The interstitial cells of Cajal (ICC) are well known for their pacemaker activity in the gastrointestinal (GI) tract.^1-3 ICC generate spontaneous rhythmic depolarizations that are passively conducted to adjacent smooth muscle cells (SMCs) through gap junctions. Such potential oscillations, also known as slow waves, are eventually recorded in smooth muscle and are thought to underlie phasic contractions. The cooperation of the SIP syncytium, consisting of SMCs, ICC, and platelet-derived growth factor receptor alpha-positive cells, with the enteric nervous system (ENS) is essential for GI motility.^4,5

Abnormal slow waves caused by loss or dysfunction of ICC are associated with motor disorders of the GI tract, such as constipation and gastroparesis.^6-12 Our earlier findings suggest that the rescue of ICC and their pacemaker activity is thought to benefit recovery from GI motility disorder.^13-15 However, the manipulation of rhythmic pacemaker activity to achieve arbitrary control of colonic contractions has not yet been demonstrated.

Several chemical agents have been shown to modulate pacemaker currents in ICC.^16-18 However, the lack of spatiotemporal precision due to the inherent characteristics of the dynamics of the chemicals, which are often persistent and not adjustable, makes this approach difficult to achieve. Optogenetics has been widely used to specifically monitor and modulate the activity of a group of excitable cells, and several recent studies have reported its application in SMCs or certain enteric neurons to promote GI motility.^19-24 The generation and propagation of slow waves depend on the structured network and electrophysiological mechanisms of the ICC. Therefore, we attempt to stimulate the pacemaker activity of ICC by optogenetics, aiming to reveal whether the rhythmic contraction of the mouse colon could be regulated by light stimulation.

Here, we report an optogenetics-based mouse model in which the light-sensitive protein channelrhodopsin-2 (ChR2) was expressed in ICC. The effects of ChR2 activation on colonic motor activity were assessed by measuring isometric contractions. The results showed that 470 nm light in a specific illumination mode caused changes in the colonic motor patterns of mouse muscle strips. A single light stimulus evoked premature low-frequency and high-amplitude (LFHA) contractions, while periodic light stimulation enhanced the inherent frequency of LFHA contractions in these mice.

Animals

C57BL/6-Gt(ROSA)26Sor^{em1(CAG-LSL-ChR2(H134R)-tdTomato-WPRE-polyA)Smoc} and B6;129-Kit^{tm1(CreERT2)Smoc} mice were purchased from Shanghai Model Organisms Center, Inc (Shanghai, China). Mice from the 2 strains were crossbred to obtain offspring with the ChR2(H134R)-tdTomato fusion gene inserted into cells expressing Kit and Cre recombinase as the case group (Kit^CreERT2/+;ROSA^{ChR2(H134R)/tdTomato/+}). Littermates were used as controls in all experiments. These specific pathogen-free animals were housed under a 12-hour light-dark cycle and given free access to water and food.

All offspring mice from both groups were injected with tamoxifen (TAM) at 6-8 weeks of age. TAM (80 mg; T5648; Sigma-Aldrich, St. Louis, MO, USA) was dissolved in corn oil (4 mL; C7030; Solarbio, Beijing, China) to create a 20 mg/mL solution. All mice were injected with 0.1 mL of TAM solution for 3 consecutive days and were used for experiments at least 7 days after the last injection. The fusion and expression of ChR2(H134R)-tdTomato were confirmed by genotyping and immunofluorescence, respectively.

All animal procedures were conducted in compliance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines and approved by the Laboratory Animal Welfare and Ethics Committee of the Third Military Medical University (AMUWEC2020935).

Tissue Preparation

Mice were sacrificed by cervical dislocation after sedation with an inhaled substance, and a middle abdominal incision was made. The entire colon was removed immediately and placed in cold Krebs-Ringer bicarbonate (KRB) solution with the following composition (in mM)²²: NaCl 118, KCl 4.7, NaH₂PO₄ 1, NaHCO₃ 25, MgCl₂ 1.2, D-glucose 11, and CaCl₂ 2.5. The solution had a pH of 7.2-7.4 at 37°C when bubbled to equilibrium with 95% O₂ + 5% CO₂.

