The cells and conductance mediating cholinergic neurotransmission in the murine proximal stomach

Enteric neurotransmission is essential for gastrointestinal (GI) motility, although the cells and conductances responsible for post‐junctional responses are controversial. The calcium‐activated chloride conductance (CaCC), anoctamin‐1 (Ano1), was expressed by intramuscular interstitial cells of Cajal (ICC‐IM) in proximal stomach and not resolved in smooth muscle cells (SMCs). Cholinergic nerve fibres were closely apposed to ICC‐IM. Conductances activated by cholinergic stimulation in isolated ICC‐IM and SMCs were determined. A CaCC was activated by carbachol in ICC‐IM and a non‐selective cation conductance in SMCs. Responses to cholinergic nerve stimulation were studied. Excitatory junction potentials (EJPs) and mechanical responses were evoked in wild‐type mice but absent or greatly reduced with knockout/down of Ano1. Drugs that block Ano1 inhibited the conductance activated by carbachol in ICC‐IM and EJPs and mechanical responses in tissues. The data of the present study suggest that electrical and mechanical responses to cholinergic nerve stimulation are mediated by Ano1 expressed in ICC‐IM and not SMCs.


Introduction
The smooth muscle/interstitial cells of Cajal (ICC)/ platelet-derived growth factor receptor α positive (PDGFRα + ) cell (SIP) syncytium generates spontaneous electrical activity and regulates the excitability of gastrointestinal (GI) smooth muscles (Sanders et al. 2014a), although organ-level motility patterns are co-ordinated by the enteric nervous system. An example of this is the gastric accommodation reflex that is mediated by enteric motor neurons innervating the SIP syncytium of the proximal stomach (Desai et al. 1991;Tack et al. 2002). Previous studies have suggested that motor innervation of the proximal stomach by nitrergic (inhibitory) and cholinergic (excitatory) neurons occurs, in part, through transduction mechanisms expressed by ICC (Burns et al. 1996;Ward et al. 2000). However, other studies have contested this hypothesis, based on studies of Kit mutants in which ICC are developmentally impaired and reduced in numbers, and concluded that ICC are not important for enteric motor neurotransmission (Huizinga et al. 2008;Zhang et al. 2011).
We have also reported that cholinergic neurotrans mission persists in Kit mutants, and contractile responses to cholinergic neurotransmission can actually be enhanced in amplitude vs. responses in wild-type muscles (Sanders et al. 2014b). However, assays of post-junctional Ca 2+ sensitization pathways demonstrate that loss of ICC causes recruitment of Ca 2+ sensitization mechanisms that do not appear to be activated by cholinergic neurotransmission in wild-type animals (Bhetwal et al. 2013). Thus, although fundus muscles deprived of most ICC respond to cholinergic nerves, these responses are abnormal in nature. The mechanisms responsible for augmented cholinergic responses in Kit mutants probably leads to abnormal contractile responses to other hormones, neurotransmitters and paracrine substances because changing the gain of Ca 2+ sensitivity mechanisms would tend to affect contractile responses to all excitatory and inhibitory agonists. Our studies also showed that the Ca 2+ sensitization pathway (i.e. CPI-17 phosphorylation) activated in wild-type mice depends upon activation of a Ca 2+ -dependent protein kinase C (PKC), which could be regulated by a SIP syncytial pathway including: (i) acetylcholine binds to muscarinic receptors on ICC; (ii) activation of an inward current; (iii) conduction of the depolarization response to smooth muscle cells (SMCs); (iv) stimulation of Ca 2+ entry; and (v) activation of PKC. A better understanding of the post-junctional mechanisms responsible for neuroeffector responses may provide ideas for novel therapies for gastric emptying disorders, gastroparesis and functional dyspepsia.
A prominent CaCC (encoded by Ano1) has been identified in ICC of GI muscles, including humans (Gomez-Pinilla et al. 2009;Hwang et al. 2009;Zhu et al. 2009Zhu et al. , 2011Rhee et al. 2011;Blair et al. 2012). Ano1 is expressed in Kit + ICC, and its gene products, Ano1 channels, have been implicated in the pacemaker activity of GI muscles (Hwang et al. 2009;Zhu et al. 2009;Singh et al. 2014;Cobine et al. 2017;Malysz et al. 2017). In the murine stomach, pacemaker activity, attributed to ICC in the plane of the myenteric plexus (ICC-MY), is resolved in the gastric corpus and antrum but typically not observed in the fundus (Burns et al. 1996;Ward et al. 2000;Beckett et al. , 2017 where only intramuscular ICC (ICC-IM) are found (Burns et al. 1996;Ward et al. 2000;. ICC-IM in the fundus make close, synapse-like anatomical contacts with varicosities of enteric motor neurons and form gap junctions with neighbouring SMCs (Horiguchi et al. 2003;Beckett et al. 2005;Sanders et al. 2014a). Thus, responses activated in ICC-IM would be expected to conduct to SMCs. We found previously that cholinergic responses in the small intestine are linked to activation of a CaCC (Zhu et al. 2011). However, the results of that study were based on pharmacological blockade of CaCC using traditional blockers of this conductance, such as niflumic acid, which can have non-specific effects (Hartzell et al. 2005).
In the present study, we tested the hypothesis that a CaCC, rather than a NSCC, is the primary conductance activated in post-junctional cells in the proximal stomach during cholinergic neurotransmission. We examined the type(s) of conductances activated by cholinergic nerve stimulation in cells of the murine fundus and then studied cholinergic responses in muscles with Ano1 deactivated genetically. The results obtained demonstrate that a CaCC-Ano1 mediates cholinergic excitatory junction potentials (EJPs) and contractions and that knockdown of Ano1 inhibits electrical and mechanical responses to cholinergic excitatory neurotransmission.

