Miniature endoscope for high resolution electrophysiological recordings from the colon of live mice

Miniature endoscope for high resolution electrophysiological recordings from the colon of live mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Miniature endoscope for high resolution electrophysiological recordings from the colon of live mice Aleksander Sobolewski, Arielle Planchette, Karol Wójcicki, Yoseline Cabara, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6916016/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Feb, 2026 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract A major weakness in the field of neurogastroenterology research has been lack of technology to determine the spatial and temporal coordination of electrical activity along the gastrointestinal (GI) tract in-vivo without requiring a surgical procedure. To overcome this weakness we developed a miniaturized endoscope consisting of 128 iridium oxide recording sensors that allowed us to make high resolution intraluminal electrophysiological recordings in-vivo from the mucosal surface of the terminal large intestine of anesthetized mice. Recordings revealed discharges of smooth muscle action potentials organized into complex spatiotemporal patterns. The patterns were modified by pharmacological agents donepezil and atropine that stimulated or suppressed cholinergic neurotransmission, respectively. The patterns were also ablated by benzalkonium chloride, known to disrupt the function of the enteric nervous system. The endoscope was further validated under ex-vivo recording conditions, where blocking enteric neural activity with tetrodotoxin (TTX) again altered spontaneously occurring action potential patterns. This novel approach offers a unique opportunity to easily characterize normal and dysfunctional patterns of GI electrical activity in genetically modified and/or diseased mouse models, including drug discovery and high-throughput studies. Health sciences/Gastroenterology/Colonoscopy Biological sciences/Biological techniques/Electrophysiology Biological sciences/Biological techniques/High-throughput screening Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Preclinical research in small animal models is advanced mainly by increasing the number of tools for detailed characterization of neurobiology spanning from immunohistochemistry to advanced high-resolution microscopy. Ex-vivo studies have been crucial in dissecting the fundamental mechanisms that govern colonic function in the absence of systemic influence such as hormonal or extrinsic innervation (Corsetti et al., 2019 ; Johnson et al., 2020 ). Whole colon preparations in an organ bath also facilitate targeted pharmacological manipulations without systemic effects and allow video monitoring of (micro)motion of the entire specimen (Hoogerwerf et al., 2010 ; Maxton & Whorwell, 1990 ; Parkar et al., 2024 ). However, functional in-vivo assessment of the colon, including the enteric nervous system (ENS) is still limited to very simplified approaches like total transit time (Koester et al., 2021 ; Schonkeren et al., 2023 )or bead expulsion assay (Han et al., 2025 ; Raffa et al., 1987 ). Hence, the underlying, complex electrophysiological patterns of gastrointestinal motor activity that drives propulsion of content along the GI-tract, and the modulations of those patterns by functional gastrointestinal disease (FGID) models or preclinical pharmacological interventions, remain hidden from researchers. Most electrical recordings from the gastrointestinal (GI) tract of live animals have been made after surgical implantation of recording electrodes in the outer muscle layers (the circular and longitudinal coats), known as the muscularis externa, in a variety of species, including dogs (Fioramonti, Bueno, Sarna, et al., 1980; Fioramonti, Garcia-Villar, et al., 1980 ; Fioramonti & Bueno, 1980a ), rats (Fioramonti & Bueno, 1980a ), rabbits and pigs (Fioramonti & Bueno, 1980b ), sheep and cows (Fioramonti & Hubert, 1980 ) and humans (Fioramonti, Bueno, & Frexinos, 1980 ). Notable studies have also correlated the electrical activity with motor activity in the upper GI-tract of anesthetized animals (Kuruppu et al., 2022 ), for review see: O’Grady et al., 2019 . In previous studies intraluminal suction electrodes have been used to record the myoelectric activity from the GI-tract of a variety of animal models, including human large intestine (Altaparmakov & Wienbeck, 1984 ; Couturier et al., 1969 ; Taylor et al., 1975 ). These studies used low-resolution recordings, such that only single or small numbers of recording sites were obtained. A downfall of using low-resolution data acquisition from organs like the GI tract is that the directionality and degree of spatial coordination of unique patterns of electrical activity is difficult to discriminate. This is partly because smooth muscle action potentials propagate over short distances (Spencer et al., 2002 ; Stevens et al., 2000 ) due to the low spatial constant of the syncytial properties of smooth muscle (Tomita, 1967 ). It would be a major step forward to be able to record in high resolution the electrical activity along the smooth muscle of the GI tract in live animals without requiring invasive surgical procedures, e.g. laparotomy. Improved resolution recordings have been made, on the other hand, of the mechanical activity of the intestine using fiber Bragg grating technology (Arkwright et al., 2011 ), however, these recordings were based on slow mechanical distortion of the gut wall, rather than the underlying electrophysiological function driving it. Two recent studies made intraluminal recordings from the intestine of rabbits (Xue et al., 2025 ), and another in pigs and mice (Srinivasan et al., 2024 ). Both approaches share similar, very interesting design concepts focused on recording from larger luminal diameters, clearly prioritizing future translatability to human medical devices. For this reason, however, they necessarily compromise on the resolution, ease of manufacture and ease of use of a device specifically needed and designed for high-resolution, high-throughput preclinical (mouse) medical research using modern technology. Thus, to advance the state-of-the-art, we have developed a novel miniaturized endoscope patterned with a matrix of 128 electrodes, each capable of resolving single smooth muscle action potentials, and together offering unprecedented exhaustive visualization of complex patterns of activity those action potentials form. Despite the high resolution, the endoscope remains small enough to be introduced per rectum into the colon of anesthetized mice with minimal workload burden on the experimenter and high repeatability, thus facilitating high-throughput investigations into functional GI changes in the vast array of disease models, including transgenic and genetically modified mice. Materials and Methods Mini-endoscope for high resolution electrophysiology recording The developed device consists of a semi-rigid ⌀2 mm and 30 mm-long cylindrical endoscope (Fig. 1 ). The substrate of the endoscope is a transparent nylon tube with a hemispherical distal end cap. The tube is wrapped in an electrode matrix that is a 10 µm-thick polyimide film bearing 128 tissue contacts connected to external readout pads through isolated conducting paths (Fig. 1 A). The electrode matrix is designed in Python using the KLayout integrated circuit (IC) layout library ( www.klayout.de ) and custom Python modules, and manufactured in an ISO-7 level cleanroom on 10 inch wafer. The tissue contacts, readout pads and conducting paths are made of platinum. The tissue contacts are circular with a 200 µm diameter. They are additionally coated with iridium oxide for improved impedance and charge injection capacity. The tissue contacts are laid out on a 32-by-4 grid with a 0.8 mm pitch along the endoscope shaft (thus longitudinally covering 24.8 mm of the colon) and 1.57 mm pitch (= 2 mm × π / 4) on the circumference of the shaft. The manufactured polyimide electrode matrix is L-shaped (Fig. 1 A), so that after wrapping the longer arm of the L longitudinally around the nylon tube, the shorter arm of the L forms a freely floating tab bearing readout pads for connection to a custom printed circuit board (PCB). The tab has alignment holes for precise positioning over the connection pads of the PCB, which has matching alignment holes. Connection pads of the electrode matrix are bonded to the connection pads of the custom PCB with conductive silver epoxy applied through a matching stencil. The custom PCB pins out the 128 channels to four 36 Position Dual Row Male Nano-M Connector (Omnetics Connector Corporation, USA) for connection to downstream recording / stimulating equipment (here we used the Grapevine Nomad, Ripple, LLC, USA). The extreme pins on each side of each Omnetics connector are fused and connected via the PCB to two 30 AWG (0.254 mm) ~ 10 cm-long insulated silver-plated copper wires terminated with ⌀0.22 13 mm-long gold-plated subdermal needles forming ground and reference electrodes. The PCB is housed in a custom 3D-printed enclosure, which also clamps the substrate nylon tube. The nylon tube contains a central channel for introduction of optional optical devices, e.g. for imaging of tissue motion. The electrode matrix wrapped around the tube is transparent enough to allow this. The design was optimized in repeated bench recordings in a saline bath, as well as in-vivo recordings in C57BL/6J mice (n = 20 in total), until the final design was converged upon as presented in Fig. 1 . In-vivo experimental recordings in anesthetized mice All animals were raised under specific pathogen free conditions and handled in compliance with Swiss Veterinary Law guidelines. The procedures were approved by the Veterinary Office of the Canton of Geneva (approval No. GE241D). During the recordings the animals were anaesthetized with a mixture of ketamine (100mg/kg dose, KETANARKON 100 ad us. Vet. Streuli Pharma) and medetomidine (10mg/kg dose, DORBENE® ad us. vet., Dr. E. Graeub AG) by intraperitoneal injection. Prior to anesthesia induction, animals were handled for ~ 5 minutes to naturally expel any feces. Once anesthesia was established, the distal colon was additionally flushed with 2 ml of warm saline using a slightly lubricated ⌀2 mm plastic tubing attached to a syringe, inserted to a depth of 30 mm, and vitamin A ointment (VITAMINE A Blache ong opht ,Bausch & Lomb Swiss AG) was applied to the eyes for hydration. The endoscope was inserted per rectum into the distal colon of the mouse up to the depth of 35 mm. Subdermal ground and reference needle electrodes were inserted under the skin of each of the hind legs of the mouse (Fig. 1 ). Endoscope electrode signals were monitored in real-time and recorded for up to 90 minutes with a Grapevine Nomad neural signals processor (Ripple, LLC, USA) at 2 kHz sampling rate with a 0.1 Hz high pass-filter and an antialiasing low-pass filter. In-vivo pharmacological manipulations To ascertain the physiological origin of the observed signals in-vivo, we tested their response to intraperitoneal injections of several compounds known to modulate the activity of the enteric nervous system and/or the intestinal smooth muscle, specifically: donepezil (3 