Insights on gastrointestinal motility through the use of optogenetic sensors and actuators

Abstract

The muscularis of the gastrointestinal (GI) tract consists of smooth muscle cells (SMCs) and various populations of interstitial cells of Cajal (ICC), platelet-derived growth factor receptor α⁺ (PDGFRα⁺) cells, as well as excitatory and inhibitory enteric motor nerves. SMCs, ICC and PDGFRα⁺ cells form an electrically coupled syncytium, which together with inputs from the enteric nervous system (ENS) regulate GI motility. Early studies evaluating Ca²⁺ signalling behaviours in the GI tract relied upon indiscriminate loading of tissues with Ca²⁺ dyes. These methods lacked the means to study activity in specific cells of interest without encountering contamination from other cells within the preparation. Development of mice expressing optogenetic sensors (GCaMP, RCaMP) has allowed visualization of Ca²⁺ signalling behaviours in a cell specific manner. Additionally, availability of mice expressing optogenetic modulators (channelrhodopsins or halorhodospins) has allowed manipulation of specific signalling pathways using light. GCaMP expressing animals have been used to characterize Ca²⁺ signalling behaviours of distinct classes of ICC and SMCs throughout the GI musculature. These findings illustrate how Ca²⁺ signalling in ICC is fundamental in GI muscles, contributing to tone in sphincters, pacemaker activity in rhythmic muscles and relaying enteric signals to SMCs. Animals that express channelrhodopsin in specific neuronal populations have been used to map neural circuitry and to examine post junctional neural effects on GI motility. Thus, optogenetic approaches provide a novel means to examine the contribution of specific cell types to the regulation of motility patterns within complex multi-cellular systems.

Graphical Abstract

Optogenetic activators and sensors can be used to investigate the complex multi-cellular nature of the gastrointestinal (GI tract). Optogenetic activators that are activated by light such as channelrhodopsins (ChR2), OptoXR and halorhodopsinss (HR) proteins can be genetically encoded into specific cell types. This can be used to directly activate or silence specific GI cells such as various classes of enteric neurons, smooth muscle cells (SMC) or interstitial cells, such as interstitial cells of Cajal (ICC). Optogenetic sensors that are activated by different wavelengths of light such as green calmodulin fusion protein (GCaMP) and red CaMP (RCaMP) make high resolution of sub-cellular Ca²⁺ signalling possible within intact tissues of specific cell types. These tools can provide unparalleled insight into mechanisms underlying GI motility and innervation.

The SIP (SMCs, ICC and PDGFRa⁺ cells) syncytium

The muscularis of the gastrointestinal (GI) tract exhibits regular patterns of contraction and relaxation that underlie peristalsis, segmentation, and tone, facilitating normal transport and digestion of contents within the GI lumen (Bayliss & Starling, 1899; Bayliss & Starling, 1901). These contractile patterns result from excitation-coupling of smooth muscle cells (SMCs) (Sanders et al., 2012). In addition to SMCs, GI tissues contain interstitial cells of Cajal (ICC), readily identified from SMCs by their lack of myosin and abundant expression of the tyrosine kinase receptor Kit (Maeda et al., 1992), and the Ca²⁺-activated-Cl⁻ channel Ano1 (Gomez-Pinilla et al., 2009). In addition, another population of interstitial cells are present within the GI tract, distinguished from ICC and SMCs by expression of small conductance Ca²⁺-activated K⁺ (SK3) channels (Fujita et al., 2003) and platelet-derived growth factor receptor alpha (PDGFRα⁺ cells) (Iino et al., 2009; Kurahashi et al., 2011; Kurahashi et al., 2012). Together, SMCs, ICC and PDGFRα⁺ cells form an electrically coupled syncytium, known as the SIP syncytium (Sanders et al., 2014). GI motility patterns are modulated by excitatory and inhibitory enteric motor neurons that interact with cells of this SIP syncytium (Kunze & Furness, 1999; Ward & Sanders, 2006; Sanders et al., 2010; Furness, 2012; Kurahashi et al., 2014).

Given that the muscularis of the GI tract is an amalgam of multiple cell types including, but not limited to, SMCs, ICC, PDGFRα⁺ cells, enteric neurons, glia and muscularis macrophages (MMs), it is challenging to study physiological functions of individual cell types or cellular pathways within intact GI tissues. This is made more complex by the fact that there are several sub-populations of these cells each occupying a unique anatomical and functional niche in different GI organs. For example, distinct subtypes of ICC are electrical pacemakers (Smith et al., 1987b, a; Ward et al., 1994; Huizinga et al., 1995; Ordog et al., 1999). Pacemaker ICC generate propagating depolarizing events (electrical slow waves) via activation of Ca²⁺-activated Ano1 channels (Hwang et al., 2009; Zhu et al., 2009; Hwang et al., 2016). Slow waves are transduced from ICC to electrically coupled SMCs via gap junctions, resulting in activation of voltage-gated L-type Ca²⁺ channels, leading to contraction (Sanders et al., 2014). ICC generating slow waves typically form networks at the myenteric plexus (ICC-MY) or in the case of the proximal colon, at submucosal borders (ICC-SM). Other ICC, such as intramuscular ICC (ICC-IM) do not typically generate slow waves in most regions of the GI tract, however a subpopulation of these cells act as pacemakers in the internal anal sphincter (IAS) (Hall et al., 2014; Hannigan et al., 2020).

ICC-IM serve as intermediary neuroeffector cells between enteric nerves and SMCs (Ward & Sanders, 2006; Sanders et al., 2010). While development of functional ICC does not rely on enteric neurons (Ward et al., 1999), ICC-IM are in close association with cholinergic and nitrergic nerves (Ward et al., 1998; Toma et al., 1999; Wang et al., 1999, 2000; Ward et al., 2006; Cobine et al., 2011; Blair et al., 2012b; Sung et al., 2018). Nitrergic and cholinergic responses are diminished in the absence of ICC-IM (Burns et al., 1996; Ward et al., 2000) and cholinergic responses are absent in Ano1^−/− mice (Sung et al., 2018). Similarly, PDGFRα⁺ cells are positioned near end terminals of enteric (purinergic) and extrinsic (adrenergic) motor neurons (Cobine et al., 2011; Kurahashi et al., 2011; Kurahashi et al., 2012; Baker et al., 2013; Baker et al., 2015; Kurahashi et al., 2020). Stimulation of purinergic P2Y1 receptors or adrenergic α1a receptors in PDGFRα⁺ cells leads to activation of apamin sensitive SK3 channels, leading to K⁺ efflux, hyperpolarization and a net decrease in SIP syncytium excitability (Kurahashi et al., 2011; Kurahashi et al., 2014; Kurahashi et al., 2020) (Fig. 1).