Animals
Mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) or where specific strains were used, generated in house at the University of Nevada (Reno, NV, USA) or University of California San Francisco (San Francisco, CA, USA). Several strains were used, including: (i) Ano1 (tm1Bdh)(/tm1Bdh) previously termed Tmem16a (tm1Bdh)(/tm1Bdh) because these mice die as neonates (Rock et al. 2008), they were used by P5; (ii) Kit CreERT2Ejb1/+ (Kit CreERT2 ) mice were donated by Dr Dieter Saur (Technical University Munich, Munich, Germany) (Klein et al. 2013) and crossed with Ano1 tm1jrr to generate Kit CreERT2/+ ; Ano1 tm1jrr/+ and Kit CreERT2/+ ; Ano1 tm1jrr/− animals (Faria et al. 2014;Schreiber et al. 2015); (iii) Kit CreERT2/+ mice were also crossed with Gt(ROSA)26Sor tm4(ACTB-tdTomato,-EGFP)Luo /J reporter mice to produce Kit CreERT2/ ; Ano1 tm1jrr/+ ; Rosa mtmg and Kit CreERT2/ ; Ano1 tm1jrr/− ; Rosa mtmg animals; (iv) Kit copGFP/+ mice (P8-P10) were used for patch clamp and molecular expression studies because expression of the reporter allowed unequivocal identification of ICC in a mixed cell population resulting after enzymatic dispersion, as described previously Zhu et al. 2011); (v) Pdgfra tm11(EGFP)Sor /J heterozygote mice (The Jackson Laboratory), where PDGFRα + cells are constitutively labelled by expression of a transgene encoding a histone 2B-enhanced green fluorescent protein (eGFP) fusion protein driven by the endogenous, cell-specific Pdgfra promoter; these mice were used to purify PDGFRα + cells by fluorescence-activated cell sorting (FACS); and (vi) B6. eGFP) mice (Myh11 eGFP/+ ; donated by Dr Michael Kotlikoff, Cornell University, Ithaca, New York) were used to identify SMCs for comparative molecular transcript studies. (vii) Age-matched C57Bl6/6J (The Jackson Laboratory) between the ages post-embryonic day (P)3-P5 and 15 weeks were also used as control mice for morphological and physiological experiments (complete mice information and abbreviated names are provided in Table 1). The animals used in the present study were age-matched and experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The Institutional Animal Use and Care Committees at the University of Nevada, Reno and University of California, San Francisco approved the procedures used on the mice. Animals were fed ad libitum and had free access to water. Animals were humanely killed by isoflurane sedation followed by cervical dislocation and exsanguination. The investigators involved in the present study are aware of the ethical principles under which The Journal of Physiology operates and confirm that the use of animals presented here complies with the check list in Grundy (2015).
The stomachs from the oesophagus to the pyloric sphincter was removed and placed in oxygenated Krebs-Ringer buffer (KRB) for further dissection. Stomachs were opened along the lesser curvature and gastric contents were washed away with KRB. The gastric fundus was isolated by a surgical incision across the stomach along the border between fundus and corpus (as indicated by a change in the mucosa structure) and processed for morphological or physiological experiments. To induce Cre recombinase, 8-week-old mice were treated with tamoxifen (Sigma-Aldrich, St Louis, MO, USA) by I.P. injection (0.2 mg I.P. injection made up as 20 mg mL −1 solution in safflower oil). Each animal received four consecutive doses of tamoxifen given every other day and the experiments were performed from 7 weeks following the last treatment. This has been reported to be successful with respect to obtaining maximal gene knockdown in the GI tract (Groneberg et al. 2015).

Genotyping and phenotyping
Ano1 (tm1Bdh)(/tm1Bdh) mice were generated by replacing exon 12 of Tmem16a with a phosphoglycerate kinase-neomycin cassette by homologous recombination in embryonic stem cells (Rock et al. 2008). Genomic DNA was isolated from transgenic mice tails using standard procedures. DNA (0.5 μL) was amplified in each PCR reaction to determine the genotypes of the transgenic mice. A 393 bp PCR fragment was amplified from the Tmem16a +/+ allele with primers that bind within and spanning exon 12. The Ano1 -/allele (350 bp) was amplified with primers that bind to the PGK-neomycin cassette as described previously (Hwang et al. 2009).
Kit  were generated by inserting CreERT2 under control of the mouse proto-oncogene receptor tyrosine kinase (Kit) promoter/enhancer regions on the BAC transgene. Genomic DNA was isolated from transgenic mice tails using standard procedures. DNA (0.5 μL) was amplified in each PCR reaction to determine the genotypes of the transgenic mice. A 685 bp PCR fragment was amplified from the Kit CreERT2+/+ allele with primers that span the insertion region. The 330 bp PCR fragment was amplified from the Kit CreERT2+/allele with primers that bind to the CreERT2 insert (Klein et al. 2013). When Kit CreERT2+/mice are bred with mice containing loxP-flanked sequence, tamoxifeninducible, Cre-mediated recombination results in deletion of the floxed sequences in Kit cells of the offspring.

Morphological studies
Whole mounts were prepared after removing the mucosa from fundus by sharp dissection. The remaining strips of tunica muscularis were pinned to the base of a dish filled with Sylgard elastomer (Dow Corning Corp., Midland, MI, USA) with the circular muscle layer facing upward and stretched to 110% of their resting length. Tissues were fixed in either acetone (4°C for 10 min) or paraformaldehyde [4% w/v in 0.1 M phosphate buffer (PB) for 15 min at 4°C]. Following fixation, preparations were washed overnight in PBS (0.01 M, pH 7.4). Incubation of tissues in BSA (1%) for 1 h at room temperature containing Triton X-100 (0.3%) was used to reduce non-specific antibody binding. For double-labelling, tissues were incubated sequentially in a combination of primary antibodies ( Table 2). The first incubation was carried out for 48 h at 4°C; tissues were subsequently washed in PBS before being incubated in a second antibody for an additional 48 h at 4°C. The combinations of antibodies used were goat/rabbit and chicken/rabbit. Following incubation in primary antibodies, tissues were washed and incubated separately in secondary antibodies (Alexa Flor 488 and 594; Thermo Fisher Scientific Inc., Waltham, MA, USA, diluted to 1:1000 in PBS for 1 h at room temperature). Control tissues were prepared by either omitting primary or secondary antibodies from the incubation solutions. Tissues were examined with an LSM 510 Meta confocal microscope (Carl Zeiss, Jena, Germany) with appropriate excitation wavelengths. Confocal micrographs were digital composites of Z-series scans of 10-20 optical sections through a depth of 2-40 μm. Final images were constructed and montages were assembled using LSM 5 Image Examiner (Carl Zeiss) and converted to Tiff files for processing in Photoshop CS5 (Adobe Co., Mountain View, CA, USA) and Corel Draw X4 (Corel Corp., Ottawa, ON, Canada).

Cell purification and cell-specific RNA isolation and quantitative RT-PCR (qRT-PCR)
Strips of gastric fundus muscles (25 ± 5 mg tissue weight) were equilibrated in Ca 2+ -free Hanks' solution at 4°C consisting of (mM): 125 NaCl, 5.36 KCl, 15.5 NaHCO 3 , 0.336 Na 2 HPO 4 , 0.44 KH 2 PO 4 , 10 glucose, 2.9 sucrose and 11 Hepes, adjusted to pH 7.2 with NaOH, for 30 min. Cells were dispersed as described previously ). Briefly, muscle strips were incubated for 25 ± 2 min at 37°C in an enzyme solution containing (per mL): 1.0 mg of collagenase (Worthington Type II; Worthington Biochemical, Lakewood, NJ, USA), 2.0 mg of BSA (Sigma, St Louis, MO, USA), 2.0 mg of trypsin inhibitor (Sigma) and 0.13 mg of ATP (Sigma). Strips were washed with Ca 2+ -free Hanks' solution to remove the enzyme and isolated cells were obtained by triturating the digested tissues. eGFP-SMCs, CopGFP-ICC and eGFP-PDGFRα cells were purified by FACS (FACSAriaII; Becton-Dickinson, Franklin Lakes, NJ, USA) using the blue laser (488 nm) and the GFP emission detector (530/30 nm). Expression of genes in each sorted cell type was compared against expression in the total fundus cell population (TCP). TCP represents all cells dispersed from the tunica muscularis from fundus and was prepared from each cell-specific reporter mouse strain. Gene transcript expression was averaged from three mice of each reporter strain.
Total RNA was isolated from SMCs, ICC and PDGFRα + cells, using an illustra RNAspin Mini RNA Isolation kit (GE Healthcare, Little Chalfont, UK). Concentration and purity of RNA was measured using a ND-1000 Nanodrop Spectrophotometer (Nanodrop, Wilmington, DE, USA), comparative amounts of RNA were used for first-strand cDNA synthesized using SuperScript III (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer's instructions. PCR was performed with specific primers (Table 3) using Go-Taq Green Master Mix (Promega Corp., Madison, WI, USA). PCR products were analysed on 2% agarose gels and visualized by ethidium bromide. qRT-PCR was performed with the same primers as PCR using Fast SYBR green chemistry (Applied Biosystems, Foster City, CA, USA) on the 7900HT Real Time PCR System (Applied Biosystems).