The intraluminal contents were then washed away with KRB solution. The proximal and mid-colon segments were harvested and opened along the mesenteric border. The segments were then placed in a petri dish coated with SYLGARD elastomer (Dow Corning, Midland, MI, USA) that was filled with cold oxygenated (95% O₂ + 5% CO₂) KRB solution. The segments were pinned to the base of the dish with the circular muscle layer facing upward and slightly stretched to facilitate exposure of the mucosa. The mucosa and submucosa were carefully removed by sharp dissection using a stereoscopic microscope.

Genotyping

When the mice were at 3 weeks of age, genomic DNA was isolated from offspring mouse tails using a commercially available Mouse Direct PCR Kit (B40013; Bimake, Shanghai, China).

All the sequences of each primer were designed by Shanghai Model Organisms Center, Inc (Shanghai, China) and purchased from Shanghai Shenggong Co, Ltd (Shanghai, China) (Table).

Immunofluorescence Staining

Whole-mount preparations were used for further analysis. The muscle strip preparations were placed into a petri dish coated with SYLGARD elastomer that was filled with cold phosphate-buffered saline (PBS; 0.01 M, pH 7.4). The tissues were fixed in 4% paraformaldehyde for 2 hours at 4°C and washed several times with PBS. The tissues were then incubated in PBS containing 3% bovine serum albumin for 1 hour at room temperature (RT), stained with rat anti-Kit (Cat No. ab65525; 1:500; Abcam, Cambridge, MA, USA) for at least 48 hours at 4°C with constant agitation, rinsed several times in PBS and then incubated for another 1 hour in the dark at RT with Alexa Fluor 488 goat anti-rat secondary antibodies (Cat No. ab150157; 1:1000; Abcam). Nuclear staining was achieved using mounting medium with DAPI (Cat No. ab104139; Abcam) for 5 minutes in the dark at RT.

All immunostained samples were examined by confocal microscopy using a Leica TCS SP8 STED microscope (Leica, Wetzlar, Germany) equipped with air objectives, including ×5/0.15, ×10/0.4, and ×20/0.75 objectives, as well as a ×63/1.4 oil-immersion objective with excitation wavelengths of 488 nm and 552 nm (appropriate for Alexa 488 and tdTomato, respectively).

Images with a frame size of 2048 × 2048 pixels and an image size of 2325 × 2325 μm (×5/0.15 air objective), 1162.5 × 1162.5 μm (×10/0.4 air objective), 581.25 × 581.25 μm (×20/0.75 air objective), and 184.52 × 184.52 μm (×63/1.4 oil-immersion objective) were collected as appropriate. The images were merged and converted with Leica LAS Application Suite X software (version 3.7.4.23463).

Colocalization of tdTomato and Kit immunoreactivity was quantified in the colonic muscularis from different sites. Images were randomly collected using a ×63/1.4 oil-immersion objective. ImageJ software (V1.52i, National Institutes of Health, Bethesda, MD, USA) was applied to perform the quantification.

Isometric Force Recordings

Isometric force recording of the circular muscle was performed to observe the contractions of colonic smooth muscle under light stimulation. Fresh muscle strips (5 × 10 mm) cut parallel to the circular muscle fibers from the proximal and mid-colon were harvested after the mucosa and submucosa were removed. The tissues were then vertically placed into organ baths (ADInstruments, Sydney, Australia) that were filled with continuously oxygenated KBS at 37℃. They were attached at one end to a fixed mount with suture thread and at the opposite end to an isometric force transducer (MLT0402; ADInstruments) connected to a PowerLab (ADInstruments). A resting force of 3 mN was applied. An equilibration period of 60 minutes was essential for capturing the spontaneous phasic contractions. The tissues were washed with fresh, oxygenated, warm (37°C) KRB solution twice every 60 minutes or 5 times after the end of the pharmacological interventions before being subjected to another equilibration period of at least 20 minutes.

The data were collected and further analyzed using LabChart software (version 8) (ADInstruments).

Light Stimulation

Light delivery was implemented using a light stimulation system (Inper-B1-470; Inper, Hangzhou, China). The blue light was delivered focally via a Mono Optogenetics Fiber (MOF-F-1.25-200-0.37-150; Inper) and controlled by custom software (Inper Studio). According to the results of the preliminary experiment, some parameters were fixed at the beginning of the experiment. The power was set to 100%, and the frequency was set to 20-30 Hz to obtain sufficient optical density in all experiments, while the pulse width was set to 5 milliseconds.