Fig. 1: The SIP syncytium:

The SIP syncytium composed of Smooth muscle cells, Interstitial cells of Cajal (ICC) and Platelet-derived growth factor receptor α⁺ (PDGFRα⁺) cells. ICC and PDGFRα⁺ cells are electrically coupled to SMCs via gap junctions. In ICC, release of Ca²⁺ from endoplasmic reticulum (ER) via inositol-triphosphate receptors (IP₃Rs) activates Ano1 channels, leading to Cl⁻ efflux, inward current and depolarization. This depolarization is transduced to SMC, leading to activation of voltage-dependent L-type Ca²⁺ channels, Ca²⁺ influx and excitation-coupling. In PDGFRα⁺ cells, release of Ca²⁺ from endoplasmic reticulum (ER) via IP₃Rs is coupled to opening small conductance K⁺ channels (SK3), K⁺ efflux, outward current and hyperpolarization. When transduced to SMC, hyperpolarization prevents opening of L-type⁺ channels, reducing SMC excitability. Activity of ICC and PDGFRα⁺ cells is modulated by enteric neurotransmission, with inhibitory purines increasing activity in PDGFRα⁺ cells while inhibitory nitric oxide and excitatory acetylcholine / substance P influence ICC activity. Together, the coordinated behaviours of cells within the SIP syncytium manifest as complex motility patterns such as peristalsis, segmentation and tone.

Enteric neurons, glia and other non-neuronal cells

While the central nervous system (CNS) is responsible for neural control of many bodily functions, the GI tract is largely controlled by a division of the autonomic nervous system (ANS) known as the enteric nervous system (ENS) (Bayliss & Starling, 1899; Mawe & Sharkey, 2016; Spencer & Hu, 2020). While neurons of the ENS function independently from the CNS, they are connected and modulated by the CNS via both parasympathetic (vagus, pelvic splanchnic) and sympathetic pathways that arise from the thoracolumbar spinal cord and synapse in the coeliac, superior mesenteric and inferior mesenteric ganglia. These postganglionic axons synapse with enteric neurons and within the SIP syncytium (Ward & Sanders, 2006; Furness et al., 2014; Spencer & Hu, 2020). Parasympathetic nerves to the GI tract are excitatory while sympathetic nerves are inhibitory to most of the GI tract but excitatory to some regions such as the IAS (Cobine et al., 2007; Spencer & Hu, 2020).

Both enteric motor and sensory neurons are present within the gut. Excitatory motor pathways include cholinergic, tachykinergic and purinergic pathways (Lee et al., 1993; Lee et al., 1995; Lee et al., 2005) while inhibitory motor pathways include nitrergic, purinergic and vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase activating polypeptide (PACAP) pathways (Sanders & Ward, 2019). Intrinsic sensory or primary afferent neurons (IPANS; Dogiel Type I and II, intestinofugal) and extrinsic nerve endings (vagal and spinal afferents) are also present within the gut (Smith et al., 2007; Spencer & Hu, 2020). Intestinofugal neurons typically have their cell bodies in the myenteric plexus but project to sympathetic neurons in the prevertebral ganglia (Hibberd et al., 2020). They are thought to influence the inhibition of contractile activity by sympathetic neurons (Kreulen & Szurszewski, 1979). Dogiel Type II or AH-neurons represent mechanosensitive IPANs that project from the myenteric plexus to the mucosa and they receive fast and slow inputs from other enteric neurons. In contrast, Dogiel Type I or S-neurons, are chemosensitive as well as mechanosensitive. Unlike Dogiel Type II neurons, Dogiel Type I neurons represent interneurons and motor neurons as well as IPANs, and do not typically project into the mucosa (Spencer & Hu, 2020). Interneurons are important in coordinating motor responses within the colon (Smith et al., 2007; Perez-Medina & Galligan, 2019).

Both enteric neurons and enteric glial cells are derived from the neural crest and are considered part of the ENS although glia are 4–6 times more abundant than neurons (Neunlist et al., 2013; Rosenberg & Rao, 2021). Enteric glia share several characteristics with astrocytes in the CNS including the expression of glial fibrillary acidic protein (GFAP) and the Ca²⁺ binding protein S100ß as well as a close association with enteric neurons (Boesmans et al., 2015; Grubisic & Gulbransen, 2017). Enteric neural crest derived cells express both Wnt1 and Sox10. However, during differentiation, Sox10 is downregulated in those cells that become neurons but retained in most glia (Young et al., 2003; Laranjeira et al., 2011). Thus, Sox10 expression can differentiate between neurons and glia in whole GI preparations (Laranjeira et al., 2011; Boesmans et al., 2015; McClain & Gulbransen, 2017). In terms of a functional interaction between enteric neurons and glia, some of the earliest studies by Brian Gulbransen and Keith Sharkey revealed that neurons signal to glia via purinergic signalling pathways (Gulbransen & Sharkey, 2009) and that glia are targets of sympathetic innervation (Gulbransen et al., 2010). In addition to their interactions with neurons, glia are associated with a number of other non-neuronal cells such as epithelial cells, enterochromaffin cells and immune cells (Neunlist et al., 2007; Bohórquez et al., 2014; Ibiza et al., 2016; Seguella & Gulbransen, 2021).

Debate on the role of specific cell types in GI motility

With these many cell types, plus some others we have not even discussed here, e.g., enterochromaffin cells, mast cells, etc., the importance of one cell type in regulating GI function over another is often a topic of debate. Such areas of controversy include the role of ICC versus SMCs and enteric neurons versus ICC. As discussed in a later section, the role of ICC as mediators of neuromuscular transmission has been a frequent topic of debate (Goyal, 2016; Sanders et al., 2016a, b) since neural responses have been shown to be diminished in some studies using animals models lacking ICC (Burns et al., 1996; Ward et al., 2000) but not others (Zhang et al., 2009). Discrepancies between studies likely arise, at least in part, because of the insufficiency of the transgenic animals utilised. For example, the W/Wv mouse which has one null Kit allele and one Kit allele with a point mutation (Iino et al., 2007; Iino et al., 2011) is commonly used to investigate how neural responses are altered in the absence of ICC. However, this is not a perfect model as several populations of ICC persist throughout the GI tract (Iino et al., 2007). Additionally, a frequent problem with constitutive knockout or mutant animals such as the W/Wv is the probability of these models acquiring compensatory mechanisms. Thus, while Kit mutant models do provide valuable insight into the roles of certain ICC populations, data obtained using these animals should be interpreted with caution.

Recent advances in the field of optogenetics have provided a unique opportunity to visualize or modify the activity of distinct populations of cells within intact preparations using light stimulation and genetic manipulation (Asano et al., 2021). Optogenetic techniques can be split into activators/inactivators (such as light activated rhodopsins) and reporters (such as genetically encoded Ca²⁺ indicators (GECIs)). The advantage of combining optical imaging and genetically encoded fluorescent sensors or activators provides the capability of targeting specific cell populations, increasing spatial and temporal resolution (Deisseroth, 2011). While initially conceived in the CNS, optogenetic techniques have proven invaluable in the study of other complex integrated cellular systems, including the GI tract (Boesmans et al., 2015, 2018; Johnson et al., 2020). In subsequent sections, we will review general optogenetic techniques and how they have been used to study the cellular systems underlying GI motility patterns.

