Activation of ENS Circuits in Mouse Colon: Coordination in the Mouse Colonic Motor Complex as a Robust, Distributed Control System.

The characteristic motor patterns of the colon are coordinated by the enteric nervous system (ENS) and involve enterochromaffin (EC) cells, enteric glia, smooth muscle fibers, and interstitial cells. While the fundamental control mechanisms of colonic motor patterns are understood, greater complexity in the circuitry underlying motor patterns has been revealed by recent advances in the field. We review these recent advances and new findings from our laboratories that provide insights into how the ENS coordinates motor patterns in the isolated mouse colon. We contextualize these observations by describing the neuromuscular system underling the colonic motor complex (CMC) as a robust, distributed control system. Framing the colonic motor complex as a control system reveals a new perspective on the coordinated motor patterns in the colon. We test the control system by applying electrical stimulation in the isolated mouse colon to disrupt the coordination and propagation of the colonic motor complex.

Control of colonic motility using electrical stimulation to modulate enteric neural activity.

Electrical stimulation of the enteric nervous system (ENS) is an attractive approach to modify gastrointestinal transit. Colonic motor complexes (CMCs) occur with a periodic rhythm, but the ability to elicit a premature CMC depends, at least in part, upon the intrinsic refractory properties of the ENS, which are presently unknown. The objectives of this study were to record myoelectric complexes (MCs, the electrical correlates of CMCs) in the smooth muscle and 1) determine the refractory periods of MCs, 2) inform and evaluate closed-loop stimulation to repetitively evoke MCs, and 3) identify stimulation methods to suppress MC propagation. We dissected the colon from male and female C57BL/6 mice, preserving the integrity of intrinsic circuitry while removing the extrinsic nerves, and measured properties of spontaneous and evoked MCs in vitro. Hexamethonium abolished spontaneous and evoked MCs, confirming the necessary involvement of the ENS for electrically evoked MCs. Electrical stimulation reduced the mean interval between evoked and spontaneous CMCs (24.6 ± 3.5 vs. 70.6 ± 15.7 s, P = 0.0002, n = 7). The absolute refractory period was 4.3 s (95% confidence interval (CI) = 2.8-5.7 s, R2 = 0.7315, n = 8). Electrical stimulation applied during fluid distention-evoked MCs led to an arrest of MC propagation, and following stimulation, MC propagation resumed at an increased velocity (n = 9). The timing parameters of electrical stimulation increased the rate of evoked MCs and the duration of entrainment of MCs, and the refractory period provides insight into timing considerations for designing neuromodulation strategies to treat colonic dysmotility.NEW & NOTEWORTHY Maintained physiological distension of the isolated mouse colon induces rhythmic cyclic myoelectric complexes (MCs). MCs evoked repeatedly by closed-loop electrical stimulation entrain MCs more frequently than spontaneously occurring MCs. Electrical stimulation delivered at the onset of a contraction temporarily suppresses the propagation of MC contractions. Controlled electrical stimulation can either evoke MCs or temporarily delay MCs in the isolated mouse colon, depending on timing relative to ongoing activity.

Calcium image analysis in the moving gut.

BACKGROUND: The neural control of gastrointestinal muscle relies on circuit activity whose underlying motifs remain limited by small-sample calcium imaging recordings confounded by motion artifact, paralytics, and muscle dissections. We present a sequence of resources to register images from moving preparations and identify out-of-focus events in widefield fluorescent microscopy. METHODS: Our algorithm uses piecewise rigid registration with pathfinding to correct movements associated with smooth muscle contractions. We developed methods to identify loss-of-focus events and to simulate calcium activity to evaluate registration. KEY RESULTS: By combining our methods with principal component analysis, we found populations of neurons exhibit distinct activity patterns in response to distinct stimuli consistent with hypothesized roles. The image analysis pipeline makes deeper insights possible by capturing concurrently calcium dynamics from more neurons in larger fields of view. We provide access to the source code for our algorithms and make experimental and technical recommendations to increase data quality in calcium imaging experiments. CONCLUSIONS: These methods make feasible large population, robust calcium imaging recordings and permit more sophisticated network analyses and insights into neural activity patterns in the gut.

An intravital window to image the colon in real time.

Intravital microscopy is a powerful technique to observe dynamic processes with single-cell resolution in live animals. No intravital window has been developed for imaging the colon due to its anatomic location and motility, although the colon is a key organ where the majority of microbiota reside and common diseases such as inflammatory bowel disease, functional gastrointestinal disorders, and colon cancer occur. Here we describe an intravital murine colonic window with a stabilizing ferromagnetic scaffold for chronic imaging, minimizing motion artifacts while maximizing long-term survival by preventing colonic obstruction. Using this setup, we image fluorescently-labeled stem cells, bacteria, and immune cells in live animal colons. Furthermore, we image nerve activity via calcium imaging in real time to demonstrate that electrical sacral nerve stimulation can activate colonic enteric neurons. The simple implantable apparatus enables visualization of live processes in the colon, which will open the window to a broad range of studies.

Computational motility models of neurogastroenterology and neuromodulation.