The fiber was immersed into the organ bath and positioned approximately 3-5 mm above the muscle strips at an oblique angle to expose the tissue to the light over as large an area as possible.

Solutions and Drugs

PBS and all drugs used for compounding KRB were purchased from Shanghai Shenggong Co. T16Ainh-A01 and TAM were purchased from Sigma-Aldrich. Dimethyl sulfoxide and corn oil were purchased from Solarbio. All drugs were dissolved with the corresponding solvent according to the manufacturers’ protocols.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Statistical Methods

Statistical analysis was performed using SPSS (version 25.0, IBM Corp, Armonk, NY, USA). The displayed figures were generated from digitized data using GraphPad Prism 6 (GraphPad Software, Inc, La Jolla, CA, USA) after being transformed with LabChart. The data are expressed as the mean ± standard error of the mean. Statistical analysis was performed using paired or unpaired Student’s t tests and nonparametric tests, including the Mann–Whitney test and Wilcoxon test, as appropriate. The chi-squared test was performed for categorical variables. Two-way repeated-measures ANOVA was used to compare data with and without light stimulation.

In all statistical analyses, a P-value of < 0.05 was considered to indicate significance. Significance levels are denoted as follows: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. A lowercase “n” indicates the number of animals, while “c” refers to the number of colonic muscle strips analyzed in that same data set.

Generation of Kit^CreERT2/+; ROSA^{ChR2(H134R)/tdTomato/+} Mice

To regulate the activity of ICC with light, the light-sensitive protein ChR2 first had to be genetically expressed in ICC. An inducible site-specific Cre-loxP recombination system, which is one of the most common tools applied in optogenetics,²³ was used in the present study. Kit (also known as c-kit) is the specific marker of ICC in the GI tract.²⁵ It has been reported that mast cells are also labeled by Kit. However, few mast cells are present in the tunica muscularis when mice are kept under specific pathogen-free conditions.² We obtained Kit^CreERT2/+;ROSA^{ChR2(H134R)/tdTomato/+} offspring by crossbreeding the 2 mouse strains mentioned above. Upon the administration of TAM, Cre-ERT2 translocated into the nucleus, resulting in the expression of the light-sensitive protein ChR2(H134R) and the fluorescent protein tdTomato in Kit-positive ICC in mice from the case group (Fig. 1A). According to the genotyping results, the mice with both knock-in CreERT2 and ChR2(H134R)-tdTomato alleles were defined as the cases–(Kit-ChR2[+]). And Kit^{CreERT2–/–} mice in which CreERT2 was not knocked into the Kit allele were defined as the control (Kit-ChR2[–]) (Fig. 1B).

Figure 1. Generation of Kit^CreERT2/+;ROSA^{ChR2(H134R)/tdTomato/+} (Kit-ChR2[+]) mice. (A) The genetic strategy used to generate Kit-ChR2(+) mice. (B) PCR-based genotyping of a litter of mice. Mice B1-B2 were Kit-ChR2(+) mice containing both the Kit-CreERT2 and ChR2 (H134R)-tdTomato alleles. Mouse B3 is Kit^{CreETR2–/–} (Kit-ChR2[–]) mice, in which the Kit-creERT2 allele was missing.

Channelrhodopsin-2 Was Expressed in the Colonic Tunica Muscularis and Specifically in Interstitial Cells of Cajal in Kit-ChR2(+) Mice

Two weeks after the last injection of TAM, tdTomato-positive cells were detected in the colonic muscularis in Kit-ChR2(+) mice (Fig. 2A). In contrast, tdTomato expression was not observed in Kit-ChR2(–) mice (Fig. 2B). The ChR2(H134R)-tdTomato fusion gene encodes both ChR2 and tdTomato indicates tdTomato can be a specific fluorescence marker for cells expressing ChR2. Therefore, our results suggested that ChR2 was expressed only in Kit-ChR2(+) mice.

Figure 2. Expression of channelrhodopsin-2 (ChR2) in mouse colon. (A) TdTomato-positive cells were detected in the colonic muscularis in Kit-ChR2(+) mouse colon (scale bars = 400 μm). (B) No tdTomato-positive cell was observed in Kit-ChR2(–) mouse colon (scale bars = 200 μm). (C-E) ChR2 was expressed in Kit-positive cells. Red represents tdTomato and green represents Kit (scale bars = 50 μm).