mg/kg dose, donepezil hydrochloride D6821 Sigma-Aldrich) and atropine (2 mg/kg dose, atropine sulphate A03BA01). In-vivo recordings in mice with colonic tissue lesions To assess the sensitivity of the observed signals to local lesions of the tissue we used a benzalkonium chloride (BAC) model known to disrupt colonic motility (Qin et al., 2010 ). Briefly, the treatment consisted of inserting a swab soaked in 0.2% BAC to the desired depth up to 3 cm of the colon, holding it in place for 15 s before removing it for 5 minutes and repeating the process for a total of 8 cycles. Swabbing was chosen to minimize the invasiveness of the approach, contrary to previously published methods relying on laparotomy to treat the exterior lining of the gut wall (Tamada & Kiyama, 2016 ; Yoneda et al., 2002 ) or local intrarectal injections (Lan et al., 2023 ; Qin et al., 2010 ). Ex-vivo organ bath recordings of mouse colon To validate the physiological origin and significance of activity observed in-vivo in a more controlled setting, we used excised mouse colon preparations in an organ bath. Ex-vivo experiments also provided an opportunity for measurements over a slightly larger distance, since in-vivo recordings were limited to 3.5 cm insertion to avoid tissue damage. As a result, we also created a modified version of the device with an increased longitudinal pitch of the electrodes to 1.29 mm to cover 40 mm of the excised colon. C57BL/6J mice (n = 11 in total) were euthanized by inhalation overdose of isoflurane. The procedures were approved by Animal Welfare Committee of Flinders University (approval No. 4004). The entire colon was removed via midline laparotomy and placed in a Petri dish containing Krebs solution (containing in mM: NaCl 118; KCl 4.7; NaH 2 PO 4 1; NaHCO 3 25; MgCl 2 1.2; D-Glucose 11; CaCl 2 2.5, gassed with 95% O 2 and 5% CO 2 , 36.5°C). Intraluminal content was flushed with Krebs solution via syringe and the mesentery was removed with spring scissors. Preparations were then transferred to a 100 ml organ bath where the full length of the endoscope was inserted within the colonic lumen. The colon was fixed in position by ⌀100 µm stainless steel etymology pins at the distal and proximal ends to prevent longitudinal displacement during recordings. Preparations were continuously superfused with Krebs solution (36.5°C) at ~ 3.5 mL/min. Gut movements were recorded by video camera fixed above the organ bath (1280 x 960 pixels, 9.15 f.p.s.; Dino-Lite AM7515MZT, AnMo Electronics Corporation, Taiwan). Video was transformed into maps of circumferential gut diameter (diameter maps) with the spatiotemporal mapping technique described by Hennig et al. ( 1999 ) using custom-made software in Matlab (MathWorks, Inc., USA). Regions of minimal diameter (i.e. contraction) are represented as lightest pixels and maximal diameter (i.e. dilatation) is represented by darkest pixels. Ex-vivo pharmacological manipulations During ex-vivo validation, we investigated effects of application on the tissue preparation of tetrodotoxin (TTX, neuronal voltage-gated sodium channel blocker), BAYK8644 (L-type voltage-gated calcium channel agonist) and nicardipine (calcium channel blocker). Drugs were dissolved as stock solutions in water or dimethylsufoxide before dilution to the final concentrations in organ baths: tetrodotoxin (1 µM; T-550, Alomone Laboratories, Israel), nicardipine (3 µM, N7510) and BAYK8644 (0.1 µM, B112, Sigma Chemical Co., USA). Statistical and signal processing procedures One of the most conspicuous features of the recordings from our endoscope are smooth muscle action potentials. To visualize and quantify the intensity of this spiking activity we use RMS (root mean square) “envelope” of the signals, after first high-pass filtering them above 10 Hz. We use either 0.1 or 1 s-long windows for RMS calculation, depending on the time frame of interest (minutes or tens of minutes, respectively.) The derivation of this metric envelope timeseries is illustrated in Fig. 2 B. To capture and quantify any rhythmicity in the spiking activity, we calculate spectra of the RMS envelope using the short-time Fourier transform in 1- or 2-minute Hamming windows. As RMS are not zero-mean signals, we detrend them prior to spectrum calculation. To test the statistical significance of changes in action potential activity in-vivo across all channels - as measured by the RMS - brought on by our pharmacological manipulations presented in Fig. 3 , we compared each post-first-stimulus window of the RMS to the distribution of the RMS from baseline windows in the corresponding channel. This results in a very large number of singular statistical tests, requiring rigorous control for multiple comparisons (the false discovery error). To this end we used a two-dimensional (time / channel) implementation of the cluster-based permutation test (Sobolewski et al., 2011 ), first devised for one-dimensional time series by Maris & Oostenveld ( 2007 ). In brief: we ran a t-test on every time RMS window on every channel. Individual significant (p < 0.001) windows which were adjacent – either in time or across neighboring channels - were clustered. The data were then randomly permuted (mixing post-stimulus values with pre-stimulus baselines) 1000 times, with the above clustering and summing repeated for each permutation. The largest cluster size (window count) was taken from each random permutation to obtain an empirical random distribution of the sizes of the clusters. Finally, only those of the original clusters whose sizes exceeded 99.9% (equivalent to corrected p < 0.001) of the random permutation results were retained as truly significant. For any other results presented in this article, the more straightforward data analysis methods used are identified where applicable. Results Contact impedance characterization Bench tests of the endoscope in a saline bath demonstrated median functional electrode impedances of 23.65 kΩ (9.90 kΩ interquartile range Fig. 1 F). Notably, one major concern we had, pertaining to the entire concept of intraluminal recordings, was whether such values would carry over to in-situ (in-vivo), i.e. whether the endoscope electrodes would establish sufficient electrical contact with the tissue when simply inserted into the colon. Median in-situ impedances were 32.75 kΩ (12.20 kΩ interquartile range; Fig. 1 F), significantly higher than the preceding saline bath measurement (p < 0.001, paired two-sided Wilcoxon signed rank test), but still within a range for electrophysiological recordings for such small footprint contacts. Individual electrode impedances correlated strongly between saline and in-situ conditions (Pearson's r = 0.76; Fig. 1 F), suggesting that it is the manufacturing variability that explains much of the in-situ variance, ruling out a concern of some uncontrolled confounding factors (e.g. anatomy-related) systemically compromising intraluminal recordings. In-vivo recordings of intraluminal electrophysiology with high spatial resolution We present recordings with the final iteration of the device, which were performed in six animals (three healthy and three with colonic tissue lesions described below). A hallmark feature of our intraluminal colonic recordings was the detection of prominent action potentials (“spikes”, Fig. 2AB). These spikes had a mean half-width of 9.5 ms (no variance across three healthy mice; Fig. 2 C) and mean peak-to-peak amplitude of 285, 294 and 315 µV in the three animals; Fig. 2 C). A critical point regarding intraluminal recordings was whether the electrodes could maintain spatial selectivity or whether physiological fluid between them would create a common electrical environment, blurring spatial resolution. However, action potentials were recorded in neighboring contacts of the endoscope with less than half the amplitude (Fig. 2 D). Thus, with such steep spatial decay, we can be certain of the intraluminal recording’s ability to spatially capture signals of very local origin. Simultaneously, this also verifies optimal contact pitch of the endoscope: since spatially the sources of electrophysiological activity are somewhat oversampled, this ensures no blind spots. Interestingly, on average the detected action potentials exhibited not only amplitude attenuation but - along the circumferential axis - also phase reversals (Fig. 2 D), suggesting some possible non-trivial interplay of electrophysiological sources and sinks across the colonic wall. The recorded spikes formed readily discernible and consistent spatiotemporal patterns (Fig. 3 ). In ketamine/medetomidine anesthetized animals, the most prominent pattern consisted of ~ 10 s-long bursts of spiking activity observed primarily in the oral channels ~ 2 times per minute, covering the distal ~ 10 mm of the endoscope (black stars in Fig. 3 A). Each recorded burst typically had an internal pattern of activity, composed of periodic (~ 1 per second) burstlets (black diamonds in Fig. 3 B), frequently forming propagating anterograde waves. From the aboral sections of the colon we recorded different patterns of colonic spike patterns, frequently with a predominant retrograde propagation of action potentials (black triangles in Fig. 3 A). To quantify these periodicities in spiking activity, as well as capture and less conspicuous rhythms, we performed Fourier analysis of the RMS (calculated with at 100 ms window) of 30- minute signals (Fig. 3 D). This spectral analysis showed that the orally located anterograde bursts occur at 2.2 c.p.m. (black star in Fig. 3 D), whereas anterograde burstlets occur indeed at 60 c.p.m. (black diamond in Fig. 3 D). These findings were consistent across wildtype mice (Fig. 4 ). Spectral representation of the signals also uncovers other rhythmicity hotspots (e.g. oral 18 c.p.m. and aboral 30 c.p.m. in Figs. 3 D and 4 A), that are not easily distinguished with the naked eye in the raw electrophysiology signal. In-vivo pharmacological modulation of colonic electrophysiological activity To demonstrate in real-time the sensitivity of the device to pharmacological interventions, we modulated the activity by intraperitoneal injections of well-known pharmacological agents (Fig. 3 E). Donepezil, a cholinesterase inhibitor used to enhance cholinergic tone, rapidly and significantly upregulated spiking activity (Fig. 3 E, t = 7 min), while atropine, a cholinergic antagonist, had a predictable reversing effect and produced a rapid, short-lived significant reduction of spiking activity (Fig. 3 E, t = 17 min). Areas of statistically significant change in activity are outlined in cyan (upregulation) and orange (downregulation). The rapid onset and decay of pharmacological effects were most evident in the oral regions that primarily exhibit ~ 2.2 c.p.m. bursts of activity (black stars in Fig. 3 A), with the exception of a prolonged upregulation of activity at the aboral end of the colon (black triangles in Fig. 3 A). Furthermore, the decay of atropine downregulation was followed by a localized (across topmost seven channels) increase in spiking activity. In-vivo aberrant electrophysiological patterns in gut-lesioned animal model To verify the capability of the device to detect aberrant patterns between healthy and disrupted organs, we implemented a benzalkonium chloride (BAC) model known to