Patch clamp experiments on identified cell phenotypes
Whole-cell patch clamp configuration was used to record currents from ICC that were identified by copGFP fluorescence and SMCs identified by their typical long spindle-shaped morphology. After sharp dissection of the mucosa, the smooth muscle layer was cut into small strips and equilibrated in Ca 2+ -free Hanks' solution at 4°C (see above). Muscle strips were incubated in the same enzyme solution over a time period similar to that used for FACS sorting (see above). Strips were washed with Ca 2+ -free Hanks' solution to remove the enzyme, and isolated cells were obtained by triturating the strips with smooth muscle growth medium (SMGM; Clonetics Corp., San Diego, CA, USA) using a glass pipette. The cell suspension was placed on to glass coverslips coated with murine collagen (2.5 mg mL −1 ; Becton-Dickinson) in 35 mm culture dishes. SMGM supplemented with 2 % antibiotic-anti-mycotic (Gibco, Grand island, NY, USA) and stem cell factor (5 ng ml −1 ; Sigma) was added 20 min after the cells were settled. The cells were used from 1 h after incubating at 37°C in a 95% O 2 -5% CO 2 incubator. An Axopatch 200B patch clamp amplifier (Axon Instruments, Union City, CA, USA) and a 12-bit A/D converter (Digidata 1320A; Axon Instruments) were used to voltage clamp the cells at -80 mV. Micropipettes used for recordings had a resistance of 4-6 M . All data were digitized and acquired using pClamp , version 10.0.0.61 (Axon Instruments) and analysed using Clampfit (Axon Instruments) and Prism (Graphpad Software Inc., San Diego, CA, USA) software. All experiments were performed using a calcium containing physiological salt solution (PSS) solution at 30°C with a CL-100 bath heater (Warner Instruments, Hamden, CT, USA). J Physiol 596.9

Electrophysiological and contractile experiments on intact muscles
The fundus region of stomachs was pinned as a sheet to the base of a Sylgard silicone elastomer (Dow Corning Corp.) dish and the mucosa removed by sharp dissection. Strips of muscle (4-8 mm) were isolated from along the greater curvature and placed in a recording chamber with the submucosal surface of the circular muscle layer facing upward. Circular muscle cells were impaled with glass microelectrodes (resistances of 80-120 M ) and electrical activity was recorded as described previously (Burns et al. 1996). Briefly, transmembrane potentials were recorded with a high impedance amplifier (Axon Instruments) and stored on a PC running the data acquisition software AxoScope, version 10 (Axon Instruments). Images were prepared using Clampfit (Axon Instruments) and Corel Draw.
Parallel platinum electrodes were placed on either side of the muscle strips to elicit neural responses with square pulses of electrical field stimulation (EFS) (0.3 ms pulse duration, 1-20 Hz, train durations of 1 s, 10-15 V) delivered by a Grass S48 stimulator (Grass Instrument Company, Quincy, MA, USA). Isometric force measurements were performed using tissues (3-6 mm) prepared as described previously (Burns et al. 1996). Tissues were placed between two ring electrodes fixed at one end and mounted onto a Gould force transducer at the other end. EFS using similar parameters (1-30 s duration) as in electrophysiological experiments were used to elicit motor responses.

Statistical analysis
For morphological studies, the numbers of ICC-IM were counted in six random fields of view of fundus muscles from Ano1 +/+ and Ano1 −/− mutants taken at 40× magnification. The number of ICC-IM in each muscle layer was calculated as the number of cells crossing a 100 μm transactional line drawn perpendicular to the axis of the circular or longitudinal muscle layer.
For analysis of molecular studies, gene transcript expression was compared between eGFP + -SMCs, CopGFP + -ICC and eGFP + -PDGFRα + , and each cell type was also compared with total cell population of fundus tunica muscularis. Regression analysis of the mean values of three multiplex qPCRs for the log 10 diluted cDNA was used to generate standard curves. Unknown amounts of mRNA were plotted relative to the standard curve for each set of primers and graphically plotted using Excel (Microsoft Corp., Redmond, WA, USA). Primer efficiencies of 90-110% were only accepted for analysis. This gave transcriptional quantification of each gene relative to the endogenous Gapdh standard after log transformation of the corresponding raw data. In pilot studies, Gapdh was tested on all three cell types used in the present study and represents an appropriate control for qPCR analyses. Normalized values and SDs were calculated in differences of relative gene expression from four dilutions of technical duplicates from each animal. The data are shown as the mean ± SD of triplicate samples (n = 3). For the fold change of genes with, P < 0.05 was considered statistically significant. An unpaired Student's t test was used to determine P values in the parametric analysis.
Data are expressed as the means ± SEM. Differences between the reported means of mouse groups were evaluated using an unpaired Student's t test. P < 0.05 was considered as a statistically significant difference.
Differences between the means of three or more measured parameters were evaluated using repeated measures ANOVA where appropriate in conjunction with the Dunnett's multiple comparison test. Again, P < 0.05 was considered statistically significant. Statistical tests were performed using Prism, version 5.03 (GraphPad Software Inc.). The reported n values refer to the number of animals used for each experimental protocol.

Ano1-like immunoreactivity in ICC-IM and close apposition to cholinergic nerves
Double-labelling immunohistochemistry using antibodies against Kit and Ano1 showed that spindle-shaped Kit + ICC-IM within the circular and longitudinal muscle layers of the gastric fundus were immunopositive for Ano1 (Ano1 + ) ( Fig. 1A-C). Kit and Ano1 labelling was also performed on gastric fundus muscles from P3-P5 animals to test whether a similar relationship existed in younger animals. We used animals in this age range during the course of the present study because most global knockouts of Ano1 (Ano1 −/− mice) die within 1 week after birth (Rock et al. 2008). Similar to adults, fundus ICC-IM were Ano1 + in P3-P5 animals ( Fig. 1D-I).
ICC-IM were spindle shaped and closely associated with varicose motor nerve fibres, as shown by labelling with the pan neuronal marker protein-gene product 9.5, as reported previously (Burns et al. 1996;Ward et al. 2000). Ano1 expression was not resolved in Kit − cells. Double-labelling studies with antibodies against vesicular acetylcholine transporter (VAChT) and Kit were performed to highlight specifically the morphological relationship between cholinergic nerve fibres and ICC-IM. Close contacts between VAChT immunopositive (VAChT + ) varicose nerve fibres and Kit + ICC-IM were observed for >250 μm along the surfaces of ICC-IM ( Fig. 2A-F). vAChT and Kit immunohistochemistry was also performed on fundus muscles from P3-P5 mice, and similar close alignments were observed between VAChT + nerve fibres and ICC-IM in these young animals ( Fig. 2G-L).