To verify the specific expression of ChR2 in ICC, Kit antibodies were used to label ICC, and immunofluorescence was used to detect the immunoreactivity of tdTomato-positive cells. Colocalization of Kit and tdTomato suggested that ChR2 was explicitly expressed in ICC (Fig. 2C-E).

Colocalization of tdTomato and Kit immunoreactivity was quantified according to 28 randomly collected images from 8 Kit-ChR2(+) mice. To obtain a satisfactory visual effect, 2 colonic layers (circular muscularis and myenteric plexus) were the main sites used for image acquisition. Our results showed that 65.07 ± 6.07% of Kit-positive cells were tdTomato-positive cells, and 84.64 ± 3.46% of tdTomato-positive cells were Kit-positive cells.

A Single Light Stimulus Evoked Premature Low-frequency and High-amplitude Contraction of Muscle Strips in Kit-ChR2(+) Mice

Spontaneous low-frequency and high-amplitude contractions

To determine whether ChR2 was functional in ICC, particularly during the activity of ICC, isometric force recording was performed on mouse colonic circular muscles since slow waves are known to underlie phasic contractions.²⁶ Ongoing phasic contractions, including high-frequency and low-amplitude (HFLA) contractions (Fig. 3A and 3B, green arrow), as well as intermittent LFHA contractions (Fig. 3A and 3B, golden arrow), were recorded in muscle strips in mice in both groups.

Figure 3. Light stimulation evoked premature low-frequency and high-amplitude (LFHA) contraction of muscle strips in Kit-ChR2(+) mice. (A, B) Light stimulation induced premature LFHA contractions in muscle strips in Kit-ChR2(+) mice, but not in Kit-ChR2(–) mice. The green arrow represented the high-frequency and low-amplitude (HFLA) contractions and the golden arrow represented LFHA contractions. The blue rectangles represented the light stimuli. (C, D) Quantification and comparison of characteristics of spontaneous LHFA in mice of both groups. (E-G) The ratio of LFHA contractions/light stimuli in mice of both groups. *P < 0.05, ****P < 0.0001.

To quantify the characteristics of these spontaneous LFHA contractions, 17 mice, including 11 Kit-ChR2(+) mice and 6 Kit-ChR2(–) mice, were used to analyze forty-eight 10-minute isometric force recordings without any intervention. The frequency was 0.50 ± 0.04 (range 0.20-0.70) times/minute, the amplitude was 4.19 ± 0.68 (range 1.37-13.69) mN, and the duration was 30.50 ± 1.35 (range 19.38-42.17) seconds. Further results showed that the frequency of LFHA contractions in Kit-ChR2(+) mice was lower than that in Kit-ChR2(–) mice (0.45 ± 0.04 times/min vs 0.61 ± 0.05 times/minute) (P = 0.026), while no differences were detected in amplitudes (4.23 ± 1.04 mN vs 4.13 ± 0.46 mN, P = 0.950) or durations (31.43 ± 2.02 sec vs 28.79 ± 0.85 sec, P = 0.248) between the 2 groups (Fig. 3C-E).

Light-evoked low-frequency and high-amplitude contractions

In the present study, light stimulation did not alter the HFLA contractions in any mice. In contrast, a total of 76 (45.5%) LFHA contractions were detected during 167 light illuminations in mice in both groups. The ratio of LFHA contractions/light stimuli was further investigated in each group. We found that the ratio was significantly higher in Kit-ChR2(+) mice (n = 10) than in Kit-ChR2(–) mice (n = 7) (0.91 ± 0.06 vs 0.15 ± 0.05, P < 0.0001) (Fig. 3F).

To further rule out the possibility that these light-related LFHA contractions were spontaneous, we placed specific restrictions on the stimulation mode of the light. According to the characteristics of spontaneous LFHA contractions, an LFHA contraction rarely occurred within 40 seconds after the end of the previous spontaneous LFHA contraction. Therefore, we fixed the interval between the end of the last spontaneous LFHA contraction and the light stimulus to be 20 seconds. Moreover, considering the duration of LFHA (ranging between 19.38-42.17 seconds), we limited the duration of the light stimulus to 20 seconds. In this context, we again evaluated the correlation of light stimuli with LFHA contractions. The results still showed that a higher ratio of LFHA contractions/light stimuli existed in Kit-ChR2(+) mice (n = 5) than in Kit-ChR2(–) mice (n = 4) (1.00 ± 0.00 vs 0.13 ± 0.05, P < 0.0001) (Fig. 3G).