disrupt colonic motility through tissue damage in three mice. None of the treated mice exhibited outward signs of distress or other spontaneous behavioral alterations. However, in all of them endoscopic intraluminal recording of the colon was altered from that described above in untreated wild type mice. We still recorded action potentials in all animals after BAC treatment (although their morphology appeared altered: Fig. 2 C) but the typical spatiotemporal patterns they formed in untreated animals formed were abolished (Fig. 4 BC). Instead, they formed different configurations of activity in each BAC-lesioned animal. Spiking frequency spectral analysis revealed the loss of the frequency bands seen commonly in healthy mice (18–30 c.p.m. and 60 c.p.m. bands), as well as broadening of the ~ 2 c.p.m. band (indicating “arrythmia”) as seen in Fig. 4 B. Various different patterns of electrical activity were recorded, for example trains of spikes with steady linear decrease in firing rate (Fig. 4 D, bottom). At the oral end of the endoscope, where the propagating waves of spike bursts occur at ~ 2 c.p.m. in healthy controls, summed spiking activity plots reveal the global loss of rhythmicity and amplitude of spike bursts in BAC-lesioned animals (Fig. 4 C, orange plots) compared to unlesioned controls (Fig. 4 C, blue plots). In BAC animals, a larger spread of spiking frequency with no prominent peaks at ~ 2 c.p.m. as was seen in Fourier frequency plots comparing the two groups (Fig. 4 C). Interestingly, each BAC animal exhibit different profiles of dysfunction, most easily observed in Fig. 4 B; this is likely due to the variation in the effects that BAC exposure can have on tissue. Ex-vivo colonic electrophysiological patterns To validate our endoscope, we used an established ex-vivo setup to characterize the intrinsic neural and muscular properties of the colon in isolation (Spencer, Costa, et al., 2021 ; Spencer et al., 2020 ; Spencer, Travis, et al., 2021 ). Resected intact sections of the colon were threaded over the endoscope shaft to mimic intraluminal recording. The ex-vivo set-up was slightly modified to allow the endoscope and the colon to be submerged in the bath at an angle to protect the electronics. Additionally, a benchmark suction pipette electrode was attached to the colon from the serosal side (see photo in Fig. 5 A). In the ex-vivo setup we readily recorded spikes. Moreover, the spikes were confirmed to be directly related to motor activity of the colon. Instantaneous variations of colon diameter along the entire length of the preparation extracted from the video demonstrate precise relationship between patterns of observed spikes and colonic (micro)motion (Fig. 5 B-D). Spike bursts were markedly different than those in anesthetized animals, as they appeared only every ~ 5 minutes (compared to twice per minute in-vivo), lasted much longer (up to 90 s compared to ~ 10 s in vivo) and travelled the extent of the entire colon preparation of 40 mm (Fig. 5 A). To confirm the role of enteric neurons in regulating colonic muscle activity(Spencer, Costa, et al., 2021 ) using our device, the sodium channel-blocker tetrodotoxin (TTX), was applied to the preparation (Fig. 5 A) and repeated in four biological samples. TTX did not abolish action potentials, however the pattens of electrical activity were modified such that complex spike bursts were replaced with single spike waves that discharged rhythmically at an increased frequency (4.6 times per minute in Fig. 5 ACD), specifically in the aboral region of the colon. To characterize the effects of increased action potential discharge following abolition of enteric nerve conduction, we applied the Ca 2+ channel opener, BAYK8644. This agonist did not restore the propagating waves of activity that occurred prior to TTX application. The global effects of these pharmacological interventions are evident in the RMS plots (Fig. 5 A). Finally, we confirm that signals recorded with the endoscope are L-type Ca 2+ channel-dependent action potentials by applying nicardipine to the colon from another animal, resulting in the complete removal of action potentials (Fig. 5 F). In another experiment, we compared the action potentials recorded with the suction pipette electrode, which was been established as a benchmark approach in numerous foundational studies (Costa et al., 2021 ; Hibberd et al., 2017 ; Spencer et al., 2018 ; Wood, 1973 )to the signals recorded from the contact of our endoscope nearest to it. The resemblance between the two signals was very close (Fig. 5 G), with Pearson’s coefficients of correlation of their RMS envelopes at 0.87 and 0.91 for 0.1 and 1 s RMS windows (precision levels), respectively (correlations were significant at p = 0 to the level of machine precision). Lastly, to confirm the spatial origin of the signals, the colon was opened longitudinally and the mucosa was peeled off from the distal one third of the preparation. The tissue was mounted lumen-side down over the endoscope shaft (see photo in Fig. 5 G). Electrical activity was recorded in all regions (Fig. 5 G) and the propagating waves of action potentials were observed in all preparations. Partial lengthwise surgical removal of the mucosa enabled selective recording with the tissue in direct contact with the circular muscle layer while at the same time recording from intact mucosa confirming that the endoscopes records electrophysiological signals originating below the mucosa that propagate through the full thickness of the gut wall. Discussion We have developed a miniature, easy-to-use endoscope capable of recording in high resolution the propagation of action potentials over tens of millimeters simultaneously along the rostro-caudal and circumferential axis of colonic smooth muscle in live mice. We believe that this new device can uncover anomalies and offer new insight into mechanistic understanding of gut function using disease models and genetically modified mice. Because recordings can be made minimally invasively, we can use this technique to provide repeated and rapid high-throughput experimentation without surgical intervention. Previous electrophysiological recordings from GI-smooth muscles in live animals have mostly – with the exception of the approaches discussed below - involved surgically invasive techniques from the outmost gut layers, often with low spatial resolution. Commonly such electrodes also used special attachment to tissue in order to ensure sufficient electric contact and create an isolated electric compartment. Thus, one justifiable concern with intraluminal recordings using an endoscope was tissue contact, particularly with no features for enhancing tissue adhesion through e.g. suction, distension (Xue et al., 2025 ), or adhesion (Srinivasan et al., 2024 ). We found, however, that a well-chosen endoscope diameter and specialized low-impedance coating on the sensors was sufficient for high-signal-to noise ratio thus enabling consistent capture single action potentials. However, it is possible that our approach works well in mice because their colons have no haustration and would not work as well in other species. Nevertheless, such approach is far less cumbersome and possible to use at scale, as required e.g. for drug discovery in preclinical research. It is possible the distension provided by the endoscope induces patterns of myoelectric activity recorded in live animals. However, the diameter of the endoscope was made at 2 mm which is similar to that of a murine fecal pellet and consistent with a view that the patterns recorded are physiologically relevant. Another concern of intraluminal recordings with individual sensors bearing no isolating attachment features could be the spatial selectivity of the electrodes. Immersion of all contacts within a shared conductive fluid environment could, in principle, lead to signal spread and reduce the ability to resolve localized activity. However, our results demonstrate that this is not the case when the electrode design is properly optimized. In developing our device, we took inspiration from recent advances in micro-electrocorticography (µECoG, Shokoueinejad et al., 2019 ). µECoG electrode arrays achieve high spatial resolution recording from the cerebral cortex - even sufficient for brain-computer interfacing - despite no compartmentalization, relying only on passive tissue adhesion. Similarly to µECoG, we made the footprint of the electrodes very small, but coated them with a low-impedance iridium oxide layer. As a result we found that the amplitude of action potentials declined significantly across neighboring contacts (Fig. 2 D), indicating that the signals originate from highly localized sources. Moreover, the precise electrode geometry and slight spatial oversampling of our device, open the door to applying advanced computational methods. These methods will allow advances toward localization and extraction of underlying signal sources. Electrophysiological recordings from contracting tissue are susceptible to motion artifacts. These artifacts may introduce spurious waveforms into the data, with time courses that reflect the physical displacement of tissue rather than genuine neural or myogenic activity. Ensuring that the contact is well attached to tissue is not enough to counter this: in any case the downstream signal path (whether realized as a cable or otherwise) must change its geometry (move, bend) and generate a triboelectric artifact (Symeonidou et al., 2018 ). To mitigate this, we restricted our analysis to high-frequency events, specifically action potentials, with time constants in the range of 10-20ms, much faster than the motion of the gut. We filtered slower activity (< 10 Hz) that can be seen in raw data (Fig. 2 A). A recent study(Xue et al., 2025 ) used a scalable balloon catheter which enabled electrical recordings from the colon of anesthetized rabbits. That study also recorded action potentials through the mucosa in the form of periodic spike bursts. That device had longitudinal resolution limited to two locations. This additionally prompted us to develop the current device, which provides high spatial resolution of the spatial spread and coordination of action potentials, and direct assessment of the complex patterns they form. A second recent publication proposed a device tested in pigs, as well as a version used in mice (Srinivasan et al., 2024 ). Not much information, however, was provided on the mouse device (which would be relevant to contrast here), nor data recorded with it, thus it is difficult to draw comparisons. The dimensions of the device are comparable (20-by-2 mm), though the channel count was limited to16 electrodes. The validation of our new device presented here also included recordings of intraluminal electrophysiological activity in excised mice colon in an organ bath combined with simultaneous video imaging of colonic wall movements using spatiotemporal mapping. This approach confirmed that the patterns of action potentials were directly related to motor activity of the colon This allows us to infer a similar relationship occurs in-vivo, where large coverage recording of colonic motility and other micromotion at high temporal- and spatial-resolution would otherwise not be possible. In conclusion, we believe that our novel simple-to-use device, with its ability to record colonic electrical activity in live mice at high resolution – down to singular action potentials - and high spatial coverage offers a unique opportunity to study GI dysfunction in disease models for drug discovery, as well as fundamental research. Declarations Acknowledgements: This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No 853378). Experiments in this study were supported by an Australian Research Council (ARC) grant # DPEI250100648 to NJS. References Altaparmakov, I., & Wienbeck, M. (1984). Local inhibition of myoelectrical activity of human colon by loperamide. Digestive Diseases and Sciences , 29 (3), 232–238. https://doi.org/10.1007/BF01296257 Arkwright, J. W., Blenman, N. G., Underhill, I. D., Maunder, S. A., Spencer, N. J., Costa, M., Brookes, S. J., Szczesniak, M. M., & Dinning, P. G. (2011). A fibre optic catheter for simultaneous measurement of longitudinal and circumferential muscular activity in the gastrointestinal tract. Journal of Biophotonics , 4 (4), 244–251. https://doi.org/10.1002/jbio.201000056 Corsetti, M., Costa, M., Bassotti, G., Bharucha, A. 