CCh activates inward currents in ICC and SMCs of murine fundus
Cholinergic neurotransmission in GI muscles, mediated by muscarinic receptors, generates depolarization of post-junctional cells known as EJPs (Bennett, 1966;Ward et al. 2000;Matsuyama et al. 2013). We investigated the conductances activated by muscarinic stimulation in isolated and identified ICC-IM and SMCs from the murine fundus using the whole-cell patch clamp technique. A Cs + -rich pipette solution with E Cl = −40 mV was used to study the properties of CCh-induced inward currents in ICC-IM and SMCs. Cell capacitance averaged 5.5 ± 0.4 pF (n = 28) for ICC and 25.0 ± 1.6 pF (n = 21) for SMCs. Holding currents were −7.0 ± 1.1 pA pF -1 and −0.5 ± 0.1 pA pF -1 at -80 mV for ICC and

Nature of the conductance activated in ICC-IM and SMCs
A CaCC mediated by Ano1 is expressed by ICC but not resolved in SMCs in GI muscles (Gomez-Pinilla et al. 2009;Hwang et al. 2009;Zhu et al. 2009Rhee et al. 2011Blair et al. 2012). EJPs in the fundus in response to cholinergic neurotransmission may be mediated by activation of CaCC. Spontaneous transient inward currents (STICs) were observed in many ICC held at −80 mV. STICs reversed between −30 and −20 mV (before correction of the junction potential calculated to be 14.6 mV in our experiments) using a Cs + -rich pipette solution with E Cl = -40 mV ( Fig. 4A and B). NPPB (10 μM), decreased holding currents from −13.9 ± 2.8 pA pF -1 to −2.5 ± 0.7 pA pF -1 and inhibited STICs (P < 0.05; n = 5) ( Fig. 4C and D), suggesting that that STICs and part of the holding current were a result of CaCC. NPPB added 5 min before CCh application blocked the inward currents evoked by CCh (−5.0 ± 1.6 pA pF -1 ; n = 5; P > 0.05 when comparing NPPB and NPPB after CCh addition; n = 5) ( Fig. 4E-G).
Although Ano1 was not resolved in SMCs of the fundus (Figs 1, 8 and 9), experiments aiming to examine the effects of the Ano1 inhibitors were also performed on isolated SMCs. At -80 mV, SMCs generated small holding currents and T16A inh -A01 (10 and 30 μM) did not affect these currents. As described above, CCh activated small amplitude noisy inward currents in SMCs and T16A inh -A01 did not affect these responses (−1.6 ± 0.1 pA pF -1 at 10 μM and −1.6 ± 0.3 pA pF -1 at 30 μM, respectively; n = 5 for each concentration) ( Fig. 5I-K).
We also tested whether the inward current evoked by CCh in ICC-IM might be mediated partially by a NSCC by testing the effects of La 3+ pretreatment. La 3+ (10 μM) tended to reduce holding currents slightly, although this effect did not reach statistical significance. La 3+ also had little effect on STICs in ICC-IM. In the presence of La 3+ , CCh (10 μM) evoked large amplitude inward currents (i.e. to −176.3 ± 40.5 pA pF -1 ; P < 0.01; n = 5) ( Fig. 6A-C). These results, in accordance with complete block of responses by CaCC blockers, suggest that NSCC does not contribute significantly to CCh responses in CCh (10 µM) CCh (10 µM) Atropine (1 µM ICC-IM of the fundus. The inward currents induced by CCh in SMCs were probably the result of activation of NSCC, as reported previously ). Therefore, we tested the effects of lanthanum (La 3+ ) on the CCh-induced currents in SMCs ( Fig. 6D-F). Ramp protocols applied in the presence of CCh revealed a reversal potential of 0 mV (E cl − = -40 mV) (Fig. 6E). La 3+ (10 μM), added 10 min before application of CCh (10 μM), reduced inward currents evoked by CCh significantly (to −0.7 ± 0.1 pA pF -1 in the presence of La 3+ and −0.9 ± 0.2 pA pF -1 in the presence of La 3+ after CCh addition; P > 0.05 when comparing La 3+ with La 3+ after CCh addition; n = 5) (Fig. 6F). These data suggest that CCh activates a NSCC in fundus SMCs.

Cholinergic responses in intact muscles require Ano1
CCh activates different conductances in ICC-IM and SMCs, and so the suppression of currents carried by Ano1 channels may provide evidence of whether ICC are the cells primarily responsible for post-junctional electrical responses to cholinergic neurotransmission. We measured electrophysiological responses of intact fundus muscles to electrical field stimulation (EFS) of intrinsic neurons in mice with global deactivation of Ano1 (known formerly as Tmem16a, so that the strain name of these mice is Tmem16a (tm1Bdh)(/tm1Bdh) ; Rock et al. 2008). In this part of the study, fundus muscles were harvested from P5 mice as a result of the short life span of these animals. Post-junctional responses to

