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

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 PBN^Glu-NTS^Glu-DMV^ChAT 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 function^2,3,4, 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 signals^12,13. The above information suggests that acupuncture could potentially modulate gastric motility might be related to the PBN^Glu-NTS^Glu-DMV^ChAT 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 PBN^Glu-NTS^Glu-DMV^ChAT 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 nL^16,17.
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 opt...

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 ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Virus
rAAV-CaMKIIα-CRE-WPRE-hGH polyA (AAV2/9)	BrainVTA	Cat#PT-0020
rAAV-CaMKIIα-DIO-ChrimsonR-WPREs (AAV2/9)	BrainVTA	Cat#PT-12637
rAAV-ChAT-GCamp6s-WPRE-hGH-polyA (AAV2/9)	BrainVTA	Cat#PT-10973
Other
1 μL microinjector	Hamilton	80135
3M EPSP Single Bond Universal Adhesive	Dental World Official	41269
Acupuncture Needle	ZhongYanTaiHe	0.25/13s
CED Micro1401-4	CED(England)	1401-4
Dental cement/Room temperature hardened resin	YAMAHACHI DENTAL	N/A
Fine Forceps	RWD	F11028-13
Glass tube	World Precision Instruments	504949
HANS-200A	HANS Institute	HANS-200
Inper Signal (software)	Inper	N/A
Inper Studio(Venus)	Inper	B1502
Latex balloon	Harward Apparatus	BS4 73-2787
Multichannel optical fiber recording system(Vista Pro)	Inper	R5268
Neurolog digitimer Model	Digitimer	NL900D
Optical fiber ferrule	Inper	V8475
Spike 2(software)	CED(England)	N/A
Stereotaxic instruments (with coordinates calculator)	David Kopf Instruments	KOPF 942
Tribromoethanol	Sigma	T48402
tribromoethanol	Sigma	T48402