Optogenetic activators

The most well-known optogenetic activators are the channelrhodopsin (ChR) proteins, light-gated cation channels, originally isolated from the cell membrane of Halobacterium in the 1970s. ChR1 & 2 were successfully cloned from algae Chlamydomonas reinhardtii (Crick, 1999) and their functional expression in oocytes of Xenopus laevis and mammalian cells (Nagel et al., 2002; Nagel et al., 2003; Li et al., 2005; Nagel et al., 2005; Bi et al., 2006; Ishizuka et al., 2006) revealed that blue-light stimulation results in a net influx of positive ions (mainly Na⁺) and depolarization. This occurs as ChRs undergo a conformational change when activated by blue-light leading to opening of the channel pore (Hegemann & Nagel, 2013) (Fig. 2). Several reports in smooth muscle research have utilized optogenetic approaches to examine cellular functions and control. For instance, modulation of bladder SMCs via the expression of ChR2 was employed to actively control urinary bladder contraction with spatial and temporal accuracy (Park et al., 2017). Optical actuators have been expressed in vascular SMCs to control cerebral blood flow (Abe et al., 2021). Use of ChR2 in neurons is well documented and by using ChR2 with different excitation kinetics (such as the CsCrimson red shifted opsin) expressed in different neuronal cells, it is possible to independently activate different neural populations within the same preparation by changing the excitation light wave length (Klapoetke et al., 2014).

Fig. 2: Optogenetic activators:

Channelrhodopsins (ChR2), OptoXR and halorhodopsinss (HR), Guillardia theta channel rhodopsins (GtACR) and archaerhodopsins (AR) proteins can be genetically encoded into specific cell types within the GI tract. ChR2 are cation channels (Na⁺ shown for example) that are activated by blue light. OptoXR are G-protein coupled receptors (GPCR) that are activated by blue light and can be used to activate a range of cellular signalling pathways. HR and GtACR are families of Cl⁻ channels or pumps that when activated by light affect movement of Cl⁻ ions. ARs are proton pumps that move H⁺ ions out of cells. In enteric neurons, activation of ChR2 would lead to Na⁺ influx and depolarization, resulting in neurotransmitter release that would lead to downstream effects on cells within the SIP syncytium. In contrast, activation of HR, GtACR or AR in enteric neurons, would lead to Cl⁻ influx or H⁺ efflux and neural silencing. OptoXR activation could lead to numerous intracellular signalling cascades depending on the GPCR that it is linked to.

Other channelrhodopsins can be used in neuronal silencing, instead of activation. Anion channelrhodopsins isolated from the alga Guillardia theta (GtACR1 and GtACR2) and halorhodopsins (NpHr) are light activated Cl⁻ channels activated by excitation light at 515, 470 and 580 nm respectively (Deisseroth, 2011; Hegemann & Nagel, 2013). When neurons expressing GtACR1, GtACR2 or NpHr are exposed to the appropriate light excitation, it leads to the influx of Cl⁻ ions into these cells, hyperpolarizing and inactivating them (Zhang et al., 2007; Mohamed et al., 2017; Mohammad et al., 2017) (Fig. 2). Neuronal inactivation can also be accomplished by activation of archaerhodopsins, which are membrane bound light activated proton pumps. When activated (550 nm excitation wavelength), archaerhodopsins pump protons out of neurons leading to hyperpolarization and neuronal silencing (Chow et al., 2010). Use of archaerhodopsins is sometimes preferred over halorhodopsins as they spontaneously recover from light induced inactivation, unlike halorhodopsins which can enter long lasting light induced inactivation states (Chow et al., 2010). Optogenetic tools used to modulate cellular activity are not only limited to rhodopsins as several other light-activated proteins that can modulate intracellular signaling pathways are also available. Most of these proteins are coupled to G-proteins such as melanopsin which is coupled to G_q (Koizumi et al., 2013). There are also genetically engineered proteins, such as OptoXRs, a set of G-protein modulating opsins that that can be coupled to different signaling pathways (Airan et al., 2009; Rost et al., 2017) (Fig. 2).

Optogenetic reporters

The availability of optogenetic reporters has made it possible to study specific cell types more selectively over traditional methods such as microelectrode recording and loading of whole tissues with Ca²⁺ dyes. Expression of Cre recombinases driven off cell-specific promoters with Cre-lox P recombination techniques (Sauer & Henderson, 1988; Gu et al., 1994) and the FLP-FRT recombination strategy (Sadowski, 1995), has allowed for fast and efficient expression of optogenetic reporters in specific cell types. Advancements in optical methods, including 2-photon, confocal and light-sheet microscopy combined with improvements of GECIs, allowed for in-depth cellular spatial resolution, resulting in a reduction of background fluorescence contamination (Diliberto et al., 1994; Hayashi & Miyata, 1994; Bolton & Gordienko, 1998; Deisseroth et al., 2006; Pittet & Weissleder, 2011; Boesmans et al., 2015; Drumm et al., 2019a).

Optogenetic reporters are now used in many different applications including monitoring cellular Ca²⁺ (Nakai et al., 2001; Ji et al., 2004; Tallini et al., 2006; Chen et al., 2013) detecting membrane voltage (Siegel & Isacoff, 1997), observing metabolite production (Rogers & Church, 2016), and RNA sensing (Ying et al., 2017). Expression of GECIs such as GCaMP (a fusion protein of green fluorescent protein (GFP) and calmodulin (Nakai et al., 2001)) can be driven off promoters expressed by specific cells in the GI SIP synticium such as smooth muscle myosin heavy chain, Acta, PDGFRα or c-Kit. When Ca²⁺ is bound to the calmodulin domain, GCaMP undergoes a conformational change to expose GFP which has a peak excitation of 488 nm and a peak emission of 510 nm (Fig. 3). Throughout the following sections, we will review how the optogenetic tools described above have been applied to the complex multi-cellular systems that make up the muscularis of the GI tract.

Fig. 3: Optogenetic reporter (GCaMP):

GCaMP is a genetically encoded Ca²⁺ reporter that emits light >510 nm when excited with blue light at 488 nm. GCaMP is a fusion protein of green fluorescent protein (GFP), calmodulin (CaM) and a peptide sequence from myosin light chain kinase (M13). In the absence of Ca²⁺, the CaM and M13 domains of GCaMP are physically separated, resulting in exposure of GFP to water molecules which prevent its ability to fluoresce by keeping the GFP in a protonated state. In the presence of Ca²⁺, CaM undergoes a conformational change which allows it to bind to the M13 domain. This conformational change prevents water molecules from accessing the GFP, allowing it to deprotonate and fluoresce in its native anionic state.