. The CaCC blocker NPPB inhibited CCh activation of inward currents in ICC-IM
A, spontaneous transient inward currents (STICs) in ICC-IM at voltage steps from -80 to +20 mV. Large STICs were observed in ICC-IM held at potentials between -80 mV and -40 mV. B, STICs reversed at potentials between -30 and -20 mV (before correction of junction potential; calculated junction potential = 14.6 mV) using a Cs + -rich pipette solution with E cl − = -40 mV. C and D, CaCC channel blocker NPPB (10 µM) blocked the sustained inward current and STICs, suggesting that these events were a result of CaCC. NPPB decreased holding currents from −13.9 ± 2.8 pA pF -1 to −2.5 ± 0.7 pA pF -1 (P < 0.05; n = 5). D, currents recorded in response to voltage ramps under control conditions (a) were reduced significantly by NPPB (b). Voltage ramps were applied at the time points indicated in (C). E-G, NPPB and CCh-induced inward currents in ICC-IM. E, NPPB (10 µM) reduced STICs and holding currents in ICC-IM. CCh, in the presence of NPPB, added 5 min before CCh application, failed to activate inward current in ICC-IM. F, voltage ramp protocols performed before (a) and after addition of NPPB (b) and after addition of CCh (c) (10 µM), at the time points indicated in (E). Control voltage ramps were reduced by NPPB and CCh failed to activate inward current in response to voltage ramps in the presence of NPPB. G, summary of currents activated in response to voltage ramps under control conditions (white bar) and reduction in currents after addition of NPPB (10 µM; grey bar) and CCh (10 µM) in the continued presence of NPPB (black bar; n = 5; * P < 0.05, one-way ANOVA).
cholinergic neurotransmission in muscles of Ano +/+ and Ano1 -/animals were compared. EFS was applied and responses were recorded: (i) under control conditions with no drugs present; (ii) in the presence of the nitric oxide synthase inhibitor L-NNA (100 μM) to unmask EJPs; (iii) in the continued presence of L-NNA and after addition of an acetylcholinesterase (AChE) inhibitor, neostigmine (1 μM) to determine whether ACh activates post-junctional receptors not normally stimulated when ACh is being metabolized; and (iv) in the presence of L-NNA, neostigmine and atropine (1 μM) to confirm that EJPs were evoked via muscarinic receptors. Membrane potentials recorded from circular muscle cells of Ano1 +/+ muscles averaged −45.3 ± 1.4 mV (n = 8). Fundus muscles in the mouse do not display slow waves (Burns et al. 1996) but, instead, generate small amplitude, noisy oscillations, known as spontaneous transient depolarizations (STDs) or unitary potentials (Burns et al. 1996;Edwards et al. 1999;Van Helden et al. 2000;. Under control conditions, EFS (0.3 ms pulses, 1-20 Hz, 1 s) evoked a biphasic response consisting of an EJP that averaged 4.1 ± 2.2 mV in amplitude and 179 ± 52 ms (half-maximal amplitude duration; 1 Hz; 0.3 ms pulse duration) followed by a more sustained hyperpolarization or inhibitory junction potential (IJP) (3.5 ± 1.1 mV; n = 8). EJPs and IJPs were frequency-dependent and increased in amplitude and duration as the stimulus frequency increased (e.g. EJP = 5.2 ± 2.8 mV in amplitude and 229 ± 66 ms in half-maximal amplitude duration; IJP = 7.3 ± 1.3 mV in amplitude at 10 Hz stimulation) (Fig. 7A).
RMP was unchanged by L-NNA (100 μM; -46.1 ± 1.9 mV; P > 0.05 vs. control) but EJPs increased to 8.2 ± 2.6 mV in amplitude and 304 ± 43 ms (half-maximal amplitude duration) at 1 Hz and 10.4 ± 2.8 mV in amplitude and 860 ± 86 ms at 10 Hz after addition of L-NNA (Fig. 7B). In the continued presence of L-NNA, addition of neostigmine caused membrane depolarization to −40.2 ± 1.9 mV (P < 0.05 vs. L-NNA and control) and EFS evoked a biphasic response consisting of a fast EJP (i.e. 10.4 ± 1.5 mV at 1 Hz and 12.4 ± 0.9 mV at 5 Hz) and a secondary, slower and longer-lasting depolarization that often merged with the fast EJP. The slow depolarization averaged 6.4 ± 1.0 mV and the half-maximal amplitude duration of 5586 ± 809 ms at 1 Hz and 14 ± 2.7 mV and 9161 ± 911 ms at 5 Hz (Fig. 7C). The fast and slow components of the responses to EFS in the presence of neostigmine were inhibited by atropine (Fig. 7D), confirming that both were mediated by muscarinic receptors. Cells of fundus muscles from Ano1 −/− mice had resting potentials averaging −45.3 ± 1.5 mV (n = 15; P > 0.05 compared to Ano1 +/+ ) and the small oscillations in membrane potential (STDs or unitary potentials) (Burns et al. 1996;Edwards et al. 1999;Van Helden et al. 2000;) observed after impalement of cells in Ano1 +/+ muscles were greatly attenuated or not resolvable in Ano1 −/− muscles. EFS (1-20 Hz) failed to elicit EJPs before or after L-NNA (P > 0.05 vs. control) (Fig. 7E and F). However, neostigmine (in the continued presence of L-NNA) continued to cause depolarization (to −39.0 ± 1.3 mV; P < 0.001) and EFS caused slowly developing depolarization responses, averaging 2.6 ± 0.6 mV in amplitude and 3410 ± 882 ms in duration at half-maximal amplitude at 1 Hz and 6.8 ± 1.5 mV in amplitude and 10500 ± 1196 ms in duration at half-maximal amplitude at 5 Hz (Fig. 7G). Responses elicited after neostigmine were blocked by atropine (Fig. 7H). Fully developed myenteric ganglia (not shown) and innervation of circular muscles by vAChT + (cholinergic) motor neurons were observed in P5 mice (Fig. 2), suggesting that reduced cholinergic neurotransmission in these young animals was not the result of a paucity of motor neurons. Furthermore, there was no loss of vAChT + neurons in Ano1 −/− mice compared to Ano1 +/+ controls (Fig. 2).
Ano1 is reported to be necessary for the development or proliferation of ICC (Stanich et al. 2011). We investigated

in response to EFS
A, neural responses in Ano1 +/+ fundus muscles under control conditions in response to EFS, 1 Hz (delivered at arrow, left) and 5 Hz (horizontal bar, right) respectively (n = 8). EFS evoked a frequencydependent biphasic motor response consisting of an initial fast transient EJP followed by a more sustained IJP that was often followed by a secondary depolarization in membrane potential before it returned to pre-stimulus levels. B, in the presence of L-NNA (100 µM), the EFS evoked IJP was abolished and the EJP increased in amplitude. C, in the continued presence of L-NNA, neostigmine (1 µM) depolarized membrane potential, evoked an EJP and revealed a slower developing and more sustained depolarization in membrane potential (dashed lines) to EFS. D, atropine (1 µM) abolished both the initial and sustained depolarization at all EFS frequencies examined. E, in Ano1 −/− mutants, EFS evoked little or no post-junctional responses to EFS under control conditions at 1 Hz and a small hyperpolarization at 5 Hz (n = 15). F, L-NNA (100 µM) produced little change to EFS at 1 pulse but attenuated the slight hyperpolarization in membrane potential at 5 Hz. G, in the continued presence of L-NNA, neostigmine (1 µM) caused membrane depolarization and produced a slowly developing and sustained depolarization response to EFS, as in Ano1 +/+ animals. H, atropine repolarized membrane potential and blocked the nerve evoked depolarization observed in the presence of neostigmine at all frequencies tested. Note also the difference in basal electrical activity between Ano1 +/+ and Ano1 −/− mutants. whether ICC were reduced in Ano1 −/− mutants, which could also explain the compromised post-junctional responses to cholinergic neurotransmission (Burns et al. 1996;Ward et al. 2000). Densities of ICC-IM in the fundus of Ano1 +/+ and Ano1 −/− mice were compared using Kit immunohistochemistry. P5 Ano1 +/+ mice had an average of 9.8 ± 0.4 ICC-IM and Ano1 −/− mutants had an average of 11.3 ± 0.5 ICC-IM in the circular layer and 5.4 ± 0.5 ICC-IM and 7.2 ± 0.2 ICC-IM in the longitudinal layers of Ano1 +/+ and Ano1 −/− mutants per 100 μm cross-sectional transaction line. Differences in ICC-IM were not significant in the circular and longitudinal muscle layers of Ano1 −/− mice in comparison to Ano1 +/+ age-matched controls (P > 0.1; when comparing the number of ICC in Ano1 +/+ and Ano1 −/− circular or  Figure 8. Numbers of Kit + ICC-IM were not different in the gastric fundus of Ano1 +/+ and Ano1 −/− mice A and B, Kit labelling of ICC-IM in the circular (cm) and longitudinal (lm) muscle layers of a P5 Ano1 +/+ control and an age-matched Ano1 −/− mutant in two random fields of view. In a 100 µm cross-transection line perpendicular to the respective muscle layer, there was an average of 9.8 ± 0.4 ICC-IM in the cm and 5.4 ± 0.5 ICC-IM in the lm of fundus from Ano1 +/+ controls. In Ano1 −/− animals, there was an average of 11.3 ± 0.5 ICC-IM in the cm and 7.2 ± 0.2 ICC-IM in the lm, respectively (n = 5). C, there was no statistical significant difference between the number of ICC-IM in both muscle layers between Ano1 +/+ controls and Ano1 -/mutants (six random fields of view from five Ano1 +/+ and 5 Ano1 −/− mutants).