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Sensors and Actuators B: Chemical , 424 , 136955. https://doi.org/10.1016/j.snb.2024.136955 Yoneda, A., Shima, H., Nemeth, L., Oue, T., & Puri, P. (2002). Selective chemical ablation of the enteric plexus in mice. Pediatric Surgery International , 18 (4), 234–237. https://doi.org/10.1007/s003830100681 Additional Declarations There is NO Competing Interest. Cite Share Download PDF Status: Published Journal Publication published 04 Feb, 2026 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6916016","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":481617312,"identity":"54984124-e370-4c74-977e-9da0a25e1caf","order_by":0,"name":"Aleksander Sobolewski","email":"data:image/png;base64,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","orcid":"","institution":"Wyss Center for Bio and Neuroengineering","correspondingAuthor":true,"prefix":"","firstName":"Aleksander","middleName":"","lastName":"Sobolewski","suffix":""},{"id":481617313,"identity":"b6c26024-f88e-4862-8ef9-2623e797e02b","order_by":1,"name":"Arielle Planchette","email":"","orcid":"","institution":"Wyss Center for Bio and Neuroengineering","correspondingAuthor":false,"prefix":"","firstName":"Arielle","middleName":"","lastName":"Planchette","suffix":""},{"id":481617314,"identity":"f0cc2f65-447c-4917-b46b-1559317ec563","order_by":2,"name":"Karol Wójcicki","email":"","orcid":"","institution":"Wyss Center for Bio and Neuroengineering","correspondingAuthor":false,"prefix":"","firstName":"Karol","middleName":"","lastName":"Wójcicki","suffix":""},{"id":481617315,"identity":"10258705-9711-4edf-8379-10574c384b61","order_by":3,"name":"Yoseline Cabara","email":"","orcid":"","institution":"Wyss Center for Bio and Neuroengineering","correspondingAuthor":false,"prefix":"","firstName":"Yoseline","middleName":"","lastName":"Cabara","suffix":""},{"id":481617316,"identity":"959c19c1-ceff-4c4a-981b-4a10b9f275c8","order_by":4,"name":"Timothy Hibberd","email":"","orcid":"https://orcid.org/0000-0002-3747-140X","institution":"Flinders University","correspondingAuthor":false,"prefix":"","firstName":"Timothy","middleName":"","lastName":"Hibberd","suffix":""},{"id":481617317,"identity":"98369797-34b6-422e-a2dc-261aae2d2b9d","order_by":5,"name":"Lee Travis","email":"","orcid":"https://orcid.org/0000-0003-1770-7486","institution":"Flinders University","correspondingAuthor":false,"prefix":"","firstName":"Lee","middleName":"","lastName":"Travis","suffix":""},{"id":481617318,"identity":"5ff0e6fa-18b1-4021-87d8-53177d1ce1ea","order_by":6,"name":"Nick Spencer","email":"","orcid":"https://orcid.org/0000-0003-2190-5303","institution":"Flinders University","correspondingAuthor":false,"prefix":"","firstName":"Nick","middleName":"","lastName":"Spencer","suffix":""},{"id":481617319,"identity":"0fde2eeb-e8b1-42c1-ac03-ee580f08fbde","order_by":7,"name":"Michalina Gora","email":"","orcid":"https://orcid.org/0000-0002-1200-3511","institution":"Wyss Center for Bio and Neuroengineering","correspondingAuthor":false,"prefix":"","firstName":"Michalina","middleName":"","lastName":"Gora","suffix":""}],"badges":[],"createdAt":"2025-06-17 15:45:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6916016/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6916016/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-026-69144-2","type":"published","date":"2026-02-04T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":86666790,"identity":"6b255838-c220-4c6f-bc5c-ad93b1bdec9d","added_by":"auto","created_at":"2025-07-14 11:09:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":402055,"visible":true,"origin":"","legend":"\u003cp\u003eThe electrophysiological endoscope presented in this paper. \u003cstrong\u003eA: \u003c/strong\u003eThe polyimide electrode matrix with: 1 - tissue contacts, 2 - conductive paths and 3 - PCB connection pads. \u003cstrong\u003eB: \u003c/strong\u003eExploded view of constituent parts of the endoscope: 1 - printed circuit board (PCB) routing signal paths to connectors, 2 – nylon tube, the substate of the endoscope, 3-5 - housing components, 6 – polyimide film bearing electrode contacts, connection pads and conducting paths, which is wrapped around the nylon tube, 7,8 – reference and ground wires terminating in gold-plated subdermal needle electrodes.\u003cstrong\u003e C: \u003c/strong\u003eRendering of the endoscope and its positioning when inserted into the animal. \u003cstrong\u003eD:\u003c/strong\u003eMicroscope image of the endoscope shaft wrapped in the electrode matrix. Several tissue contacts have been marked with arrows. \u003cstrong\u003eE: \u003c/strong\u003ePhoto of the entire setup. \u003cstrong\u003eF:\u003c/strong\u003e Box plot of impedances of the endoscope’s electrodes in-situ, as well as in a saline bath immediately preceding and following the in-situ recording in one animal. Boxes denote the median, the lower and upper quartiles; the whiskers denote the minimum and maximum values that are not outliers. P-values denote significance in median differences in a paired two-sided Wilcoxon signed rank test; r-values denote Pearson’s coefficients of correlation across the electrodes between measurements. The few channels broken during device manufacturing were excluded from this data.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6916016/v1/e98c219ff1d4731432d99ad1.png"},{"id":86666762,"identity":"a0a415ff-e788-44e8-885d-1b4e80ca2335","added_by":"auto","created_at":"2025-07-14 11:09:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":284604,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA:\u003c/strong\u003eExample of a raw signal from one electrode of the endoscope showing the spikes, which constitute the predominant feature of the recordings. A single spike is magnified on the inset. \u003cstrong\u003eB:\u003c/strong\u003e The blue trace is the signal from panel A high-pass filtered above 10 Hz, the blue histograms are an RMS quantification (“envelope”) of the spiking activity in that example in 0.1 and 1 s sliding windows. Since RMS is a metric of the spiking activity we rely on frequently herein, we include this illustration of how it is derived. \u003cstrong\u003eC:\u003c/strong\u003e Mean (n from 14951 to 39881) spike traces from six mice, demonstrating the time course of waveform of an average isolated action potential. The spike pool used for the average was obtained by finding all negative peaks in data with a defined minimal prominence (maximum value of -150 µV and no neighbors within 50 ms) and extracting and averaging epochs centered on those peaks. \u003cstrong\u003eD:\u003c/strong\u003e Spatial spread of an average (n=35299) isolated spike from an example mouse (iv13wt3). Median, the lower and upper quartile traces are drawn. On the inset below, the same spread is presented but in both spatial dimensions on one plot and the temporal dimension across consecutive plots (timestamps are relative to the timing of main negative peak of the spike).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6916016/v1/f85f5e53ccf02966021e3dac.png"},{"id":86666801,"identity":"3610b199-1b1d-47ba-83c7-425551771389","added_by":"auto","created_at":"2025-07-14 11:09:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":878510,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA: \u003c/strong\u003eExample 3 minutes of signals recorded from all electrodes of the endoscope in a ketamine / medetomidine anesthetized wild type mouse (animal ID: iv13wt3) covering 24.8 mm of the distal colon. The low frequency component, visible in Figure 2A, has been removed with 10 Hz high-pass filter, a common-average spatial filter has been applied to reduce any remaining power line noise, and a few (n=4) broken channels have been interpolated. \u003cstrong\u003eB:\u003c/strong\u003e Example 15 s of signals from panel A at a higher temporal magnification. \u003cstrong\u003eC:\u003c/strong\u003e RMS heatmap representation of the spiking activity of the signal in panel A. \u003cstrong\u003eD:\u003c/strong\u003eSpectra of RMS of all the channels of 30 min data from panel E, showing various rhythmic spiking patterns discernible in that data. The power spectral density has been log-transformed to better visualize power in higher frequencies. \u003cstrong\u003eE: \u003c/strong\u003eFull 30-minute recording from same mouse (iv13wt3). Cyan and orange outlines denote significant (cluster-based permutation test, see Methods for details) increases and drops of spiking activity due to donepezil and atropine application (respectively) compared to the baseline before the donepezil application. The histogram above the signals shows summed RMS of spiking activity in from the oral (distal) half of the channels; the black curve overlaid on the histogram traces a 200 s moving mean trend, with the dashed line indicating the mean pre-donepezil baseline. The blue rectangle outlines the time span zoomed in on in panel A.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6916016/v1/e05dbe47892d9cf21df0a249.png"},{"id":86666800,"identity":"1af7b3f8-d319-4e64-874e-f7b0e7231784","added_by":"auto","created_at":"2025-07-14 11:09:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":651895,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA:\u003c/strong\u003e3-minute examples of signals from all channels from three wildtype mice and power spectra of the RMS envelopes of full 30-minute recordings. (Narrow harmonic bands seen in high frequencies in oral channels of mouse iv14wt1 are artifacts.) \u003cstrong\u003eB:\u003c/strong\u003e 3-minute examples of signals from all channels from three BAC-lesioned mice and log-transformed power spectra of the RMS envelopes of full 30-minute recordings. (Narrow harmonic bands seen in high frequencies in oral channels of mouse iv14bac3-1 are artifacts.) \u003cstrong\u003eC:\u003c/strong\u003e Aggregate spiking activity (summed RMS envelopes) from oral (distal) half of the channels in the three wild-type and three BAC-lesioned mice over 30 minutes (left), and their spectra (right). \u003cstrong\u003eD:\u003c/strong\u003eAn example of aberrant activity patterns in a BAC-lesioned mouse, consisting of trains of spikes with steady linear decrease in rate, likely representing spasms.