Application of optogenetics to cells of the SIP syncytium

The conductances in ICC (Ano1 channels) and PDGFRα⁺ cells (SK3 channels) that regulate SMC contractility are both Ca²⁺ dependent (Fig. 1), and activation of pacemaker conductances in ICC relies on Ca²⁺ release from the endoplasmic reticulum (ER) (Zhu et al., 2015). This suggests that Ca²⁺ signalling in interstitial cells is fundamental for the regulation of GI motility. Through traditional loading of GI tissues from several species, with cell-permeable Ca²⁺ indicators such as Fura or Fluo, various investigators have attempted to visualize Ca²⁺ activity in ICC or PDGFRα⁺ cells (Park et al., 2006; Lee et al., 2007b, a; Lee et al., 2009; Lowie et al., 2011; Baker et al., 2013; Singh et al., 2014; Baker et al., 2015; Chevalier et al., 2020). These techniques were also used to determine how ICC respond to stimulation of enteric nerves (Lee et al., 2009; Huizinga et al., 2014). However, these approaches had inherent limitations such as indiscriminate loading of dye, resulting in contaminating signal from non-interstitial cells. Furthermore, these dyes lack photo-stability resulting in photobleaching, therefore these tissues could only be visualized for short durations at low magnifications. During faster image acquisitions, the duration of cell exposure to excitation light is shorter per image, thus to boost signal to noise ratios during such recordings, excitation intensity must be increased to compensate for the shorter exposure time. This leads to an increased risk of photobleaching and cellular phototoxicity, which is exacerbated at higher magnifications (Bayguinov et al., 2010b, a; Bayguinov et al., 2012; Okamoto et al., 2012). GCaMP however, is much more photostable than traditional Ca²⁺ dyes. The neutral configuration (no bound Ca²⁺) of the GCaMP molecule does not absorb excitation light and thus the Ca²⁺ sensor is only susceptible to photobleaching when Ca²⁺ is bound to it (Fig. 3), as opposed to traditional Ca²⁺ dyes that are constantly absorbing light under excitation (Hennig et al., 2015; Barnett et al., 2017; Kleist et al., 2017). The availability of mice with cell-specific expression of GCaMP (Fig. 3) has allowed direct monitoring of Ca²⁺ activity of SIP cells in situ. This activity can be recorded at high magnifications, with fast rates of acquisition, capturing temporally brief and spatially restricted Ca²⁺ signals in a manner not possible previously.

Ca²⁺ imaging of SIP cell activity with cell-specific GCaMP mice

Using Cre recombinase-loxP technology, GCaMP can be expressed in specific SIP cells (Fig. 4). This was first accomplished when GCaMP3 was expressed in ICC using a Cre recombinase driven off the Kit promoter within ICC located at the deep muscular plexus (ICC-DMP) of the mouse small intestine (Baker et al., 2016). In a similar manner, GCaMP6f was expressed in ICC and these mice have been used to examine Ca²⁺ signalling within ICC-IM of the proximal colon (Drumm et al., 2019b; Drumm et al., 2020a; Drumm et al., 2020b; Baker et al., 2021b) and lower esophageal sphincter (LES) (Drumm et al., 2022). Utilization of in situ GCaMP imaging at high frame rates (>33-100 frames per second) and high magnifications (40-100x, spatial resolution of 0.27-0.2μm per pixel), enabled the characterization of Ca²⁺ activity in ICC-DMP and ICC-IM. Spatio-temporal mapping and particle analysis were used to quantify this activity (Drumm et al., 2019a; Leigh et al., 2020), revealing stochastic firing patterns and pharmacological inhibition profiles similar to spontaneous transient depolarizations (STDs) or ‘unitary potentials’ recorded from several GI muscles (Edwards et al., 1999; Hirst & Edwards, 2001; Beckett et al., 2002; Kito et al., 2002; Kito & Suzuki, 2003; van Helden & Imtiaz, 2003). This suggested that STDs represent the electrophysiological signature of ICC-DMP and ICC-IM Ca²⁺ signals. This activity occurred at multiple sites, hundreds of times per minute in a single ICC-DMP or ICC-IM, summating to generate a net inward current (via activation of Ano1 channels) that set the resting membrane potential of the SIP syncytium (Baker et al., 2016; Drumm et al., 2019b; Drumm et al., 2022).

Fig. 4: Cell-specific Cre recombinase mice breeding:

Cre recombinases can be used to express optogenetic proteins in a cell-specific manner within the GI tract. The breeding strategy to generate mice expressing GCaMP6f specifically in ICC is shown for example. Mice expressing a Cre recombinase driven by a cell-specific promoter (c-Kit⁺/Cre-ER^T2, c-Kit is a tyrosine kinase receptor expressed in ICC) are crossed with mice expressing floxed GCaMP6f driven by a Rosa26 promoter. Resulting c-Kit⁺/Cre-ER^T2-GCaMP6f mice are injected with tamoxifen at 8–10 weeks of age to induce recombination of lox P sites in c-Kit⁺ cells; Rosa26 can then drive expression of GCaMP6f in these cells. Similar breeding strategies can be utilized with different cell specific drivers of Cre recombinase (e.g., Acta, GFAP, Nos1, Wnt1, Sox10, etc.) with different optogenetic proteins (GCaMP, RCaMP, CHr2, HR, OptoXR).

While most optogenetic work on SIP cells has focused on ICC (detailed in subsequent sections), similar approaches have been used to study PDGFRα⁺ cells. The functions of PDGFRα⁺ cells in the GI tract were first examined in mice expressing a histone 2B–eGFP fusion protein confined to nuclei of PDGFRα⁺ cells ((PDGFRα-eGFP)(Kurahashi et al., 2011)). PDGFRα⁺ cells highly express transcripts for P2Y1 receptors and SK3 channels and generate outward currents in response to purines. To examine Ca²⁺ signaling in these cells within the gastric fundus and colon, preparations of PDGFRα-eGFP mice were loaded with the Ca²⁺ dye Oregon Green 488 BAPTA-2 AM (Baker et al., 2013). These studies revealed that exogenous addition of purines or stimulation of purinergic signalling pathways with electrical field stimulation enhanced Ca²⁺ signaling within PDGFRα⁺ cells in situ. More recently, Ca²⁺ signalling was examined in colonic PDGFRα⁺ cells using PDGFRα-GCaMP6f mice (Kurahashi et al., 2020). In this study, Ca²⁺ signalling was enhanced by activating α1a-adrenoceptors (noradrenaline, phenylephrine) or P2Y1 receptors. Addition of agonists resulted in activation of apamin-sensitive outward currents, hyperpolarization and inhibition of phasic contractions, revealing that PDGFRα⁺ cells play an important role in adrenergic regulation of colonic motility.