Effects of Ano1 knockdown on mechanical responses to fundus muscles
Cholinergic EJPs were lost in Ano1 −/− mice and in muscles of iAno1 −/− mice, suggesting that post-junctional electrical responses are generated by ICC-IM and not by SMCs. However, mechanisms involving Ca 2+ sensitization of the contractile apparatus also contribute to cholinergic responses (Mori et al. 2011;Bhetwal et al. 2013) and might be mediated directly in SMCs. Therefore, we also investigated contractile responses of fundus muscles evoked by EFS to determine how loss, reduction and block of Ano1 affects excitation-contraction coupling.
Fundus muscles from P5 Ano1 +/+ and Ano1 −/− mice were relatively small in size and generated contractile responses that were smaller in amplitude than in muscles of adult animals. Nevertheless, experiments were performed on these muscles to compare motor responses to EFS. Because the muscle tissues were delicate, only short durations (1 s) were tested (i.e. 1-20 Hz, 0.3 ms pulses). Under control conditions, EFS evoked frequency-dependent contractile responses in Ano1 +/+ muscles, averaging 0.03 ± 0.02 mN at 1 Hz and 0.09 ± 0.04 mN at 10 Hz (Fig. 11A). Contractile responses to EFS in muscles of Ano1 −/− mice averaged 0.01 ± 0.01 mN and 0.04 ± 0.04 mN at 1 Hz and 10 Hz, respectively. Possibly as a result of the masking of responses by inhibitory neurotransmitters, responses to EFS were not statistically significant (P > 0.5) under control conditions in the two groups of animals. After addition of L-NNA (100 μM), contractile responses were enhanced in Ano1 +/+ but not in Ano1 −/− muscles. For example, contractions of Ano1 +/+ fundus muscles averaged 0.18 ± 0.06 mN and 0.33 ± 0.12 mN at 1 and 10 Hz, respectively (Fig. 11C), and responses to EFS of Ano1 −/− muscles averaged 0.01 ± 0.01 mN at 1 Hz and 0.05 ± 0.02 mN at 10 Hz (P < 0.05 when comparing Ano1 +/+ and Ano1 −/− tissues at 1 and 10 Hz, respectively) (Fig. 11F). Neostigmine (1 μM), in the continued presence of L-NNA, further increased contractile responses evoked by EFS in both animal groups and there was no statistical difference in the responses at all frequencies tested. At 1 Hz contractions of Ano1 +/+ tissues averaged 0.33 ± 0.09 mN and at 10 Hz 0.74.2 ± 29 mN, whereas, in Ano1 −/− muscles, contractions averaged 0.33 ± 0.18 mN at 1 Hz and 0.8 ± 0.38 mN at 10 Hz (P > 0.05 when comparing Ano1 +/+ and Ano1 −/− mutants at both frequencies) (Fig. 11C and G). In the presence of L-NNA and neostigmine, atropine (1 μM) blocked excitatory contractile responses at all frequencies ( Fig. 11D and H).
A summary of the contractile responses of Ano1 +/+ and Ano1 −/− mutants under different experimental conditions is provided in Fig. 11I-L. The role of Ano1 in cholinergic motor responses evoked by EFS was also characterized in muscles from adult animals before and after tamoxifen treatment (i.e. in muscles of iAno1 −/− and in muscles of iAno1 +/controls). Age-matched wild-type (+/+) fundus tissues was also examined for comparison (Fig. 12A-D). EFS (1-20 Hz for 30 s) under control conditions (i.e. absence of antagonists) evoked small excitatory responses followed by dominant inhibitory responses in muscles of iAno1 +/mice ( Fig. 12E-H). L-NNA (100 μM) blocked the inhibitory responses and EFS evoked excitatory responses at all frequencies tested. For example, EFS (5 Hz) evoked tonic contraction averaging 0.66 ± 0.24 mN and, at 20 Hz, the increase in tone averaged 1.81 ± 0.56 mN (n = 8) (Fig. 12F). Addition of neostigmine (1 μM) in the presence of L-NNA caused an increase in basal tone and a marked increase in the contractions evoked by EFS. At 5 Hz, the amplitude of contractions averaged 5.4 ± 1.1 mN and, at 20 Hz, 9.3 ± 0.8 mN vs. 7.9 ± 1.6 mN (Fig. 12G). Atropine (1 μM) inhibited the contractile responses evoked by EFS at all frequencies tested (Fig. 12H). These responses to EFS were essentially similar to that recorded from (+/+) fundus muscles (Fig. 12A-D).
EFS of fundus muscles of iAno1 -/mice under control conditions produced smaller amplitude relaxations at all frequencies tested (Fig. 12I). L-NNA (100 μM) inhibited the relaxation responses and converted responses to small amplitude tonic contractions that were significantly smaller than those in iAno1 +/mice (Fig. 12J). For example, at 5 Hz, the amplitude of contractions averaged 0.27 ± 0.08 mN and, at 20 Hz, the responses averaged 0.70 ± 0.16 mN (P < 0.05 and P < 0.01 for 5 and 20 Hz, respectively, compared to iAno1 +/animals; n = 20). As in iAno1 +/mice, addition of neostigmine (1 μM) in the presence of L-NNA caused an increase in tone and an increase in nerve evoked contractile responses in iAno1 -/mice. With neostigmine, there was no significant difference in the amplitude of EFS evoked contractions in iAno1 +/controls and iAno1 -/mice. At 5 Hz, the amplitude of contractions averaged 5.3 ± 1.1 mN (P = 0.91) and, at 20 Hz, 7.9 ± 1.6 mN (P = 0.54) compared to iAno1 +/mice (n = 20) (Fig. 12K). Atropine inhibited contractile responses at all frequencies tested (Fig. 12L). A summary of nerve evoked responses of gastric fundus muscles from tamoxifen treated iAno1 +/controls and iAno1 -/mutants is provided in Fig. 12M-P.

Pharmacological effects on EJPs of wild-type muscles
Pharmacological studies were also performed to further test the role of Ano1 in cholinergic excitatory responses. The small molecule Ano1 channel blocker benzbromorone, as recently identified by highthroughput screening, was tested (Huang et al. 2012) because this compound inhibited CCh-induced CaCC in isolated ICC-IM (Fig. 5) and was also recently shown to inhibit pacemaker activity in the stomach and small intestine (Hwang et al. 2016). Generation of slow waves in these organs are a result of activation of Ano1 (Hwang et al. 2009).
Mechanical responses elicited by EFS were also sensitive to benzbromarone. The dominant response of adult gastric (+/+) fundus muscles to EFS under control conditions (i.e. absence of drugs) was excitation followed by relaxation at 1 Hz and relaxation followed by excitation at higher frequencies. Relaxations were abolished by L-NNA (100 μM), unmasking frequency-dependent, atropine-sensitive contractions. Contractile responses evoked by EFS (1-10 Hz; 30 s) in the presence of L-NNA were dose-dependently inhibited by benzbromarone. Neural responses were attenuated at 1 μM (Fig. 14C) and almost completely blocked at 3-5 μM (n = 6) ( Fig. 14D and E). Benzbromarone also caused a dose-dependent reduction in basal tone. Tone was reduced 0.73 ± 0.2 mN at 1 μM, 1.54 ± 0.4 mN at 3 μM and 1.64 ± 0.4 mN at 5 μM. When neurally evoked responses to EFS were inhibited in benzbromarone (5 μM), exogenous ACh (1 μM) still caused contraction of fundus muscles (Fig. 14F). A summary of the effects of benzbromarone on responses to EFS is provided in Fig. 14G (n = 7).