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6916016/v1/b68c09393bf8880982cd12d9.png"},{"id":86666774,"identity":"93d19bb3-a44e-4189-8971-4c672963070c","added_by":"auto","created_at":"2025-07-14 11:09:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":611936,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA:\u003c/strong\u003eExample 50-minute of signals recorded from all electrodes in an ex-vivo preparation of 40 mm of the distal colon, showing spontaneous activity, as well as effects of application of tetrodotoxin (TTX, neuronal voltage-gated sodium channel blocker) and subsequent application of BAYK8644 (L-type voltage-gated calcium channel agonist). The blue histogram above the signals shows mean spiking activity (RMS envelope) across all channels. \u003cstrong\u003eB,C,D:\u003c/strong\u003e Example 2-minute signals zoomed in on blue boxes from panel A, showing magnification of spontaneous activity and activity after TTX and BAYK8644 application, as well as concurrent instantaneous micromotions of the preparation (variations of colon diameter) extracted from video, corresponding in time and location to the signals. \u003cstrong\u003eE:\u003c/strong\u003e Example 20-minute signals from another ex-vivo preparation, where mucosa was removed from distal third of the colon (see inset photo). Zoomed in 20-second sections of the signals are also shown separately from the intact and mucosa-stripped segments. \u003cstrong\u003eF:\u003c/strong\u003e 40-minute example of spiking activity (RMS envelope) of signals recorded from a different ex-vivo sample, showing the effects of a calcium channel blocker (nicardipine). G: 15-minute, 100-second and 5-second examples of signals from the benchmark state-of-the-art external suction pipette electrode and one channel of the endoscope nearest to it.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6916016/v1/f1d8bc8238dc809b73c01475.png"},{"id":104469238,"identity":"d73938b6-c189-471c-aa39-6561506b4990","added_by":"auto","created_at":"2026-03-12 07:06:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3246513,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6916016/v1/6377f97c-4b36-4113-b501-f199c993f81c.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Miniature endoscope for high resolution electrophysiological recordings from the colon of live mice","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePreclinical research in small animal models is advanced mainly by increasing the number of tools for detailed characterization of neurobiology spanning from immunohistochemistry to advanced high-resolution microscopy. Ex-vivo studies have been crucial in dissecting the fundamental mechanisms that govern colonic function in the absence of systemic influence such as hormonal or extrinsic innervation (Corsetti et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Johnson et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Whole colon preparations in an organ bath also facilitate targeted pharmacological manipulations without systemic effects and allow video monitoring of (micro)motion of the entire specimen (Hoogerwerf et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Maxton \u0026amp; Whorwell, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Parkar et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, functional in-vivo assessment of the colon, including the enteric nervous system (ENS) is still limited to very simplified approaches like total transit time (Koester et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Schonkeren et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e)or bead expulsion assay (Han et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Raffa et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). Hence, the underlying, complex electrophysiological patterns of gastrointestinal motor activity that drives propulsion of content along the GI-tract, and the modulations of those patterns by functional gastrointestinal disease (FGID) models or preclinical pharmacological interventions, remain hidden from researchers.\u003c/p\u003e\u003cp\u003eMost electrical recordings from the gastrointestinal (GI) tract of live animals have been made after surgical implantation of recording electrodes in the outer muscle layers (the circular and longitudinal coats), known as the muscularis externa, in a variety of species, including dogs (Fioramonti, Bueno, Sarna, et al., 1980; Fioramonti, Garcia-Villar, et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1980\u003c/span\u003e; Fioramonti \u0026amp; Bueno, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1980a\u003c/span\u003e), rats (Fioramonti \u0026amp; Bueno, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1980a\u003c/span\u003e), rabbits and pigs (Fioramonti \u0026amp; Bueno, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1980b\u003c/span\u003e), sheep and cows (Fioramonti \u0026amp; Hubert, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1980\u003c/span\u003e) and humans (Fioramonti, Bueno, \u0026amp; Frexinos, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1980\u003c/span\u003e). Notable studies have also correlated the electrical activity with motor activity in the upper GI-tract of anesthetized animals (Kuruppu et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), for review see: O\u0026rsquo;Grady et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eIn previous studies intraluminal suction electrodes have been used to record the myoelectric activity from the GI-tract of a variety of animal models, including human large intestine (Altaparmakov \u0026amp; Wienbeck, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1984\u003c/span\u003e; Couturier et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1969\u003c/span\u003e; Taylor et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1975\u003c/span\u003e). These studies used low-resolution recordings, such that only single or small numbers of recording sites were obtained. A downfall of using low-resolution data acquisition from organs like the GI tract is that the directionality and degree of spatial coordination of unique patterns of electrical activity is difficult to discriminate. This is partly because smooth muscle action potentials propagate over short distances (Spencer et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Stevens et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) due to the low spatial constant of the syncytial properties of smooth muscle (Tomita, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1967\u003c/span\u003e). It would be a major step forward to be able to record in high resolution the electrical activity along the smooth muscle of the GI tract in live animals without requiring invasive surgical procedures, e.g. laparotomy. Improved resolution recordings have been made, on the other hand, of the mechanical activity of the intestine using fiber Bragg grating technology (Arkwright et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), however, these recordings were based on slow mechanical distortion of the gut wall, rather than the underlying electrophysiological function driving it.\u003c/p\u003e\u003cp\u003eTwo recent studies made intraluminal recordings from the intestine of rabbits (Xue et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), and another in pigs and mice (Srinivasan et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Both approaches share similar, very interesting design concepts focused on recording from larger luminal diameters, clearly prioritizing future translatability to human medical devices. For this reason, however, they necessarily compromise on the resolution, ease of manufacture and ease of use of a device specifically needed and designed for high-resolution, high-throughput preclinical (mouse) medical research using modern technology.\u003c/p\u003e\u003cp\u003eThus, to advance the state-of-the-art, we have developed a novel miniaturized endoscope patterned with a matrix of 128 electrodes, each capable of resolving single smooth muscle action potentials, and together offering unprecedented exhaustive visualization of complex patterns of activity those action potentials form. Despite the high resolution, the endoscope remains small enough to be introduced per rectum into the colon of anesthetized mice with minimal workload burden on the experimenter and high repeatability, thus facilitating high-throughput investigations into functional GI changes in the vast array of disease models, including transgenic and genetically modified mice.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eMini-endoscope for high resolution electrophysiology recording\u003c/p\u003e\u003cp\u003eThe developed device consists of a semi-rigid ⌀2 mm and 30 mm-long cylindrical endoscope (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The substrate of the endoscope is a transparent nylon tube with a hemispherical distal end cap. The tube is wrapped in an electrode matrix that is a 10 \u0026micro;m-thick polyimide film bearing 128 tissue contacts connected to external readout pads through isolated conducting paths (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The electrode matrix is designed in Python using the KLayout integrated circuit (IC) layout library (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.klayout.de\" target=\"_blank\"\u003ewww.klayout.de\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.klayout.de\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and custom Python modules, and manufactured in an ISO-7 level cleanroom on 10 inch wafer. The tissue contacts, readout pads and conducting paths are made of platinum. The tissue contacts are circular with a 200 \u0026micro;m diameter. They are additionally coated with iridium oxide for improved impedance and charge injection capacity. The tissue contacts are laid out on a 32-by-4 grid with a 0.8 mm pitch along the endoscope shaft (thus longitudinally covering 24.8 mm of the colon) and 1.57 mm pitch (=\u0026thinsp;2 mm\u0026thinsp;\u0026times;\u0026thinsp;π / 4) on the circumference of the shaft. The manufactured polyimide electrode matrix is L-shaped (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), so that after wrapping the longer arm of the L longitudinally around the nylon tube, the shorter arm of the L forms a freely floating tab bearing readout pads for connection to a custom printed circuit board (PCB). The tab has alignment holes for precise positioning over the connection pads of the PCB, which has matching alignment holes. Connection pads of the electrode matrix are bonded to the connection pads of the custom PCB with conductive silver epoxy applied through a matching stencil. The custom PCB pins out the 128 channels to four 36 Position Dual Row Male Nano-M Connector (Omnetics Connector Corporation, USA) for connection to downstream recording / stimulating equipment (here we used the Grapevine Nomad, Ripple, LLC, USA). The extreme pins on each side of each Omnetics connector are fused and connected via the PCB to two 30 AWG (0.254 mm)\u0026thinsp;~\u0026thinsp;10 cm-long insulated silver-plated copper wires terminated with ⌀0.22 13 mm-long gold-plated subdermal needles forming ground and reference electrodes. The