Elucidation of physiological mechanisms in ICC by optogenetic reporters

Slow waves are rhythmic electrical events generated by pacemaker ICC (ICC-MY and ICC-SM). Slow waves consist of an upstroke and plateau phase and are dependent upon Ano1 channels activated by intracellular Ca²⁺ signalling. It was previously assumed that activation of excitatory conductances during the plateau phase (the duration of which ultimately determines the force of downstream SMC contraction) was due to a uniform rise in intracellular Ca²⁺ in ICC (Park et al., 2006; Lee et al., 2007a). This has now been dispelled by high magnification Ca²⁺ imaging of ICC-MY and ICC-SM in Kit-GCaMP mice. In these ICC, Ca²⁺ transients cluster asynchronously across multiple intracellular sites at the onset of slow waves (Drumm et al., 2017; Zheng et al., 2020; Baker et al., 2021a; Baker et al., 2021b). These Ca²⁺ transients are brief, lasting only a few hundred milliseconds and may occur multiple times during the plateau of a slow wave.

Simultaneous imaging of multiple cell types in the same preparation is possible by using a dual imaging system with tissues from mice expressing two separate optogenetic reporters that emit fluorescence at distinct wavelengths. This has been accomplished in intact colonic preparations where GCaMP ‘green’ is expressed in ICC and a ‘red’ Ca²⁺ indicator (RCaMP) is expressed in SMCs (driven off Acta, a specific SMC promoter), thus allowing cellular activity to be recorded and correlated across multiple cells (Baker et al., 2021b). This approach demonstrated that clustering of Ca²⁺ transients in ICC-SM leads to activation of SMCs and the duration of the ‘clustering period’ corresponds to the duration and force of SMC contraction. While it was previously thought that this pacemaker mechanism also required Ca²⁺ handling by mitochondria, recent Kit-GCaMP imaging has called this into question owing to the non-specific effects of mitochondrial inhibitors (Drumm et al., 2018).

As discussed in an earlier section, the question of whether ICC-IM mediate enteric neurotransmission has proven controversial. With Kit-GCaMP mice, this question can be addressed in a more specific manner. Activation of enteric neurons with electrical field stimulation in imaging studies of Kit-GCaMP mice show that small intestine ICC-DMP (Baker et al., 2018a; Baker et al., 2018b), colonic ICC-IM (Drumm et al., 2020a) and subserosal ICC (ICC-SS) of the proximal colon (Drumm et al., 2020b) are indeed innervated by both excitatory and inhibitory nerves. These findings corroborate previous studies that suggested a role for ICC as mediators of nitrergic and cholinergic neurotransmission (Burns et al., 1996; Ward et al., 2000; Sung et al., 2018).

Other physiological questions regarding the role and control of SIP cells have also been answered using Kit-GCaMP mice. For example, in the proximal colon, nitrergic nerves suppress spontaneous electrical and contractile activity, a phenomenon known as tonic inhibition (Christensen et al., 1978; Middleton et al., 1993; Rae et al., 1998; Spencer et al., 1998; Dinning et al., 2006; Spencer et al., 2016). This aids in generating orderly, propulsive contractions of the smooth muscle (Wood, 1972; Mule et al., 1999; Dinning et al., 2006). Impairment of tonic inhibition is linked to chronic constipation and intestinal pseudo-obstruction (Wood et al., 1986). The post-junctional effector cells and mechanisms responsible for mediating tonic inhibition were unknown until in situ imaging studies were carried out in the proximal colon of Kit-GCaMP mice (Drumm et al., 2019c). These studies revealed that Ca²⁺ transients in ICC-IM were increased in frequency by addition of tetrodotoxin (TTX), L-NNA (an inhibitor of nitric oxide synthase) or ODQ (an inhibitor of soluble guanylate cyclase). Furthermore, addition of TTX, L-NNA or ODQ increased the frequency and amplitude of colonic contractions, and this effect was reversed when Ca²⁺ signalling in ICC-IM was inhibited with pharmacological agents that disrupt Ca²⁺ release (thapsigargin) or Ca²⁺ influx via Orai channels (GSK 7975A) (Drumm et al., 2019c). These data indicate that nitrergic nerves tonically inhibit contractile activity via inhibiting Ca²⁺ transients, and subsequently Ano1 channels in ICC-IM.

Novel functions of SIP cells as revealed by optogenetic reporters

Ano1-positive ICC-IM are found within the various muscle bundles of the IAS but ICC-MY and ICC-SM are absent (Hall et al., 2014; Cobine et al., 2017). Using Kit-GCaMP mice it was revealed that there are two distinct sub-populations of ICC-IM within the IAS (Hannigan et al., 2020). Type I ICC-IM behaved as ICC-IM described elsewhere in the GI tract, in that they had localized Ca²⁺ transients whereas Type II ICC-IM exhibited rhythmic whole cell Ca²⁺ transients that occurred at a similar frequency to slow waves. Thus, Type II ICC-IM are likely pacemaker cells within the IAS whereas Type I ICC-IM may be involved in mediating neural responses. Additional studies in mice expressing GCaMP6f in SMCs (SM-GCaMP6f) revealed rhythmic high frequency Ca²⁺ transients in SMCs of the IAS (Cobine et al., 2020). These Ca²⁺ transients were highest in frequency at the distal edge of the IAS declining in frequency in the proximal direction, consistent with IAS slow wave activity (Hall et al., 2014). These rhythmic Ca²⁺ transients in IAS SMCs likely contribute to the generation of tone to maintain faecal continence (Keef & Cobine, 2019).

A unique population of stellate-shaped ICC connected to the longitudinal muscle (LM) exist at the level of the subserosa in the proximal colon (ICC-SS) (Toma et al., 1999; Vanderwinden et al., 2000; Aranishi et al., 2009; Rumessen et al., 2013). However, apart from their anatomical location and their expression of Ano1 channels (Gomez-Pinilla et al., 2009; Blair et al., 2012a), little was known about how these cells affect LM-SMC function until recently. Using SMC-GCaMP6f mice to image Ca²⁺ transients within SMCs (Drumm et al., 2020b), it was found that Ca²⁺ flashes and LM contractions were abolished by either inhibiting Ano1 channels or Ca²⁺ influx pathways essential for Ca²⁺ release in ICC (such as via Orai channels (Zheng et al., 2018)). Imaging activity of ICC-SS in situ with Kit-GCaMP mice demonstrated that these cells fired Ca²⁺ transients in a similar manner to ICC-DMP and ICC-IM, suggesting that ICC-SS contributed to the regulation of SMC motor activity. The emergent property of regular LM-SMC contraction in this region appeared to arise from ICC-SS firing Ca²⁺ transients that activated Ano1 channels causing depolarization, which was transduced to LM-SMCs to trigger L-type Ca²⁺ channel opening, Ca²⁺ influx and contraction. Due to the restricted anatomical niche that ICC-SS occupy, traditional Ca²⁺ dye imaging would likely not have yielded the high-resolution recordings required to investigate these mechanisms.

