only browse the calcium signal changes within 10 min in real time, and there are certain time limitations when comparing the data before and after. Another limitation of this experiment is that the virus injected into PBN is colorless, thus lacking verification of the virus expression level and expression site. This will be modified in subsequent experiments. Meanwhile, this experiment lacks non-acupoint control experiments, and the results of calcium signal and gastric motility function changes caused by EA-ST36 need to be further verified. After virus injection, PBN/NTS or DMV will project to different brain regions by themselves. Therefore, the theory that EA-ST36 regulates gastric motility might be related to the PBNGlu-NTSGlu-DMVChAT circuit, which remains to be further verified. It can be controlled by controlling the injection Angle, speed, and total amountin later experiments. The experimental operation of gastric motility requires opening the abdomen of mice, and the recording of visceral functions cannot be completed in the awake state. This is also the technical drawback of this experiment. We will continue to explore the techniques for recording visceral functions and neuronal activities in a waking state. However, all the stimuli were completed under the same anesthetic drugs and anesthetic doses, so the data were comparable. But this experiment only established the technique and did not conduct statistics.

Collectively, we present a robust methodology for concomitant monitoring of gastric motility and neural events in anesthetized mice, coupled with optogenetic modulation of neurons. This approach enables systematic investigation into how electroacupuncture stimulation orchestrates visceral regulation through engagement of specific neural circuitry, particularly those bridging somatosensory nuclei and autonomic control centers. Furthermore, the neuronal subtypes within these nuclei were delineated. This technology holds promise for investigating the neural circuit mechanisms modulated by acupuncture in various visceral functions.

Disclosures

The authors declare no conflicts of interest.

Acknowledgements

This study was funded by the National Natural Science Foundation of China (No. 82174518, 82474661), the Fundamental Research Funds for the Central Public Welfare Research Institutes (ZZ-2023008), and the Scientific and Technological Innovation Project of China Academy of Chinese Medical Sciences (CI2021A03402).

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Virus
rAAV-CaMKIIα-CRE-WPRE-hGH polyA (AAV2/9)BrainVTACat#PT-0020
rAAV-CaMKIIα-DIO-ChrimsonR-WPREs (AAV2/9)BrainVTACat#PT-12637
rAAV-ChAT-GCamp6s-WPRE-hGH-polyA (AAV2/9)BrainVTACat#PT-10973
Other
1 μL microinjectorHamilton80135
3M EPSP Single Bond Universal AdhesiveDental World Official41269
Acupuncture NeedleZhongYanTaiHe0.25/13s
CED Micro1401-4CED(England)1401-4
Dental cement/Room temperature hardened resinYAMAHACHI DENTALN/A
Fine ForcepsRWDF11028-13
Glass tubeWorld Precision Instruments504949
HANS-200AHANS InstituteHANS-200
Inper Signal (software)InperN/A
Inper Studio(Venus)InperB1502
Latex balloonHarward ApparatusBS4 73-2787
Multichannel optical fiber recording system(Vista Pro)InperR5268
Neurolog digitimer Model DigitimerNL900D
Optical fiber ferrule InperV8475
Spike 2(software)CED(England)N/A
Stereotaxic instruments (with coordinates calculator)David Kopf InstrumentsKOPF 942
TribromoethanolSigmaT48402
tribromoethanolSigma T48402