The success of neuromodulation therapies, particularly in the brain, spinal cord, and peripheral nerves, has been greatly aided by computational, biophysical models. However, treating gastrointestinal disorders with electrical stimulation has been much less explored, partly because the mode of action of such treatments is unclear, and selection of stimulation parameters is often empirical. Progress in gut neuromodulation is limited by the comparative lack of biophysical models capable of simulating neuromodulation of gastrointestinal function. Here, we review the recently developed biophysical models of electrically-active cells in the gastrointestinal system that contribute to motility. Biophysical models are replacing phenomenologically-defined models due to advancements in electrophysiological characterization of key players in the gut: enteric neurons, smooth muscle fibers, and interstitial cells of Cajal. In this review, we explore existing biophysically-defined cellular and network models that contribute to gastrointestinal motility. We focus on recent models that are laying the groundwork for modeling electrical stimulation of the gastrointestinal system. Developing models of gut neuromodulation will improve our mechanistic understanding of these treatments, leading to better parameterization, selectivity, and efficacy of neuromodulation to treat gastrointestinal disorders. Such models may have direct clinical translation to current neuromodulation therapies, such as sacral nerve stimulation.

Opportunities and Challenges for Single-Unit Recordings from Enteric Neurons in Awake Animals.

Advanced electrode designs have made single-unit neural recordings commonplace in modern neuroscience research. However, single-unit resolution remains out of reach for the intrinsic neurons of the gastrointestinal system. Single-unit recordings of the enteric (gut) nervous system have been conducted in anesthetized animal models and excised tissue, but there is a large physiological gap between awake and anesthetized animals, particularly for the enteric nervous system. Here, we describe the opportunity for advancing enteric neuroscience offered by single-unit recording capabilities in awake animals. We highlight the primary challenges to microelectrodes in the gastrointestinal system including structural, physiological, and signal quality challenges, and we provide design criteria recommendations for enteric microelectrodes.

A gut-brain neural circuit for nutrient sensory transduction.

The brain is thought to sense gut stimuli only via the passive release of hormones. This is because no connection has been described between the vagus and the putative gut epithelial sensor cell-the enteroendocrine cell. However, these electrically excitable cells contain several features of epithelial transducers. Using a mouse model, we found that enteroendocrine cells synapse with vagal neurons to transduce gut luminal signals in milliseconds by using glutamate as a neurotransmitter. These synaptically connected enteroendocrine cells are referred to henceforth as neuropod cells. The neuroepithelial circuit they form connects the intestinal lumen to the brainstem in one synapse, opening a physical conduit for the brain to sense gut stimuli with the temporal precision and topographical resolution of a synapse.

Aldolase B-Mediated Fructose Metabolism Drives Metabolic Reprogramming of Colon Cancer Liver Metastasis.

Cancer metastasis accounts for the majority of cancer-related deaths and remains a clinical challenge. Metastatic cancer cells generally resemble cells of the primary cancer, but they may be influenced by the milieu of the organs they colonize. Here, we show that colorectal cancer cells undergo metabolic reprogramming after they metastasize and colonize the liver, a key metabolic organ. In particular, via GATA6, metastatic cells in the liver upregulate the enzyme aldolase B (ALDOB), which enhances fructose metabolism and provides fuel for major pathways of central carbon metabolism during tumor cell proliferation. Targeting ALDOB or reducing dietary fructose significantly reduces liver metastatic growth but has little effect on the primary tumor. Our findings suggest that metastatic cells can take advantage of reprogrammed metabolism in their new microenvironment, especially in a metabolically active organ such as the liver. Manipulation of involved pathways may affect the course of metastatic growth.

Electrical stimulation of gut motility guided by an in silico model.

OBJECTIVE: Neuromodulation of the central and peripheral nervous systems is becoming increasingly important for treating a diverse set of diseases-ranging from Parkinson's Disease and epilepsy to chronic pain. However, neuromodulation of the gastrointestinal (GI) tract has achieved relatively limited success in treating functional GI disorders, which affect a significant population, because the effects of stimulation on the enteric nervous system (ENS) and gut motility are not well understood. Here we develop an integrated neuromechanical model of the ENS and assess neurostimulation strategies for enhancing gut motility, validated by in vivo experiments. APPROACH: The computational model included a network of enteric neurons, smooth muscle fibers, and interstitial cells of Cajal, which regulated propulsion of a virtual pellet in a model of gut motility. MAIN RESULTS: Simulated extracellular stimulation of ENS-mediated motility revealed that sinusoidal current at 0.5 Hz was more effective at increasing intrinsic peristalsis and reducing colon transit time than conventional higher frequency rectangular current pulses, as commonly used for neuromodulation therapy. Further analysis of the model revealed that the 0.5 Hz sinusoidal currents were more effective at modulating the pacemaker frequency of interstitial cells of Cajal. To test the predictions of the model, we conducted in vivo electrical stimulation of the distal colon while measuring bead propulsion in awake rats. Experimental results confirmed that 0.5 Hz sinusoidal currents were more effective than higher frequency pulses at enhancing gut motility. SIGNIFICANCE: This work demonstrates an in silico GI neuromuscular model to enable GI neuromodulation parameter optimization and suggests that low frequency sinusoidal currents may improve the efficacy of GI pacing.