Application of optogenetics to studies of enteric neurons

GECIs such as GCaMP and genetically encoded optical actuators such as ChRs have been utilised extensively to study the functions of enteric neurons and glial cells. This has been made possible by the availability of various CreER^T2 mice that drive recombinase expression in specific neural or glial cells such as Wnt1-CreER^T2 (wingless-related MMTV integration site 1; neural crest derived cells), Sox10-CreER^T2 (glial cells transcription factor), GFAP-CreER^T2 (Glial fibrillary acidic protein⁺ cells), Nos1-CreER^T2 (nitric oxide synthase⁺ nerves) and ChAT-CreER^T2 (cholinergic nerves) mice and the availability of chemogenetic models such as the GFAP-hM3Dq mouse where hM3Dq, a modified DREADD receptor (designer receptor exclusively activated by designer drugs) is driven by GFAP.

The different CreER^T2 mice listed above have been crossed with various GCaMPs to generate animals that can be used to selectively examine Ca²⁺ transients in particular cells of interest. For example, one study (Hennig et al., 2015) utilized mice with different cell specific Cre driver lines (as outlined above) to selectively express GCaMP3 in either enteric glia, all enteric nerves, or nitrergic / cholinergic nerves. Using this methodology, Ca²⁺ transients in specific sub-populations of cells in the myenteric and sub-mucosal plexus were examined. These studies revealed that between peristaltic contractile waves in the colon, i.e., colonic migrating motor complexes (CMMCs), nNOS⁺ nerves were spontaneously active. This activity was reduced during CMMCs while the activity of interneurons was increased during this same period. Conversely cholinergic motor neuronal activity often increased during CMMCs.

Optogenetic ChR2 activators and GCaMP reporters have also been used to study CMMCs and tonic inhibition of colonic muscles in mice. One study using mice with GCaMP3 expressed in both nNOS⁺ and choline acetyltransferase (ChAT)⁺ neurons (Gould et al., 2019), demonstrated that Ca²⁺ activity in nNOS⁺ nerves was greatest during periods of tonic inhibition, rather than during CMMCs, while conversely ChAT⁺ neurons markedly increased their activity during CMMCs. In support of this observation (i.e, nitrergic neuronal activity being depressed during CMMCs or vice versa), this same study also found that activation of nNOS⁺ neurons with blue light in Nos1-CreER^T2-ChR2 mice, resulted in inhibition of CMMCs (Gould et al., 2019).

Optogenetic stimulation can also be used to evoke CMMCs (as opposed to those induced by elongation or brush stimulation) as has been accomplished in mice expressing ChR2 in calretinin⁺ neurons which are mostly cholinergic in nature (Hibberd et al., 2018). In these studies, an increase in CMMCs (termed CMCs in this study) was observed after blue light stimulation of calretinin⁺ nerves. This is again consistent with cholinergic neurons being excitatory motor neurons in the colon (Wang, 2018; Spencer & Hu, 2020). Furthermore, this was the first study to demonstrate that optogenetics could be used to stimulate CMMCs in the colon ex vivo and that colonic motility could be increased in vivo using wireless optogenetic activation with light emitting diodes implanted on the colon wall (Hibberd et al., 2018). It should be noted, that while experiments conducted in mice with ChR2 expressed in calretinin⁺ neurons demonstrate that optogenetics are a useful tool to induce CMMCs in colonic tissues, there has been less success in optogenetically evoking similar migrating motor patterns in the small intestine of these same mice (Spencer et al., 2020)). This suggests that investigators should not assume that optogenetic activation of cellular responses is ubiquitous across all GI tissues.

Another study that examined migrating myoelectric complexes (MMCs) in the small intestine of mice lacking pacemaker ICC (W/Wv mouse (Spencer et al., 2003)) concluded that MMCs occurred in the absence of electrical slow waves and therefore ICC-MY and slow waves were not necessary for MMC generation. However, it should be noted that other ICC populations that participate in neurotransmission within the small intestine, i.e., ICC-DMP, persist in these mice (Ward et al., 2006; Iino et al., 2007; Iino et al., 2011; Baker et al., 2018a; Baker et al., 2018b). Thus, whilst slow waves and pacemaker-type ICC-MY are not required for MMCs in the small intestine (Spencer et al., 2003), it is possible that some types of ICC (but not ICC-MY) may still play a key role in the generation of MMCs in the small bowel.

Early studies examining the mechanisms underlying generation of CMMCs suggested that enteric neurons were primarily responsible, occurring via turning off inhibitory neural pathways or activation of excitatory neurons (Lyster et al., 1995). However, these findings have been contested by more recent studies that find that CMMCs result from a pre-synaptic inhibition of inhibitory neurotransmitter release (Spencer et al., 1998) and a coordinated firing pattern in myenteric neurons (Spencer et al., 2018; Spencer et al., 2021).

Other studies in the mouse colon suggested that CMMCs are likely generated by a “synergistic interaction between neural and ICC networks” (Dickson et al., 2010) with excitatory transmission enhancing ICC pacemaker activity (Bayguinov et al., 2010a).Recently, optogenetic experiments have revealed a potential new role for ICC in the generation of propulsive contractions in the colon (CMMCs). In a variety of animal models, including human, CMMCs are reduced, but not abolished, by atropine, thus they cannot be due entirely to rhythmic firing patterns of excitatory myenteric nerves (Koh et al., 2022). In these studies, CMMCs were inhibited by ablating an ICC-specific conductance (Ano1) either pharmacologically or genetically, suggesting that ICC were responsible for generating CMMCs. Using mice expressing ChR2 in Nos1⁺ nitrergic neurons, it was demonstrated that stimulation of Nos1-Chr2⁺ nerves with blue light led to generation of CMMCs and enhanced colonic transit (Koh et al., 2022). This effect was abolished either by L-NNA or by deletion of the Nos1 gene. Furthermore, by imaging Ca²⁺ transients in the colon of Nos1-GCaMP6f mice it was demonstrated that these nerves fire during mucosal stimulation, but CMMCs only occur after the cessation of stimulation. Previous studies indicated that colonic ICC-IM are spontaneously active (Drumm et al., 2019b; Drumm et al., 2019c), and during periods of nerve stimulation in the presence of atropine ICC-IM Ca²⁺ transients were inhibited. Therefore, the role of ICC in these responses were examined in Kit-GCaMP6f mice (Koh et al., 2022). With cessation of the neuronal stimulus, ICC Ca²⁺ transients rebounded with a greater frequency than the pre-EFS period. These ICC Ca²⁺ responses were inhibited by L-NNA or ODQ. Through a series of further experiments, it was concluded that CMMCs occur via by the activation of Nos1⁺ neurons which inhibit Ca²⁺ release in ICC via an NO-cGMP dependent mechanism. During periods of nitrergic stimulation, cGMP also inhibits phosphodiesterase 3a (PDE3A). Inhibition of PDE3A leads to a buildup of cAMP and protein kinase A in ICC, resulting in increased phosphorylation of phospholamban (PLB) which would enhance Ca²⁺ activity of the sarcoplasmic/endoplasmic reticulum pump (SERCA). This summates to an overloaded ER Ca²⁺ store in ICC, which upon cessation of NO mediated inhibition of Ca²⁺ release, resulting in a post-stimulus rebound increase in ICC Ca²⁺ transient firing, and activation of large numbers of Ano1 channels, leading to large depolarizations in SMCs and propulsive CMMCs (Koh et al., 2022).