PCB is housed in a custom 3D-printed enclosure, which also clamps the substrate nylon tube. The nylon tube contains a central channel for introduction of optional optical devices, e.g. for imaging of tissue motion. The electrode matrix wrapped around the tube is transparent enough to allow this. The design was optimized in repeated bench recordings in a saline bath, as well as in-vivo recordings in C57BL/6J mice (n\u0026thinsp;=\u0026thinsp;20 in total), until the final design was converged upon as presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn-vivo experimental recordings in anesthetized mice\u003c/p\u003e\u003cp\u003e All animals were raised under specific pathogen free conditions and handled in compliance with Swiss Veterinary Law guidelines. The procedures were approved by the Veterinary Office of the Canton of Geneva (approval No. GE241D). During the recordings the animals were anaesthetized with a mixture of ketamine (100mg/kg dose, KETANARKON 100 ad us. Vet. Streuli Pharma) and medetomidine (10mg/kg dose, DORBENE\u0026reg; ad us. vet., Dr. E. Graeub AG) by intraperitoneal injection. Prior to anesthesia induction, animals were handled for ~\u0026thinsp;5 minutes to naturally expel any feces. Once anesthesia was established, the distal colon was additionally flushed with 2 ml of warm saline using a slightly lubricated ⌀2 mm plastic tubing attached to a syringe, inserted to a depth of 30 mm, and vitamin A ointment (VITAMINE A Blache ong opht ,Bausch \u0026amp; Lomb Swiss AG) was applied to the eyes for hydration. The endoscope was inserted per rectum into the distal colon of the mouse up to the depth of 35 mm. Subdermal ground and reference needle electrodes were inserted under the skin of each of the hind legs of the mouse (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Endoscope electrode signals were monitored in real-time and recorded for up to 90 minutes with a Grapevine Nomad neural signals processor (Ripple, LLC, USA) at 2 kHz sampling rate with a 0.1 Hz high pass-filter and an antialiasing low-pass filter.\u003c/p\u003e\u003cp\u003eIn-vivo pharmacological manipulations\u003c/p\u003e\u003cp\u003eTo ascertain the physiological origin of the observed signals in-vivo, we tested their response to intraperitoneal injections of several compounds known to modulate the activity of the enteric nervous system and/or the intestinal smooth muscle, specifically: donepezil (3 mg/kg dose, donepezil hydrochloride D6821 Sigma-Aldrich) and atropine (2 mg/kg dose, atropine sulphate A03BA01).\u003c/p\u003e\u003cp\u003eIn-vivo recordings in mice with colonic tissue lesions\u003c/p\u003e\u003cp\u003eTo assess the sensitivity of the observed signals to local lesions of the tissue we used a benzalkonium chloride (BAC) model known to disrupt colonic motility (Qin et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Briefly, the treatment consisted of inserting a swab soaked in 0.2% BAC to the desired depth up to 3 cm of the colon, holding it in place for 15 s before removing it for 5 minutes and repeating the process for a total of 8 cycles. Swabbing was chosen to minimize the invasiveness of the approach, contrary to previously published methods relying on laparotomy to treat the exterior lining of the gut wall (Tamada \u0026amp; Kiyama, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Yoneda et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) or local intrarectal injections (Lan et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Qin et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eEx-vivo organ bath recordings of mouse colon\u003c/p\u003e\u003cp\u003eTo validate the physiological origin and significance of activity observed in-vivo in a more controlled setting, we used excised mouse colon preparations in an organ bath. Ex-vivo experiments also provided an opportunity for measurements over a slightly larger distance, since in-vivo recordings were limited to 3.5 cm insertion to avoid tissue damage. As a result, we also created a modified version of the device with an increased longitudinal pitch of the electrodes to 1.29 mm to cover 40 mm of the excised colon.\u003c/p\u003e\u003cp\u003eC57BL/6J mice (n\u0026thinsp;=\u0026thinsp;11 in total) were euthanized by inhalation overdose of isoflurane. The procedures were approved by Animal Welfare Committee of Flinders University (approval No. 4004). The entire colon was removed via midline laparotomy and placed in a Petri dish containing Krebs solution (containing in mM: NaCl 118; KCl 4.7; NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e 1; NaHCO\u003csub\u003e3\u003c/sub\u003e 25; MgCl\u003csub\u003e2\u003c/sub\u003e 1.2; D-Glucose 11; CaCl\u003csub\u003e2\u003c/sub\u003e 2.5, gassed with 95% O\u003csub\u003e2\u003c/sub\u003e and 5% CO\u003csub\u003e2\u003c/sub\u003e, 36.5\u0026deg;C). Intraluminal content was flushed with Krebs solution via syringe and the mesentery was removed with spring scissors. Preparations were then transferred to a 100 ml organ bath where the full length of the endoscope was inserted within the colonic lumen. The colon was fixed in position by ⌀100 \u0026micro;m stainless steel etymology pins at the distal and proximal ends to prevent longitudinal displacement during recordings. Preparations were continuously superfused with Krebs solution (36.5\u0026deg;C) at ~\u0026thinsp;3.5 mL/min.\u003c/p\u003e\u003cp\u003eGut movements were recorded by video camera fixed above the organ bath (1280 x 960 pixels, 9.15 f.p.s.; Dino-Lite AM7515MZT, AnMo Electronics Corporation, Taiwan). Video was transformed into maps of circumferential gut diameter (diameter maps) with the spatiotemporal mapping technique described by Hennig et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) using custom-made software in Matlab (MathWorks, Inc., USA). Regions of minimal diameter (i.e. contraction) are represented as lightest pixels and maximal diameter (i.e. dilatation) is represented by darkest pixels.\u003c/p\u003e\u003cp\u003eEx-vivo pharmacological manipulations\u003c/p\u003e\u003cp\u003eDuring ex-vivo validation, we investigated effects of application on the tissue preparation of tetrodotoxin (TTX, neuronal voltage-gated sodium channel blocker), BAYK8644 (L-type voltage-gated calcium channel agonist) and nicardipine (calcium channel blocker). Drugs were dissolved as stock solutions in water or dimethylsufoxide before dilution to the final concentrations in organ baths: tetrodotoxin (1 \u0026micro;M; T-550, Alomone Laboratories, Israel), nicardipine (3 \u0026micro;M, N7510) and BAYK8644 (0.1 \u0026micro;M, B112, Sigma Chemical Co., USA).\u003c/p\u003e\u003cp\u003eStatistical and signal processing procedures\u003c/p\u003e\u003cp\u003eOne of the most conspicuous features of the recordings from our endoscope are smooth muscle action potentials. To visualize and quantify the intensity of this spiking activity we use RMS (root mean square) \u0026ldquo;envelope\u0026rdquo; of the signals, after first high-pass filtering them above 10 Hz. We use either 0.1 or 1 s-long windows for RMS calculation, depending on the time frame of interest (minutes or tens of minutes, respectively.) The derivation of this metric envelope timeseries is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB. To capture and quantify any rhythmicity in the spiking activity, we calculate spectra of the RMS envelope using the short-time Fourier transform in 1- or 2-minute Hamming windows. As RMS are not zero-mean signals, we detrend them prior to spectrum calculation.\u003c/p\u003e\u003cp\u003eTo test the statistical significance of changes in action potential activity in-vivo across all channels - as measured by the RMS - brought on by our pharmacological manipulations presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, we compared each post-first-stimulus window of the RMS to the distribution of the RMS from baseline windows in the corresponding channel. This results in a very large number of singular statistical tests, requiring rigorous control for multiple comparisons (the false discovery error). To this end we used a two-dimensional (time / channel) implementation of the cluster-based permutation test (Sobolewski et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), first devised for one-dimensional time series by Maris \u0026amp; Oostenveld (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). In brief: we ran a t-test on every time RMS window on every channel. Individual significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) windows which were adjacent \u0026ndash; either in time or across neighboring channels - were clustered. The data were then randomly permuted (mixing post-stimulus values with pre-stimulus baselines) 1000 times, with the above clustering and summing repeated for each permutation. The largest cluster size (window count) was taken from each random permutation to obtain an empirical random distribution of the sizes of the clusters. Finally, only those of the original clusters whose sizes exceeded 99.9% (equivalent to corrected p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) of the random permutation results were retained as truly significant.\u003c/p\u003e\u003cp\u003eFor any other results presented in this article, the more straightforward data analysis methods used are identified where applicable.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eContact impedance characterization\u003c/p\u003e\u003cp\u003eBench tests of the endoscope in a saline bath demonstrated median functional electrode impedances of 23.65 kΩ (9.90 kΩ interquartile range Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Notably, one major concern we had, pertaining to the entire concept of intraluminal recordings, was whether such values would carry over to in-situ (in-vivo), i.e. whether the endoscope electrodes would establish sufficient electrical contact with the tissue when simply inserted into the colon. Median in-situ impedances were 32.75 kΩ (12.20 kΩ interquartile range; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF), significantly higher than the preceding saline bath measurement (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, paired two-sided Wilcoxon signed rank test), but still within a range for electrophysiological recordings for such small footprint contacts. Individual electrode impedances correlated strongly between saline and in-situ conditions (Pearson's r\u0026thinsp;=\u0026thinsp;0.76; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF), suggesting that it is the manufacturing variability that explains much of the in-situ variance, ruling out a concern of some uncontrolled confounding factors (e.g. anatomy-related) systemically compromising intraluminal recordings.\u003c/p\u003e\u003cp\u003eIn-vivo recordings of intraluminal electrophysiology with high spatial resolution\u003c/p\u003e\u003cp\u003eWe present recordings with the final iteration of the device, which were performed in six animals (three healthy and three with colonic tissue lesions described below). A hallmark feature of our intraluminal colonic recordings was the detection of prominent action potentials (\u0026ldquo;spikes\u0026rdquo;, Fig.