Discussion
In the present study, we investigated whether ICC-IM or SMCs primarily receive and transduce inputs from cholinergic motor neurons. This has been a subject of controversy for several years Huizinga et al. 2008;Goyal & Chaudhury 2010;Sarna 2008;Zhang et al. 2011;Sanders et al. 2010Sanders et al. , 2014a and part of the conflicting information may be a result of compensatory mechanisms that sustain, but modify, cholinergic neurotransmission in mice with congenital loss of ICC (Bhetwal et al. 2013). We found that different types of ionic conductances are activated by cholinergic stimulation of ICC-IM and SMCs. SMCs are known to express NSCC responsive to muscarinic agonists in GI SMCs of many  Figure 13. Effects of benzbromarone on neurally evoked EJPs of (+/+) fundus muscles Responses to 1 Hz (arrow; left) and to 20 Hz (horizontal bars, right), 0.3 ms in duration for 1 s. A, under control conditions, prominent EFS evoked EJPs were followed by smaller, more slowly developing IJPs. B, L-NNA (100 µM) inhibited IJPs and potentiated the amplitude of EJPs. C and D, benzbromarone (1-3 µM) caused a reduction in EFS evoked EJPs. E, EJPs were inhibited at a concentration of 5 µM. Benzbromarone also caused hyperpolarization in membrane potential. F, summary of the effects of benzbromarone on EJPs (1-20 Hz) and membrane potential (solid circles; n = 7; * P < 0.05; * * P < 0.01; * * * P < 0.001, one-way ANOVA). organs and species (Benham et al. 1985;Lim & Bolton, 1988;Inoue & Isenberg, 1990a;Vogalis & Sanders, 1990;Sims, 1992;Lee et al. 1993;. As shown in the present study, ICC-IM express CaCC, and this conductance was not resolved in fundus SMCs. The CaCC is probably encoded by Ano1, a gene that is highly expressed in ICC throughout the GI tract (Chen et al. 2007;Lee et al. 2017) and translated to protein labelled by immunohistochemical techniques (Blair et al. 2012;Gomez-Pinilla et al. 2009;Hwang et al. 2009;Cobine et al. 2017). We reasoned that, if post-junctional electrical responses (EJPs) were compromised in animals made null for Ano1, this would be strong support for the innervation of ICC-IM by cholinergic motor neurons and, if post-junctional electrical and mechanical responses were reduced or blocked in fundus muscles of Ano1 −/− mice, this would suggest that ICC-IM are the dominant post-junctional cell receiving and transducing inputs from cholinergic enteric motor neurons in wild-type muscles. These hypotheses were confirmed by the results obtained in the present study. We investigated the conductance and cells responsible for mediating post-junctional responses to cholinergic, excitatory neurotransmission in the murine gastric fundus. Fundus muscles were chosen for these experiments because they have no ongoing slow wave activity that can obscure EJPs. In rhythmic portions of the GI tract, activation of cholinergic neurons initiates premature slow waves, and EJPs cannot be distinguished from the upstroke of the slow wave (Beckett et al. 2003). Fundus muscles also have only a single class of ICC (ICC-IM) that is found to be intermingled and closely associated with motor neurons in bundles of SMCs (Burns et al. 1996;Sanders, 1996;Ward et al. 2000). Distinct membrane conductances were activated in ICC-IM and SMCs by muscarinic agonists and we used this basic observation to determine which cells are activated in fundus muscles by ACh released from motor neurons. A Cl − conductance was activated in ICC-IM, and, as above, this is probably a result of the expression of Ano1. Expression of Ano1 was not resolved in SMCs and antagonists of this conductance failed to inhibit the inward currents elicited by CCh in these cells. Many previous studies report that NSCC are activated by muscarinic stimulation of GI SMCs (Benham et al. 1985;Lim & Bolton, 1988;Inoue & Isenberg, 1990a;Vogalis & Sanders, 1990;Sims, 1992;Lee et al. 1993; and such a conductance was activated by CCh in SMCs of murine fundus. CCh generated an outwardly rectifying current that reversed at 0 mV and was blocked by lanthanum. A study of small intestinal longitudinal tissues and SMCs also concluded that the conductance activated by muscarinic stimulation is the result of an NSCC encoded by Trpc4 and Trpc6 genes (Tsvilovskyy et al. 2009). When both genes were deactivated, excitatory, cholinergic responses of small intestinal muscles to EFS were reduced, suggesting that the NSCC composed of TRPC4 and TRPC6 channel proteins is essential for transducing cholinergic neurotransmission. These findings contrast with the observations of the present study in which blockers of Ano1 reduced cholinergic responses in ICC-IM, but not in SMCs, and cholinergic EJPs and contractile responses in intact muscles of the fundus were reduced in Ano1 knockouts and by Ano1 channel blocking drugs. Differences in the anatomy and innervation of the fundus and longitudinal muscle layer of the small intestine may explain the differences in mechanisms mediating post-junctional muscarinic responses. In fundus, ICC-IM are closely associated with varicosities of enteric motor neurons, which are the presumed sites of neurotransmitter release (Burns et al. 1996;Ward et al. 2000) in both the circular and longitudinal muscle layers (Fig. 2). Previous studies have suggested that this close relationship between nerve processes and ICC-IM tends to restrict overflow of ACh to SMCs as a result of the high expression of AChE by motor neurons (Worth et al. 2015;Bhetwal et al. 2013). In the mouse and other laboratory rodents, ICC-IM are not present in the longitudinal muscle layer of the small intestine (Torihashi et al. 1995;Burns et al. 1997) and innervation appears to be indirect, possibly resulting from overflow of neurotransmitter from the tertiary plexus of neurons connecting myenteric ganglia (Richardson, 1958;Llewellyn-Smith et al. 1993). However, most regions of the GI tract, particularly in larger mammals (Horiguchi et al. 2003;Blair et al. 2012) and humans (Ibba Manneschi et al. 2004), display the anatomical arrangements of motor neurons entwined with the cells of the SIP syncytium and close associations between ICC and varicosities of motor neurons, as found in the fundus. Thus, the murine fundus may be a more stereotypical example for motor innervation and neurotransmission in the GI tract than in intestinal longitudinal muscle layers of laboratory rodents.
CCh evoked large amplitude currents in ICC-IM that reversed near the Cl − equilibrium potential were inhibited by Ano1 antagonists, and not affected by La 3+ , suggesting that activation of a Cl − conductance is the primary post-junctional response that would be activated if ICC-IM mediate responses activated by ACh released from motor neurons. This idea was tested with intact muscles in which we could observe post-junctional electrical and mechanical responses to EFS. EJPs and contractile responses were generated in fundus muscles; however, when Ano1 was knocked out in global Ano1 -/animals or knocked down by tamoxifen activation of Cre recombinase in mice with double-floxed Ano1 alleles (iAno1 −/− ), EJPs and contractile responses were reduced (inducible knockout mice) or abolished (global knockout mice). Our results are consistent with the hypothesis that ICC-IM, via expression of muscarinic receptors (Epperson et al. 2000) and coupling to activation of Ano1 channels, J Physiol 596.9 elicits most of the post-junctional response to enteric excitatory regulation of the fundus.
