(G-253) Engineering an Imaging System to Study Receptive Fields of Intrinsic Primary Afferent Neurons of the Mouse Intestine

Defining and discriminating receptive fields, or the volume of tissue where a physiological stimulus can evoke a response in a sensory neuron, is key to understanding sensory neurobiology. Receptive field biology is well described in the skin of many species, but we lack understanding of receptive fields of interoreceptors, neurons that sense stimuli in internal organs. Intrinsic primary afferent neurons (IPANs) are sensory neurons that innervate the gastrointestinal (GI) tract and initiate intrinsic neural reflexes that control vascular, secretory, and motor functions of the GI tract. Few studies have described receptive fields of IPANs, and these studies were limited by low throughput approaches and the need to remove a portion of mucosa, submucosa, and circular muscle which likely contained part of the receptive field¹.  We previously described advillin as a specific marker of IPANs in the mouse and used advillin-CreERT2 mice to conditionally and inducibly drive a genetically encoded calcium indictor (GECI; GCaMP5) in this class of neurons². The goal of this study was to engineer an imaging system to record neural activity in IPAN cell bodies located in the submucosal and myenteric plexuses of the intestine while precisely probing the mucosal surface with mechanical and electrical stimuli.

Mice hemizygous for AvilCreERT2 and homozygous for floxed-STOP GCaMP6f (Ai148D) received 5 mg of tamoxifen at 7 weeks and experiments were performed at 12-20 weeks. Segments of jejunum, ileum and colon were excised into normal Krebs solution with 5μM nifedipine and 100nM atropine to minimize contractions. Tissues were opened along the mesenteric border and pinned flat on a thin silicon elastomer in a glass bottom dish that was mounted on an inverted IX70 microscope (Olympus) equipped with a ES107 motorized stage and PS3H122R focus motor with Optiscan III controller (Prior), X-Cite X-LED1 light source (Excelitas Technologies) with QuadCube and GFP filters (Semrock), 10x UPlanFL and 20x and 40x LUCPlanFLN objectives, and ORCA-Flash4.0 CMOS camera (Hamamatsu) with two parallel Camera Link outputs to an Firebird Frame Grabber (Active Silicon). A 76mm platinum iridium concentric bipolar microelectrode with a 2-3μm tip (WPI) was used to deliver focal mechanical and electrical stimuli from a S88 stimulator (Grass) and was positioned in X and Y with two stacked CONEX-SAG-LS16P closed-loop linear piezo stages with 16mm range at 25nm resolution (Newport), and in Z with a P-625.1CD closed-loop linear piezo stage with 500μm range at < 1nm resolution (Physik Instrumente). Peripherals were controlled through Clampex (v 11.2.0.59) and MetaMorph (v 7.10.5.476) with custom protocols. Image series were motion corrected via MetaMorph’s AutoAlign function and manually curated ~150μm² ROIs extracted mean intensity values that were transferred to a custom JupyterLab (v 3.5.3, Python 3 kernel) notebook for further analysis.

An experimental protocol was developed to optimize recording IPAN activity in response to mechanical and electrical stimuli of the intestinal mucosa. Images were captured at 50fps with 1024x1024pixel detector binning. At the start of an acquisition, automated commands to the X, Y and Z piezo stages maneuvered the bipolar electrode to deliver mechanical stimuli to a grid of 24 locations in four evenly spaced squares between 0.25-1mm from the center of the imaged ganglion. The Z stage at each position moved from ~400μm above the surface of intestinal villi for 25s, during which XY movements occurred, to 25μm below the surface of villi for 10s to 50μm below for 15s. At 10s into each 50μm Z-step, a 1s train of 0.5ms 6V square-wave pulses were delivered at 5Hz through the bipolar electrode, with high fidelity responses serving as a measure of nerve fiber proximity. Two subsequent 9.5min acquisitions with grid locations 0.75-1mm and 0.25-0.5mm were made while focus was maintained on a single enteric ganglion. This protocol optimized 1) acquisition settings to maximize signal to noise and minimize file size, 2) distances between stimuli and 3) stimulus timing to resolve distinct receptive fields, 4) the depth of mucosal deformation, and 5) EFS parameters to stimulate nerve fibers within a 0.2x0.2x0.3mm³ volume of tissue with the electrode tip at the center. Custom image sequence analysis resolved both slow changes in intracellular calcium and spike frequencies that were binarized to responses/non-responses of each stimulus to subsequently create visualizations of receptive fields at the single cell level and average spatial arrangements at the population level. Collectively, we have designed experimental and computational workflows to generate an imaging system that mechanically stimulates tissue and records and analyzes neuronal activity. We have applied this system to study responses to deformation and electrical stimuli in intestinal mucosa receptive fields of mouse IPANs. This system can be further adapted to deliver other stimulus modalities to receptive fields in and outside the mucosa. Thus, work utilizing this imaging system is expected to transform our understanding of IPAN receptive field biology.

We are grateful for the technical support of David Szent-Györgyi. This work was funded by NIH grants 5R01DK129315, 5R01DK123549 and DP2AT010875.

1. Furness J.B. et al., (2004) Intrinsic primary afferent neurons and nerve circuits within the intestine. Prog Neurobiol. 72:143-64.

2. Melo, C. G. S. et al., (2020) Identification of intrinsic primary afferent neurons in mouse jejunum. Neurogastroenterol. Motil. 32:e13989.