\u0026nbsp;2AB). These spikes had a mean half-width of 9.5 ms (no variance across three healthy mice; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) and mean peak-to-peak amplitude of 285, 294 and 315 \u0026micro;V in the three animals; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). A critical point regarding intraluminal recordings was whether the electrodes could maintain spatial selectivity or whether physiological fluid between them would create a common electrical environment, blurring spatial resolution. However, action potentials were recorded in neighboring contacts of the endoscope with less than half the amplitude (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Thus, with such steep spatial decay, we can be certain of the intraluminal recording\u0026rsquo;s ability to spatially capture signals of very local origin. Simultaneously, this also verifies optimal contact pitch of the endoscope: since spatially the sources of electrophysiological activity are somewhat oversampled, this ensures no blind spots. Interestingly, on average the detected action potentials exhibited not only amplitude attenuation but - along the circumferential axis - also phase reversals (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), suggesting some possible non-trivial interplay of electrophysiological sources and sinks across the colonic wall.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe recorded spikes formed readily discernible and consistent spatiotemporal patterns (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In ketamine/medetomidine anesthetized animals, the most prominent pattern consisted of ~\u0026thinsp;10 s-long bursts of spiking activity observed primarily in the oral channels\u0026thinsp;~\u0026thinsp;2 times per minute, covering the distal\u0026thinsp;~\u0026thinsp;10 mm of the endoscope (black stars in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Each recorded burst typically had an internal pattern of activity, composed of periodic (~\u0026thinsp;1 per second) burstlets (black diamonds in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), frequently forming propagating anterograde waves. From the aboral sections of the colon we recorded different patterns of colonic spike patterns, frequently with a predominant retrograde propagation of action potentials (black triangles in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo quantify these periodicities in spiking activity, as well as capture and less conspicuous rhythms, we performed Fourier analysis of the RMS (calculated with at 100 ms window) of 30- minute signals (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). This spectral analysis showed that the orally located anterograde bursts occur at 2.2 c.p.m. (black star in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), whereas anterograde burstlets occur indeed at 60 c.p.m. (black diamond in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). These findings were consistent across wildtype mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Spectral representation of the signals also uncovers other rhythmicity hotspots (e.g. oral 18 c.p.m. and aboral 30 c.p.m. in Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), that are not easily distinguished with the naked eye in the raw electrophysiology signal.\u003c/p\u003e\u003cp\u003eIn-vivo pharmacological modulation of colonic electrophysiological activity\u003c/p\u003e\u003cp\u003eTo demonstrate in real-time the sensitivity of the device to pharmacological interventions, we modulated the activity by intraperitoneal injections of well-known pharmacological agents (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Donepezil, a cholinesterase inhibitor used to enhance cholinergic tone, rapidly and significantly upregulated spiking activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, t\u0026thinsp;=\u0026thinsp;7 min), while atropine, a cholinergic antagonist, had a predictable reversing effect and produced a rapid, short-lived significant reduction of spiking activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, t\u0026thinsp;=\u0026thinsp;17 min). Areas of statistically significant change in activity are outlined in cyan (upregulation) and orange (downregulation). The rapid onset and decay of pharmacological effects were most evident in the oral regions that primarily exhibit\u0026thinsp;~\u0026thinsp;2.2 c.p.m. bursts of activity (black stars in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), with the exception of a prolonged upregulation of activity at the aboral end of the colon (black triangles in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Furthermore, the decay of atropine downregulation was followed by a localized (across topmost seven channels) increase in spiking activity.\u003c/p\u003e\u003cp\u003eIn-vivo aberrant electrophysiological patterns in gut-lesioned animal model\u003c/p\u003e\u003cp\u003eTo verify the capability of the device to detect aberrant patterns between healthy and disrupted organs, we implemented a benzalkonium chloride (BAC) model known to disrupt colonic motility through tissue damage in three mice. None of the treated mice exhibited outward signs of distress or other spontaneous behavioral alterations. However, in all of them endoscopic intraluminal recording of the colon was altered from that described above in untreated wild type mice. We still recorded action potentials in all animals after BAC treatment (although their morphology appeared altered: Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) but the typical spatiotemporal patterns they formed in untreated animals formed were abolished (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eBC). Instead, they formed different configurations of activity in each BAC-lesioned animal. Spiking frequency spectral analysis revealed the loss of the frequency bands seen commonly in healthy mice (18\u0026ndash;30 c.p.m. and 60 c.p.m. bands), as well as broadening of the ~\u0026thinsp;2 c.p.m. band (indicating \u0026ldquo;arrythmia\u0026rdquo;) as seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB. Various different patterns of electrical activity were recorded, for example trains of spikes with steady linear decrease in firing rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, bottom).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAt the oral end of the endoscope, where the propagating waves of spike bursts occur at ~\u0026thinsp;2 c.p.m. in healthy controls, summed spiking activity plots reveal the global loss of rhythmicity and amplitude of spike bursts in BAC-lesioned animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, orange plots) compared to unlesioned controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, blue plots). In BAC animals, a larger spread of spiking frequency with no prominent peaks at ~\u0026thinsp;2 c.p.m. as was seen in Fourier frequency plots comparing the two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Interestingly, each BAC animal exhibit different profiles of dysfunction, most easily observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB; this is likely due to the variation in the effects that BAC exposure can have on tissue.\u003c/p\u003e\u003cp\u003eEx-vivo colonic electrophysiological patterns\u003c/p\u003e\u003cp\u003eTo validate our endoscope, we used an established ex-vivo setup to characterize the intrinsic neural and muscular properties of the colon in isolation (Spencer, Costa, et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Spencer et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Spencer, Travis, et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Resected intact sections of the colon were threaded over the endoscope shaft to mimic intraluminal recording. The ex-vivo set-up was slightly modified to allow the endoscope and the colon to be submerged in the bath at an angle to protect the electronics. Additionally, a benchmark suction pipette electrode was attached to the colon from the serosal side (see photo in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn the ex-vivo setup we readily recorded spikes. Moreover, the spikes were confirmed to be directly related to motor activity of the colon. Instantaneous variations of colon diameter along the entire length of the preparation extracted from the video demonstrate precise relationship between patterns of observed spikes and colonic (micro)motion (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-D). Spike bursts were markedly different than those in anesthetized animals, as they appeared only every\u0026thinsp;~\u0026thinsp;5 minutes (compared to twice per minute in-vivo), lasted much longer (up to 90 s compared to ~\u0026thinsp;10 s in vivo) and travelled the extent of the entire colon preparation of 40 mm (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003eTo confirm the role of enteric neurons in regulating colonic muscle activity(Spencer, Costa, et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) using our device, the sodium channel-blocker tetrodotoxin (TTX), was applied to the preparation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) and repeated in four biological samples. TTX did not abolish action potentials, however the pattens of electrical activity were modified such that complex spike bursts were replaced with single spike waves that discharged rhythmically at an increased frequency (4.6 times per minute in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eACD), specifically in the aboral region of the colon. To characterize the effects of increased action potential discharge following abolition of enteric nerve conduction, we applied the Ca\u003csup\u003e2+\u003c/sup\u003e channel opener, BAYK8644. This agonist did not restore the propagating waves of activity that occurred prior to TTX application. The global effects of these pharmacological interventions are evident in the RMS plots (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Finally, we confirm that signals recorded with the endoscope are L-type Ca\u003csup\u003e2+\u003c/sup\u003e channel-dependent action potentials by applying nicardipine to the colon from another animal, resulting in the complete removal of action potentials (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003eIn another experiment, we compared the action potentials recorded with the suction pipette electrode, which was been established as a benchmark approach in numerous foundational studies (Costa et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Hibberd et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Spencer et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wood, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1973\u003c/span\u003e)to the signals recorded from the contact of our endoscope nearest to it. The resemblance between the two signals was very close (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG), with Pearson\u0026rsquo;s coefficients of correlation of their RMS envelopes at 0.87 and 0.91 for 0.1 and 1 s RMS windows (precision levels), respectively (correlations were significant at p\u0026thinsp;=\u0026thinsp;0 to the level of machine precision).