Previous studies using W/W V mice in which ICC-IM are developmentally repressed as a result of loss-of-function mutations in the tyrosine kinase activity of Kit, suggested a role for ICC-IM in enteric motor neurotransmission (Burns et al. 1996;Ward et al. 2000). These experiments used short duration stimuli to allow clear evaluation of junction potentials, whereas contractile experiments, performed in later studies using longer duration EFS, concluded that ICC are not necessary for nitrergic and/or cholinergic neurotransmission (Huizinga et al. 2008;Zang et al. 2010Zang et al. , 2011. After comparing contractile responses to EFS with the actual lesions in ICC in the muscles under investigation, we more recently found that fundus muscles of W/W V mice are not completely devoid of ICC, as previously assumed (Sanders et al. 2014b). Mice with profound depletion of ICC displayed loss of nitrergic inhibition (1-32 Hz of EFS), although nitrergic responses were maintained in mice retaining portions of the ICC-IM population. However, it was also interesting to note that contractile responses to cholinergic nerve stimulation could be augmented in muscles with reduced ICC populations (Sanders et al. 2014b).
Genetic manipulation of pathways involved in neuroeffector responses is a powerful means of determining the cells and mechanisms responsible for neurotransduction. However, global knockouts can produce confusing results because, throughout development, there are opportunities for compensatory processes to replace normally utilized mechanisms. Cell-specific conditional knockout approaches such as expression of inducible Cre recombinase driven by cell-specific promoters can improve on the selectiveness of gene deactivation and reduce the time available for compensation of natural mechanisms. However, studies have shown that knockdowns produced by iCre can yield less than a complete knockout of floxed alleles (Bao et al. 2013). Thus, remaining functional phenotypes can persist and be confusing in terms of the true function and importance of specific pathways. For example, a recent study utilized Cre/LoxP technology to investigate the role of Ano1 in pacemaker activity of small intestinal ICC in the myenteric plexus (Malysz et al. 2017). Quantitative PCR demonstrated only an ß50% reduction in Ano1 transcripts after tamoxifen treatment, and immunohistochemistry revealed a mosaic pattern of Ano1 protein in ICC-MY. Partial loss of Ano1 did not block pacemaker activity but, instead, resulted in slow waves of diminished duration; however, slow waves were either desynchronized or lost entirely in animals showing extensive reduction in Ano1. In the present study, we utilized both global knockouts of Ano1 and inducible knockdowns using the Cre/loxP approach. We found total loss of cholinergic electrical and mechanical responses in the global knockouts, although in only 37% of muscles were cholinergic responses absent in the inducible knockdowns of Ano1. The remaining mice showed reduced responses. These findings, however, should not be interpreted as supporting the likelihood of additional ion channels contributing to cholinergic responses in the tamoxifen treated animals because cholinergic responses were abolished in global knockouts and in muscles treated with pharmacological antagonists of Ano1. Furthermore, although fundus muscles of iAno1 −/− mice showed a −4.3-fold reduction in Ano1 transcripts, they were still detected. Tamoxifen treatment probably had no indirect effect on Ano1 transcript expression because there was no significant difference in expression between tamoxifen treated iAno1 +/and age-matched (+/+) mice.
The SIP syncytium is an integrated tissue that determines the excitability of SMCs and ultimately the nature of GI motility . We found that knockdown of Ano1 in iAno1 −/− animals led to a significant reduction in the expression of genes associated with the three cell types making up the SIP syncytium, including Myh11 (SMCs), Kit (ICC) and Pdgfra (Pdgfrα + cells). Although SIP cells are now known to interact on a moment-to-moment basis to regulate the excitability of SMCs, the present study also suggests that regulation of SIP cell phenotypes may also be interdependent. Ano1 has been identified as a regulator of cell proliferation, migration and differentiation, as well as being involved in cell apoptosis (Wanitchakool et al. 2014). Our observation that these gene transcripts were down regulated in SMCs and Pdgfrα + cells needs further investigation. Kit was also down regulated in iAno1 −/− animals, although this did not lead to a marked change in protein expression, suggesting that there may be a significant time-dependence in the reduction of Kit transcript expression vs. Kit protein in ICC. Interestingly, although Ano1 was not expressed in Ano1 -/mutants Kit expression in ICC appeared normal.
The results of the present study and those of a previous study assaying post-junctional Ca 2+ sensitization pathways activated by cholinergic neurotransmission (Bhetwal et al. 2013) suggest that ICC-IM are the primary mediators of cholinergic neurotransmission. Both studies provide no evidence that ACh released from motor neurons reaches receptors on SMCs under normal circumstances. There is no question that SMCs of the GI tract express muscarinic receptors and respond to cholinergic agonists applied to external solutions (also known as bath application) and so the question of why it is difficult to resolve responses in SMCs to cholinergic neurotransmission remains. Many investigators have assumed that neurotransmitters released from enteric motor neurons can diffuse freely and bind to receptors distributed throughout the smooth muscle syncytium (i.e. en passant innervation and volume transmission). Indeed, the close contacts made between nerve varicosities and ICC-IM may restrict the diffusion of ACh and a high concentration of the neurotransmitter in confined post-junctional volumes may facilitate rapid metabolism as a result of expression of AChE by enteric neurons (Worth et al. 2015). Thus, the 'length constant of effectiveness' of ACh released from nerve varicosities may be limited. Data from the present study and the results of a previous study (Bhetwal et al. 2013) support this idea by showing that: (i) reduced ICC-IM in W/W V mice resulted in the activation of Ca 2+ mechanisms in SMCs in response to cholinergic neurotransmission that were not activated when ICC-IM were present in wild-type mice and (ii) an inhibitor of AChE caused recruited Ca 2+ sensitization mechanisms in SMCs and evoked large amplitude, long duration electrical and mechanical responses to EFS in fundus muscles that were not affected by genetic deactivation of Ano1.
In summary, Ano1 transcripts and protein are expressed functionally in ICC-IM that lie in close association with varicose nerve processes of cholinergic motor neurons in the gastric fundus. Cholinergic stimulation of isolated ICC-IM caused activation of a Cl − conductance, presumed to be Ano1 because these channels are expressed in ICC-IM and the responses were blocked by Ano1 antagonists. CCh activated a NSCC in SMCs. Blocking Ano1, either by genetic deactivation or by Ano1 channel antagonists, significantly decreased EJPs and contractile responses in intact fundus muscles. Our data are consistent with the hypothesis that ACh released from motor neurons primarily binds to receptors on ICC-IM and does not reach muscarinic receptors expressed by SMCs. Transduction of inputs from cholinergic motor neurons and development of EJPs occur via activation of Ano1 channels expressed by ICC-IM. Depolarization responses are conveyed to SMCs via gap junctions, and depolarization of SMCs activates voltage-dependent Ca 2+ channels, Ca 2+ entry, Ca 2+ sensitization mechanisms mediated by protein kinase C, and phosphorylation of CPI-17 and contraction (Bhetwal et al. 2013;Jensen et al. 1996;Somlyo & Somlyo, 2003). Blocking ACh metabolism or disrupting the close connectivity between nerve varicosities and ICC-IM causes activation of secondary responses mediated by SMCs. This redundancy in responsiveness may be a safety mechanism to insure continued GI motility in animals with defects in ICC or a vestigial response left over from before ICC became critical components of the SIP syncytium with respect to regulating GI motility.