References

  1. Li, S., et al. Perspective of calcium imaging technology applied to acupuncture research. Chin J Integr Med. 30 (1), 3-9 (2024).
  2. Xu, X., et al. Mechanism of electro-acupuncture in alleviating intestinal injury in septic mice via polyamine-related M2-macrophage polarization. Front Immunol. 15, e1373876(2024).
  3. Yaklai, K., Pattanakuhar, S., Chattipakorn, N., Chattipakorn, S. C. The role of acupuncture on the gut-brain-microbiota axis in irritable bowel syndrome. Am J Chin Med. 49 (2), 285-314 (2021).
  4. Wang, X. Y., Wang, H., Guan, Y. Y., Cai, R. L., Shen, G. M. Acupuncture for functional gastrointestinal disorders: a systematic review and meta-analysis. J Gastroenterol Hepatol. 36 (11), 3015-3026 (2021).
  5. China Association of Acupuncture and Moxibustion. Nomenclature and location of acupuncture points for laboratory animals Part 3: Mouse. World J Acupunct Moxibustion. 35 (2), 160-166 (2025).
  6. Chen, J., et al. A vagal-NTS neural pathway that stimulates feeding. Curr Biol. 30 (20), 3986-3998 (2020).
  7. Campos, C. A., Bowen, A. J., Schwartz, M. W., Palmiter, R. D. Parabrachial CGRP neurons control meal termination. Cell Metab. 23 (5), 811-820 (2016).
  8. Zhang, T., Perkins, M. H., Chang, H., Han, W., de Araujo, I. E. An inter-organ neural circuit for appetite suppression. Cell. 185 (14), 2478-2494 (2022).
  9. Zhang, F. C., et al. A vagus nerve dominant tetra-synaptic ascending pathway for gastric pain processing. Nat Commun. 15 (1), 9824(2024).
  10. Dong, W. Y., et al. Brain regulation of gastric dysfunction induced by stress. Nat Metab. 5 (9), 1494-1505 (2023).
  11. Iwa, M., Tateiwa, M., Sakita, M., Fujimiya, M., Takahashi, T. Anatomical evidence of regional specific effects of acupuncture on gastric motor function in rats. Auton Neurosci. 137 (1-2), 67-76 (2007).
  12. Browning, K. N., Travagli, R. A. Central nervous system control of gastrointestinal motility and secretion and modulation of gastrointestinal functions. Compr Physiol. 4 (4), 1339-1368 (2011).
  13. Sun, L., et al. Parabrachial nucleus circuit governs neuropathic pain-like behavior. Nat Commun. 11 (1), 21(2020).
  14. Simpson, E., et al. Lights, fiber, action! A primer on in vivo fiber photometry. Neuron. 112 (5), 718-739 (2024).
  15. Tao, J., et al. Highly selective brain-to-gut communication via genetically defined vagus neurons. Neuron. 109 (13), 2106-2115 (2021).
  16. Yao, L., Ye, Q., Liu, Y., et al. Electroacupuncture improves swallowing function in a post-stroke dysphagia mouse model by activating the motor cortex inputs to the nucleus tractus solitarii through the parabrachial nuclei. Nat Commun. 14 (1), 810(2023).
  17. Lyu, Q., Xue, W., Liu, R., et al. A brain-to-gut signal controls intestinal fat absorption. Nature. 634 (8035), 936-943 (2024).
  18. Ma, Q. Somatotopic organization of autonomic reflexes by acupuncture. Curr Opin Neurobiol. 76, e102602(2022).
  19. Liu, S., et al. A neuroanatomical basis for electroacupuncture to drive the vagal-adrenal axis. Nature. 598 (7882), 641-645 (2021).
  20. Liu, S., et al. Somatotopic organization and intensity dependence in driving distinct NPY-expressing sympathetic pathways by electroacupuncture. Neuron. 108 (3), 436-450 (2020).
  21. Luo, L. Architectures of neuronal circuits. Science. 373 (6559), eabg7285(2021).
  22. Yuan, Z., et al. A corticoamygdalar pathway controls reward devaluation and depression using dynamic inhibition code. Neuron. 111 (23), 3837-3853 (2023).
  23. Feng, J., et al. Monitoring norepinephrine release in vivo using next-generation GRABNE sensors. Neuron. 112 (12), 1930-1942 (2024).
  24. Smith, J., et al. Regulation of stress-induced sleep fragmentation by preoptic glutamatergic neurons. Curr Biol. 34 (1), 12-23 (2024).
  25. Li, Y., Liu, Z., Guo, Q., Luo, M. Long-term fiber photometry for neuroscience studies. Neurosci Bull. 35 (3), 425-433 (2019).

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Optogenetic StimulationCalcium Signal RecordingNeural Circuit RegulationGastric MotilityVisceral FunctionGenetically Encoded IndicatorsReal Time RecordingBrain Behavior Correlation

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