Optogenetics has also enabled the specific activation of other cell types in the mouse colon that may be involved in CMMCs. Sensory enteroendocrine cells (EECs) have been proposed to play a role in generating colonic propulsive contractions but this has been disputed as there have been few attempts to directly activate these cells within intact preparations (Treichel et al., 2022). Recent experiments with mice containing EECs expressing a red-shifted ChR (ReaChR) demonstrated that optical stimulation of EECs increased the frequency (but not amplitude or duration) of propulsive contractions in the intact mouse colon (Treichel et al., 2022). This effect was linked to an optically induced release of 5-HT from EECs. While the findings of these studies require further scrutiny and exploration, their novel conclusions were made possible using optogenetic sensors and activators in nerves, ICC and EEC and provides potential new mechanisms for CMMC formation and may require a reconsideration of how fundamental motility patterns in the colon are generated.

Small intestinal motility has also been shown to be modulated in mice by activation of ChR2 in nitrergic neurons. One study examined the effects of stimulating NOS⁺ GABAergic neurons on contractility of the small intestine in Nos1-CreER^T2-ChR2 mice (Rakhilin et al., 2016). With blue light stimulation of these neurons, all contractile activity was abolished indicating that activation of nitrergic nerves by this method could regulate small intestine motility. In a similar manner, excitatory (EJPs) and inhibitory junction potentials (IJPs) have been recorded from the colon of mice expressing ChR2 in cholinergic neurons (ChAT-ChR2-YFP Bac mice; (Perez-Medina & Galligan, 2019)) after stimulation with blue light. IJPs were reduced by a nicotinic antagonist and abolished with the combined addition of purinergic and nitrergic antagonists. EJPs were abolished by addition of a muscarinic antagonist. This study indicates that cholinergic interneurons synapse with inhibitory and excitatory motor neurons to regulate colonic motility. From these studies it is apparent that ChRs can be used to study integrated enteric neural circuitry.

Optogenetic approaches have also been used in examining how extrinsic sympathetic nerves regulate GI motility (Smith-Edwards et al., 2021). These studies utilized E2aCre-GCaMP mice which globally express GCaMP as well as NPY-CreER^T2-ChR2 mice which express ChR2 in NPY⁺ sympathetic neurons. When lumbar colonic, mesenteric or hypogastric nerves were stimulated with electrical field stimulation (EFS), there was an increase in Ca²⁺ activity in sympathetic postganglionic axons which correlated to region-specific changes in colonic motility. These responses were also observed when extrinsic sympathetic nerves were activated with blue light in NPY-CreER^T2-ChR2 mice, further supporting their function in regulating colonic motility. Furthermore, these studies revealed that sympathetic nerves regulate non-neuronal cells such as glia, interstitial cells and epithelial cells (Smith-Edwards et al., 2021). However, it should be noted that NPY is also expressed in enteric neurons, so interpretation of the NPY-CreERT-ChR2 mice should be done cautiously as not all effects are necessarily due to activation of sympathetic neurons.

In addition to studying how components of the ENS regulate GI motility, optogenetic approaches have been used to study the development of the ENS. To this end, one group transplanted ChR2 expressing enteric neural stem cells into colons of adult mice (Stamp et al., 2017). These cells gave rise to neurons that formed a plexus within the circular muscle layer. Stimulation of these ChR2 expressing neurons with blue light evoked changes in electrical activity indicating that these neurons were functional.

Optogenetic recording and stimulation of non-neuronal enteric cells

From studies with Wnt1-CreER^T2-GCaMP3 mice, it was demonstrated that glia signal to enteric neurons via the release of purines through pannexin channels, as glial Ca²⁺ transients were abolished by purinergic antagonists or probenecid, a pannexin antagonist (Boesmans et al., 2019). These findings confirmed earlier studies indicating glia and neurons communicate via purinergic signaling pathways (Gulbransen & Sharkey, 2009). Other studies using Sox10-CreER^T2-GCaMP5g mice have revealed that there is an increase in intraganglionic glial Ca²⁺ transients with activation of muscarinic receptors (Delvalle et al., 2018b). These methodologies reveal that tachykinergic mediated neuroinflammation involves glial cell activity (Delvalle et al., 2018a). More recently, optogenetic approaches have been combined with chemogenetic approaches, i.e., where mice expressing hM3Dq, a modified DREADD receptor in glia via GFAP have been crossed with Sox10-CreER^T2-GCaMP5g mice (Ahmadzai et al., 2021). After activation of hM3Dq receptors with clozapine-N-oxide (CNO), there was a reduction in Ca²⁺ transients within neurons wired to descending pathways indicating that cholinergic signaling in glia is important in repressing these neurons. From these studies, it was concluded that circuit-specific glia regulate GI motility via the regulation of neural pathways. Thus, these optogenetic studies further support the notion that there is bidirectional communication between enteric glia and neurons. Interestingly, one study used mice expressing GCaMP3 in GFAP⁺ cells (Hennig et al., 2015) to demonstrate that glial cells were largely quiescent between CMMCs but that the onset of such an event, a pattern of Ca²⁺ activity spread throughout the myenteric glia and lasted long after the CMMC ceased. Whether this amounts to a true propagation of activity that contributes to CMMCs or is merely a consequence of CMMC-induced neural activity evoking glial responses remains to be determined (Hennig et al., 2015).

A combination of optogenetic and chemogenetic approaches have also been used to study to role of muscularis macrophages in GI motility (Luo et al., 2018). Cx3cr1-CreER^T2-ChR2 mice and Cxcr1-CreER^T2-Gq-DREADD mice were used to selectively activate macrophages since Cx3cr1 is abundantly expressed in these cells. Experiments from both mice revealed that the activation of TRPV4 channels in macrophages was associated with TTX-insensitive colonic contractions and the release of prostaglandin E2 (PGE2), as stimulation of macrophages with TRPV4 agonists led to an increase in PGE2 that was absent in macrophage specific TRPV4^−/− mice or under Ca²⁺ free conditions (Luo et al., 2018). This study suggests that macrophages can influence colon contractility independently of neural inputs.

Future use of optogenetic tools in GI cells

Optogenetic tools and techniques provide the means to answer questions within the field of neurogastroenterology and motility that previously could not be determined. Much work remains to be done to maximize these technologies in the study of specific cell behaviours in the GI tract. For example, while optogenetic approaches have provided opportunities for mechanistic investigation into the behaviours of different cells within the SIP syncytium (particularly ICC), this has relied almost exclusively on optogenetic reporters (such as GCaMP or RCaMP). Future experiments could utilize specific Cre lines (such as Kit, SmMHC, Acta or PDGFRα) that drive expression of ChR proteins in SIP cells. This might also be combined with a reporter GCaMP expression in the same or in different cell types. Since electrical field stimulation or bath application of agonists or antagonists indiscriminately act upon multiple cell types within the tissue, these transgenic mice would allow investigators to study the effects of activating a specific cell type directly.