These results suggested that the majority of LFHA contractions that occurred during light stimuli might not have been spontaneous but light-related. In other words, light stimulation evoked premature LFHA contractions in the muscle strips in Kit-ChR2(+) mice but not in Kit-ChR2(–) mice (Fig. 3A and 3B).

Periodic Light Stimulation Enhanced the Inherent Frequency of Low-frequency and High-amplitude Contractions in Muscle Strips in Kit-ChR2(+) Mice

As mentioned above, colonic muscle strips exhibited ubiquitous phasic contractions and intermittent LFHA contractions, giving the LFHA contractions a certain inherent frequency. In addition, light-activated ChR2 evoked premature LFHA contractions of the muscle strips in Kit-ChR2(+) mice. Therefore, whether periodic light stimulation could cause LFHA contractions to occur at an artificially designed frequency was further investigated.

The sweep interval is the period from the end of the previous light stimulus to the start of the next stimulus, and periodic light stimulation with a specific sweep interval constitutes a train of stimuli with a particular frequency. A train of 10 light stimuli with a duration of 20 seconds per single stimulus was used to form a periodic light stimulation with a frequency of 1 time/minute (sweep intervals = 40 seconds), and isometric force recordings with and without light stimulation were analyzed.

When the colonic muscle strips were exposed to periodic light stimulation, almost every stimulus evoked a premature LFHA contraction in Kit-ChR2(+) mice. In contrast, the rhythm of colonic LFHA contractions in Kit-ChR2(–) mice was unchanged (Fig. 4A and 4B). The mean number of LFHA contractions in muscle strips increased from 4.57 ± 0.43 without periodic light stimulation to 8.27 ± 0.63 with stimulation in Kit-ChR2(+) mice (n = 5) (P < 0.0001). However, the mean number of LFHA contractions was 5.96 ± 0.32 and 5.99 ± 0.37, respectively, without and with stimulation in Kit-ChR2(–) mice (n = 6) (P = 0.847). Further analyses showed that the rhythm of LFHA contractions in Kit-ChR2(+) mice was much more sensitive to light stimulation than that in Kit-ChR2(–) mice (P < 0.0001) (Fig. 4C).

Figure 4. Periodic light stimulation enhanced the inherent frequency of low-frequency and high-amplitude (LFHA) contractions of muscle strips in Kit-ChR2(+) mice. (A, B) Isometric force recordings of colonic muscle strips in response to the periodic light stimulation in mice of both groups. The blue rectangles represented the light stimuli. (C, D) Effect of the periodic light stimulation on numbers of LFHA contractions and area under the curve in different groups. (E-G) Effect of the periodic light stimulation on characteristics of LFHA contractions in different groups. *P < 0.05, ****P < 0.0001.

The area under the curve (AUC) was also used to illustrate the different effects of ChR2 on contractions in muscle strips in both groups of mice and was calculated as the mean integrated isometric force in 10 minutes (mN_·sec) in the present study. AUC_before represents the AUC without periodic light stimulation, while AUC_under represents the AUC during periodic light stimulation. To facilitate the statistical analyses, the AUC_before was normalized to 100, and the AUC_under was adjusted as follows: (AUC_under/AUC_before) × 100. The results showed that although the AUC nonsignificantly increased to 111.36 ± 4.30 mN_·sec in Kit-ChR2(+) mice (P = 0.067) and nonsignificantly decreased to 96.97 ± 3.82 mN_·sec in Kit-ChR2(–) mice (P = 0.441), the effects of 470 blue light were significantly different between mice of both groups (P = 0.048) (Fig. 4D).