\u003c/p\u003e\u003cp\u003eLastly, to confirm the spatial origin of the signals, the colon was opened longitudinally and the mucosa was peeled off from the distal one third of the preparation. The tissue was mounted lumen-side down over the endoscope shaft (see photo in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). Electrical activity was recorded in all regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG) and the propagating waves of action potentials were observed in all preparations. Partial lengthwise surgical removal of the mucosa enabled selective recording with the tissue in direct contact with the circular muscle layer while at the same time recording from intact mucosa confirming that the endoscopes records electrophysiological signals originating below the mucosa that propagate through the full thickness of the gut wall.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe have developed a miniature, easy-to-use endoscope capable of recording in high resolution the propagation of action potentials over tens of millimeters simultaneously along the rostro-caudal and circumferential axis of colonic smooth muscle in live mice. We believe that this new device can uncover anomalies and offer new insight into mechanistic understanding of gut function using disease models and genetically modified mice. Because recordings can be made minimally invasively, we can use this technique to provide repeated and rapid high-throughput experimentation without surgical intervention.\u003c/p\u003e\u003cp\u003ePrevious electrophysiological recordings from GI-smooth muscles in live animals have mostly \u0026ndash; with the exception of the approaches discussed below - involved surgically invasive techniques from the outmost gut layers, often with low spatial resolution. Commonly such electrodes also used special attachment to tissue in order to ensure sufficient electric contact and create an isolated electric compartment.\u003c/p\u003e\u003cp\u003eThus, one justifiable concern with intraluminal recordings using an endoscope was tissue contact, particularly with no features for enhancing tissue adhesion through e.g. suction, distension (Xue et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), or adhesion (Srinivasan et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). We found, however, that a well-chosen endoscope diameter and specialized low-impedance coating on the sensors was sufficient for high-signal-to noise ratio thus enabling consistent capture single action potentials. However, it is possible that our approach works well in mice because their colons have no haustration and would not work as well in other species. Nevertheless, such approach is far less cumbersome and possible to use at scale, as required e.g. for drug discovery in preclinical research.\u003c/p\u003e\u003cp\u003eIt is possible the distension provided by the endoscope induces patterns of myoelectric activity recorded in live animals. However, the diameter of the endoscope was made at 2 mm which is similar to that of a murine fecal pellet and consistent with a view that the patterns recorded are physiologically relevant.\u003c/p\u003e\u003cp\u003eAnother concern of intraluminal recordings with individual sensors bearing no isolating attachment features could be the spatial selectivity of the electrodes. Immersion of all contacts within a shared conductive fluid environment could, in principle, lead to signal spread and reduce the ability to resolve localized activity. However, our results demonstrate that this is not the case when the electrode design is properly optimized. In developing our device, we took inspiration from recent advances in micro-electrocorticography (\u0026micro;ECoG, Shokoueinejad et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). \u0026micro;ECoG electrode arrays achieve high spatial resolution recording from the cerebral cortex - even sufficient for brain-computer interfacing - despite no compartmentalization, relying only on passive tissue adhesion. Similarly to \u0026micro;ECoG, we made the footprint of the electrodes very small, but coated them with a low-impedance iridium oxide layer. As a result we found that the amplitude of action potentials declined significantly across neighboring contacts (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), indicating that the signals originate from highly localized sources. Moreover, the precise electrode geometry and slight spatial oversampling of our device, open the door to applying advanced computational methods. These methods will allow advances toward localization and extraction of underlying signal sources.\u003c/p\u003e\u003cp\u003eElectrophysiological recordings from contracting tissue are susceptible to motion artifacts. These artifacts may introduce spurious waveforms into the data, with time courses that reflect the physical displacement of tissue rather than genuine neural or myogenic activity. Ensuring that the contact is well attached to tissue is not enough to counter this: in any case the downstream signal path (whether realized as a cable or otherwise) must change its geometry (move, bend) and generate a triboelectric artifact (Symeonidou et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). To mitigate this, we restricted our analysis to high-frequency events, specifically action potentials, with time constants in the range of 10-20ms, much faster than the motion of the gut. We filtered slower activity (\u0026lt;\u0026thinsp;10 Hz) that can be seen in raw data (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003eA recent study(Xue et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) used a scalable balloon catheter which enabled electrical recordings from the colon of anesthetized rabbits. That study also recorded action potentials through the mucosa in the form of periodic spike bursts. That device had longitudinal resolution limited to two locations. This additionally prompted us to develop the current device, which provides high spatial resolution of the spatial spread and coordination of action potentials, and direct assessment of the complex patterns they form.\u003c/p\u003e\u003cp\u003eA second recent publication proposed a device tested in pigs, as well as a version used in mice (Srinivasan et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Not much information, however, was provided on the mouse device (which would be relevant to contrast here), nor data recorded with it, thus it is difficult to draw comparisons. The dimensions of the device are comparable (20-by-2 mm), though the channel count was limited to16 electrodes.\u003c/p\u003e\u003cp\u003eThe validation of our new device presented here also included recordings of intraluminal electrophysiological activity in excised mice colon in an organ bath combined with simultaneous video imaging of colonic wall movements using spatiotemporal mapping. This approach confirmed that the patterns of action potentials were directly related to motor activity of the colon This allows us to infer a similar relationship occurs in-vivo, where large coverage recording of colonic motility and other micromotion at high temporal- and spatial-resolution would otherwise not be possible.\u003c/p\u003e\u003cp\u003eIn conclusion, we believe that our novel simple-to-use device, with its ability to record colonic electrical activity in live mice at high resolution \u0026ndash; down to singular action potentials - and high spatial coverage offers a unique opportunity to study GI dysfunction in disease models for drug discovery, as well as fundamental research.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgements:\u003c/h2\u003e\u003cp\u003eThis project has received funding from the European Research Council (ERC) under the European Union\u0026rsquo;s Horizon 2020 research and innovation program (grant agreement No 853378). Experiments in this study were supported by an Australian Research Council (ARC) grant # DPEI250100648 to NJS.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAltaparmakov, I., \u0026amp; Wienbeck, M. (1984). Local inhibition of myoelectrical activity of human colon by loperamide. \u003cem\u003eDigestive Diseases and Sciences\u003c/em\u003e, \u003cem\u003e29\u003c/em\u003e(3), 232\u0026ndash;238. https://doi.org/10.1007/BF01296257\u003c/li\u003e\n \u003cli\u003eArkwright, J. W., Blenman, N. G., Underhill, I. D., Maunder, S. A., Spencer, N. J., Costa, M., Brookes, S. J., Szczesniak, M. M., \u0026amp; Dinning, P. G. (2011). 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Selective chemical ablation of the enteric plexus in mice. \u003cem\u003ePediatric Surgery International\u003c/em\u003e, \u003cem\u003e18\u003c/em\u003e(4), 234\u0026ndash;237. https://doi.org/10.1007/s003830100681\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6916016/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6916016/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA major weakness in the field of neurogastroenterology research has been lack of technology to determine the spatial and temporal coordination of electrical activity along the gastrointestinal (GI) tract in-vivo without requiring a surgical procedure. To overcome this weakness we developed a miniaturized endoscope consisting of 128 iridium oxide recording sensors that allowed us to make high resolution intraluminal electrophysiological recordings in-vivo from the mucosal surface of the terminal large intestine of anesthetized mice. Recordings revealed discharges of smooth muscle action potentials organized into complex spatiotemporal patterns. The patterns were modified by pharmacological agents donepezil and atropine that stimulated or suppressed cholinergic neurotransmission, respectively. The patterns were also ablated by benzalkonium chloride, known to disrupt the function of the enteric nervous system. The endoscope was further validated under ex-vivo recording conditions, where blocking enteric neural activity with tetrodotoxin (TTX) again altered spontaneously occurring action potential patterns. This novel approach offers a unique opportunity to easily characterize normal and dysfunctional patterns of GI electrical activity in genetically modified and/or diseased mouse models, including drug discovery and high-throughput studies.\u003c/p\u003e","manuscriptTitle":"Miniature endoscope for high resolution electrophysiological recordings from the colon of live mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-14 11:08:50","doi":"10.21203/rs.3.rs-6916016/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"783c0bb9-6a3c-44c7-8d35-4746b5586853","owner":[],"postedDate":"July 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":51133102,"name":"Health sciences/Gastroenterology/Colonoscopy"},{"id":51133103,"name":"Biological sciences/Biological techniques/Electrophysiology"},{"id":51133104,"name":"Biological sciences/Biological techniques/High-throughput screening"}],"tags":[],"updatedAt":"2026-03-12T07:06:25+00:00","versionOfRecord":{"articleIdentity":"rs-6916016","link":"https://doi.org/10.1038/s41467-026-69144-2","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2026-02-04 05:00:00","publishedOnDateReadable":"February 4th, 2026"},"versionCreatedAt":"2025-07-14 11:08:50","video":"","vorDoi":"10.1038/s41467-026-69144-2","vorDoiUrl":"https://doi.org/10.1038/s41467-026-69144-2","workflowStages":[]},"version":"v1","identity":"rs-6916016","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6916016","identity":"rs-6916016","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}