This has already been accomplished in SMCs where ChR2 expression was driven by the chicken β-actin promoter and restricted to smooth muscle α-actin⁺ cells (Vogt et al., 2021). In this study, ICC and enteric nerves were destroyed by staining with methylene blue to simulate gastroparesis within the gastric antrum. Activation of ChR2 with blue light restored regular antral contractions (albeit to a lesser degree than previous contractions). Similarly, another study examined motility effects of a mammalian neuronal tissue opsin (OPN5, activated by UV light and deactivated with red shifted light (>470 nm)) that leads to G_q receptor activation (Wagdi et al., 2022). In the small intestine of mice with OPN5 under the control of the chicken β-actin promoter, illumination with UV light led to increased intestinal contractions. In the first study using wireless optogenetic activation of enteric nerves, colons from transgenic mice with Cre-mediated expression of light sensitive ChR2 in calretinin neurons were stimulated in vivo using light emitted diodes implanted on the colon (Hibberd et al., 2018). Stimulation of these nerves using light successfully increased colonic motility (Hibberd et al., 2018). In an additional study using transgenic mice that express opsin 3 (G-coupled protein receptor stimulated by blue light) in SMCs, colonic SMC contractile activity was inhibited with blue light stimulation under ex vivo conditions (Dan et al., 2021).

These studies demonstrate that optogenetic stimulation of GI cells can induce and potentially restore regular motility patterns in animals, suggesting that it may be possible to perform similar treatments to treat GI disorders. Also, stimulation of a light activated G_q protein such as OPN5 in ICC, would enable investigators to induce or prevent Ca²⁺ release in ICC under the control of different wavelengths of light (as Ca²⁺ release in ICC is tied to G_q-PLC-IP₃ dependent pathways), allowing for more careful study of ICC signalling behaviours. Similar experiments could be used to up or downregulate ICC or PDGFRα⁺ cell activity in mouse models of GI diseases such as pseudo-obstruction, constipation or gastroparesis. Recent GCaMP imaging studies have shown that Ca²⁺ signalling in colonic ICC is downregulated in a model of diabetes-associated constipation (Jin et al., 2021). Thus, upregulation of ICC Ca²⁺ signalling through optogenetic stimulation might ameliorate such pathophysiological conditions.

In certain populations of ICC that rely on voltage-dependent Ca²⁺ entry for entrainment of pacemaker activity (ICC-MY or ICC-SM (Baker et al., 2021a; Baker et al., 2021b)), optogenetic modulation of ICC by ChRs or halorhodopsins might provide a means to modify ICC function in situ. In neurons, due to passive distribution of Cl⁻, activation of halorhodopsins leads to neural silencing (Zhang et al., 2007). In ICC however, Cl⁻ ions are actively accumulated (Zhu et al., 2016; Zheng et al., 2020) resulting in an ECl of ~ 10 mV. Thus, activation of halorhodopsins in ICC with yellow light would lead to Cl⁻ efflux, yielding a depolarizing influence on the SIP syncytium (Wiegert et al., 2017). This could provide a means to directly activate slow waves or STDs in ICC in situ while recording the effects on other SIP cells. This may prove useful when studying chronotropic disease models in which loss of ICC has been implicated such as gastroparesis, slow transit constipation or achalasia (Vanderwinden & Rumessen, 1999; He et al., 2000; Lyford et al., 2002; Furness, 2012; Muller et al., 2014).

Limitations of using optogenetic tools in GI cells

Most optogenetic studies of enteric motor neurons or glial cells direct blue or yellow light into the dish of cells or tissues that express ChRs or halorhodopsins. This approach is sufficient to activate single action potentials in neurons but it is also possible that constant light exposure could shift these neurons into a refractory period after firing a single action potential. Thus, having a system that can deliver light at suitable frequencies would be desirable. In addition, many studies have used electrical field stimulation or exogenous application of agonists while recording Ca²⁺ activity in Kit-GCaMP mice to determine the role of ICC in neurotransmission. While these studies have shown that ICC can respond to such stimuli, it is not possible to rule out that these interventions may affect other electrically coupled cells, which then in turn influence ICC. Activation of ChR2 or halorhodopsin in mice expressing these optogenetic actuators in specific neuronal pathways while simultaneously imaging Ca²⁺ activity in ICC with an optogenetic sensor (such as RCaMP to prevent constant activation of ChR2 in nerves) may allow for more careful exploration of the role of ICC in postjunctional neural responses.

Optogenetic actuators have proven to be a highly valuable research tool for studying activity in specific cell types within the GI tract, but these technologies are mostly limited to rodents, (although recent experiments in sheep have demonstrated that optogenetic stimulation of vagus nerves is possible in large mammals (Booth et al., 2021)). Therefore, it may not be entirely appropriate to apply all knowledge gained from these studies to humans given that some physiological mechanisms vary between species. In animal studies, viral transfection (Iyer et al., 2014) and transgenic mice are commonly used to express optogenetic proteins, but these approaches come with limitations also, for example, ensuring adequate expression of the targeted cell population and maintaining homogenous light distribution. Without homogenous light distribution or adequate cell targeting there would not be consistent activation of the targeted cells. There is further speculation that optogenetic techniques may ultimately transform the underlying function of targeted cells and thus permanently change its function (Heitmann et al., 2017). Additionally, the long-term consequences of optogenetic interventions on neuronal health and function remain an open question.

Genetically encoded reporters and activators are powerful tools used in GI motility research and provide in-depth information on cellular functioning. However, there are multiple limitations to their use because of their heterogeneous expression in targeted cell populations and the lack of control over their level of expression and temporal activation. Optogenetic technology has improved significantly since the earliest studies. However, improvements need to be made in signal strength, kinetics, thermal stability, sensitivity to pH, as well as limiting possible interactions of sensors with cellular molecules that may yield non-specific effects to make sensors more optimal. Given the complexity of dynamic cellular interactions, optogenetics approaches are more precise than traditional pharmacological approaches used to manipulate cell functions and will likely revolutionize the way we study GI disease.

Biography

Bernard T. Drumm is a Lecturer and investigator in the Department of Life and Health Science at Dundalk Institute of Technology (DKIT), Ireland. Caroline A. Cobine, Ph.D. is an Assistant Professor in the Dept. of Physiology and Cell Biology at the University of Nevada, Reno. Salah A. Baker, Ph.D. is an Associate Professor in the Dept. of Physiology and Cell Biology at the University of Nevada, Reno. Together, Dr. Drumm, Dr. Cobine and Dr. Baker collaborate on a range of projects that examine the role of interstitial cells and Ca²⁺ signalling in controlling smooth muscle contractility in the gastrointestinal and urinary tract with an emphasis on the colon, lower esophageal and internal anal sphincters and the urethra.