The characteristics of light-evoked LFHA contractions were also investigated. Due to the increased numbers of LFHA contractions during light exposure in Kit-ChR2(+) mice, the frequency of LFHA contractions increased synchronously from 0.46 ± 0.04 to 0.83 ± 0.06 (P < 0.0001). However, the frequency remained unchanged in Kit-ChR2(–) mice (0.60 ± 0.03 vs 0.60 ± 0.04, P = 0.852). The duration decreased from 26.47 ± 1.04 seconds to 25.03 ± 1.21 seconds (P = 0.490), while the amplitude remained unchanged (5.02 ± 2.18 mN vs 4.68 ± 1.89 mN, P = 0.369) in Kit-ChR2(+) mice. In contrast, periodic light stimulation altered neither the duration (28.65 ± 0.78 seconds vs 28.68 ± 1.01 seconds, P = 0.965) nor the amplitude (4.12 ± 0.42 mN vs 4.17 ± 0.40 mN, P = 0.583) in Kit-ChR2(–) mice. Further results revealed that periodic light stimulation led to a higher inherent frequency of LFHA contractions in Kit-ChR2(+) mice than in Kit-ChR2(–) mice (P < 0.0001) but did not cause differences in duration or amplitude between the 2 groups (P = 0.106 and P = 0.249, respectively) (Fig. 4E-G).

The Regulatory Effect of Channelrhodopsin-2 on the Low-frequency and High-amplitude Contractions in Muscle Strips Was Based on the Activity of Interstitial Cells of Cajal

It has been suggested that the Ca²⁺-activated Cl^– channel anoctamin 1 (ANO1) is crucial for the normal activity of ICC.^27-30 To determine whether the effect of ChR2 on LFHA contraction was based on its enhancement of the activity of ICC, T16Ainh-A01 (10-20 μM), an antagonist of ANO1, was used in the following study.

The phasic contractions were blocked by T16Ainh-A01 (10 µM) (Fig. 5A and 5B). The mean integrated isometric force (mN) was significantly decreased from 0.68 ± 0.14 mN to 0.25 ± 0.05 mN (P = 0.003) in all 11 mice (Kit-ChR2(+), n = 6; Kit-ChR2(–), n = 5) (Fig. 5C). In addition, the light-evoked LFHA contractions observed in Kit-ChR2(+) mice (n = 5) could not be reproduced when T16Ainh-A01 (10 µM) was administered (Fig. 5A).

Figure 5. Channelrhodopsin-2 (ChR2) regulated the low-frequency and high-amplitude (LFHA) contractions of colonic muscle strips via interstitial cells of Cajal. (A) The effect of ANO1 antagonists, T16Ainh-A01 (10-20 µM), on phasic contractions Kit-ChR2(+) mice. The light evoked LFHA contractions could not be reproduced under T16Ainh-A01 (10 µM). (B) The effect of T16Ainh-A01 on phasic contractions Kit-ChR2(–) mice. The blue rectangles represented the light stimuli, and the red lines represented the administration of T16Ainh-A01. (C) The effect of T16Ainh-A01 (10 µM) on the mean integrated isometric force. ***P < 0.001.

Colonic motor patterns are based on the cooperation of myogenic and neurogenic mechanisms.²⁶ ICC are pacemakers of the GI tract that elicit slow waves, which are the basis of myogenic phasic contractions. In the present study, we generated a mouse model utilizing the inducible Cre-loxP recombination system in which the light-sensitive protein ChR2 was expressed in Kit-labeled ICC. We found that the premature LFHA contractions in colonic muscle strips were evoked by 470 nm blue light in Kit-ChR2(+) mice. Furthermore, our results show that the inherent frequency of LFHA contractions could be enhanced almost to the artificially desired frequency when periodic light stimulation with a particular frequency was given. It is believed that the major motor pattern in the mouse colon, the colonic motor complex, is dependent upon the ENS.^22,31 However, the results from another recently published study showed that ICC in response to nitrergic neurotransmitter were also highly involved in propulsive contractions of the mouse colon.³² The current study provides some support for the idea that ICC may have an important, possibly fundamental, role in generating colonic motor patterns.

In the current study, ChR2, which is an optogenetic tool called an actuator, was genetically expressed in ICC. ChR2 is a typical actuator used in optogenetics and is derived from Chlamydomonas reinhardtii. ChR2(H134R) is a variant of ChR2 with a more robust light-induced inward current. When ChR2 is expressed in excitable cells and exposed to 470 nm blue light, it allows the passage of cations into the cell, resulting in inward currents. Subsequently, action potentials are elicited. Using this property of ChR2, the depolarization of a class of excitable cells can be specifically controlled so that the working state of the cells can be activated and inhibited by the researcher.

A recent study showed that TAM administration induced changes in contractility in isolated colons in mice, especially an increase in the number of short contractions.³³ TAM was used in both groups in our study. However, we did not compare LFHA contractions in animals that were treated with TAM and that were untreated, as both Kit-ChR2(+) and Kit-ChR2(–) mice received the same TAM injection, which ensured comparable results in isometric force recordings between the 2 groups. Therefore, the control group we set was sufficient to rule out the possibility that TAM caused light-evoked contraction of LFHA.

Our results showed that a single light stimulus evoked premature LFHA contractions in Kit-ChR2(+) mice. In contrast, a single light stimulus could not evoke any premature LFHA contraction when the ICC were disabled by T16Ainh-A01. ANO1 is one of the primary conductance regulators responsible for maintaining normal activity in ICC, and antagonism of ANO1 blocks the slow waves caused by ICC.³⁴ Therefore, the abolishment of light-evoked LFHA contractions by T16Ainh-A01 was thought to be a consequence of the inhibition of ICC by T16Ainh-A01 in Kit-ChR2(+) mice. It is generally believed that submucosal ICC and myenteric ICC are responsible for slow wave pacing in the colon, while intramuscular ICC is responsible for the transmission of neural signals. It remains unknown which subtype of ICC plays a role in light-induced LFHA contraction. We speculate that it may be the former, since the ENS in muscle strips is often incomplete. Regardless, more in-depth studies are needed to determine which subtypes of ICC are involved in light-induced LFHA contraction.

We speculate that the following mechanisms may exist. When these ChR2-expressing ICC were exposed to blue light, ChR2 caused an increase in membrane potential in the ICC. Consequently, the opening of the voltage-dependent calcium channel (Ca_v3.2)^35,36 in ICC is activated, resulting in an influx of Ca²⁺ that activates ANO1. Ultimately, the activity of ICC is enhanced, resulting in further depolarization of ICC.³⁴ The activation of colonic SMCs is then facilitated as the probability of opening of Ca_v1.2 increases when more appreciable depolarizing potentials are conducted into SMCs via gap junctions (Fig. 6).^2,27 Further studies are required to test our speculation and evaluate the potential value of this light-induced LFHA contraction for colonic transit in mice.

Figure 6. Hypotheses for possible mechanisms of this study. i) Light induces an inward current in interstitial cells of Cajal (ICC) via activation of channelrhodopsin-2 (ChR2); ii) the inward current causes depolarization of ICC; iii) some ICC have voltage-dependent inward currents (Ca_v3.2) that might amplify the depolarization and admit Ca²⁺; iv) Ca²⁺ entry causes activation of anoctamin 1 (ANO1) channels and/or Ca²⁺-induced Ca²⁺ release (CICR); v) increased intracellular Ca²⁺ concentration in ICC activates ANO1 channels; vi) Cl^- efflux through ANO1 channels is equivalent to the inward current and this causes further depolarization; vii) depolarizing currents are conducted to smooth muscle cells (SMCs) via gap junction coupling and activate L-type Ca²⁺ channels (Ca_v1.2); viii) Ca²⁺ entry in SMCs through Ca_v1.2 initiates contraction.

In this study, a potentially feasible novel approach to modulate the activity of ICC by optogenetics was shown. When the light-sensitive protein ChR2 is expressed in ICC, a single light stimulus can evoke a premature LFHA contraction, whereas periodic light stimulation enhances the frequency of LFHA contractions. This approach will help to better study the pacemaker mechanism in ICC and its contribution to colonic motility, and in-depth research may provide new insights for the clinical treatment of ICC-related motility disorders, such as constipation.

The authors would like to thank Yan Peng and Ping Li from the State Key Laboratory of Trauma, Burns and Combined Injury, Department of Occupational Disease, Daping Hospital, Army Medical University, Chongqing, China, and Xiaowei Chen from the Brain Research Center and State Key Laboratory of Trauma, Burns, and Combined Injury, Army Medical University, Chongqing, China, for their contributions to the current study.

This work was funded by the National Natural Science Foundation of China (No. 81770541) to Weidong Tong.

None.

Song Zhao and Weidong Tong conceived and designed research, and contributed new reagents or analytical tools; Song Zhao conducted experiments, analyzed data, and wrote the manuscript; and Weidong Tong substantively revised the manuscript. All authors read and approved the manuscript.