Data from: Synaptic cell adhesion molecule Cdh6 identifies a class of sensory neurons with novel functions in colonic motility
Data files
Apr 02, 2026 version files 1.03 GB
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20230522_J281_6_colon.adicht
29.62 MB
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20230523_J281_0_colon.adicht
38.80 MB
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20230524_J287_1_colon.adicht
12.12 MB
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20230524_J287_5_colon.adicht
16.76 MB
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20230525_J277_2_colon.adicht
43.11 MB
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20230526_J279_4_colon.adicht
13.11 MB
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20230529_J279_5_colon.adicht
23.45 MB
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20230530_J278_6_colon.adicht
36.82 MB
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20230531_J285_0_colon.adicht
47.12 MB
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20230601_J281_1_colon.adicht
42.30 MB
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20230605_J286A_4_colon.adicht
36.93 MB
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20230607_J278_7_colon.adicht
24.53 MB
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20230607_J286_6_colon.adicht
14.37 MB
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20230608_J287_0_colon.adicht
27.36 MB
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20230608_J287_2_colon.adicht
14.21 MB
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20230609_J281_2_colon.adicht
30.70 MB
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A210921_0000.abf
407.55 KB
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A210921_0001.abf
407.55 KB
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A210921_0002.abf
2.01 MB
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A210921_0003.abf
407.55 KB
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A210921_0004.abf
2.01 MB
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A210921_0005.abf
7.21 MB
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A210921_0006.abf
7.81 MB
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A210921_0007.abf
3.13 MB
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A220509_0000.abf
407.55 KB
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A220509_0001.abf
2.01 MB
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A220509_0002.abf
167.42 KB
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A220509_0003.abf
407.55 KB
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A220509_0004.abf
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A220509_0005.abf
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A220509_0006.abf
407.55 KB
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A220509_0007.abf
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A220509_0008.abf
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A220509_0009.abf
407.55 KB
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A220926_0000.abf
327.17 KB
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A220926_0001.abf
2.01 MB
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A221007_0000.abf
407.55 KB
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A221007_0001.abf
407.55 KB
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A221007_0002.abf
10.01 MB
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A221007_0003.abf
11.01 MB
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A221007_0004.abf
407.55 KB
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A221007_0005.abf
3.21 MB
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A221007_0006.abf
247.30 KB
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C221011_0000.abf
407.55 KB
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C221011_0001.abf
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C221011_0002.abf
17.01 MB
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C221011_0003.abf
407.55 KB
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C221011_0004.abf
3.21 MB
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C221011_0005.abf
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C221011_0006.abf
7.01 MB
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CS__20231212_01.adicht
4.44 MB
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CS__20231212_02.adicht
33.51 MB
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CS__20231212_03.adicht
3.54 MB
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CS__20231212_04.adicht
62.28 MB
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CS__20231212_05.adicht
3.79 MB
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CS_20231205_01.adicht
3.31 MB
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CS_20231205_02.adicht
26.18 MB
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CS_20231206_03.adicht
3.28 MB
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CS_20231206_04.adicht
27.59 MB
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CS_20231207_05.adicht
4.67 MB
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CS_20231207_06.adicht
36.67 MB
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README.md
12.86 KB
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SummaryEphysData_hb9.csv
1.49 KB
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ZD___Cs_Prep_specs.csv
1.20 KB
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ZD__20231207_01.adicht
3.49 MB
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ZD__20231207_02.adicht
29.26 MB
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ZD__20231208_03.adicht
8.03 MB
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ZD__20231208_04.adicht
55.97 MB
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ZD__20231211_05.adicht
3.88 MB
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ZD__20231211_06.adicht
47.02 MB
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ZD__20231211_07.adicht
6.14 MB
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ZD__20231211_08.adicht
34 MB
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ZD_20231205_01.adicht
3.16 MB
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ZD_20231205_02.adicht
31.79 MB
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ZD_20231206_03.adicht
3.20 MB
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ZD_20231206_04.adicht
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ZD_20231206_05.adicht
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ZD_20231206_06.adicht
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Abstract
Intrinsic sensory neurons are an essential part of the enteric nervous system (ENS) and play a crucial role in gastrointestinal tract motility and digestion. Neuronal subtypes in the ENS have been distinguished by their electrophysiological properties, morphology, and expression of characteristic markers, notably neurotransmitters and neuropeptides. Here, we investigated synaptic cell adhesion molecules as novel cell type markers in the ENS. Our work identifies two Type II classic cadherins, Cdh6 and Cdh8, specific to sensory neurons in the mouse colon. We show that Cdh6+ neurons demonstrate all other distinguishing classifications of enteric sensory neurons, including marker expression of Calcb and Nmu, Dogiel type II morphology and AH-type electrophysiology and IH current. Optogenetic activation of Cdh6+ sensory neurons in the distal colon evokes retrograde colonic motor complexes (CMCs), while pharmacologic blockade of rhythmicity-associated current IH disrupts the spontaneous generation of CMCs. These findings provide the first demonstration of selective activation of a single neurochemical and functional class of enteric neurons, and demonstrate a functional and critical role for sensory neurons in the generation of CMCs.
https://doi.org/10.5061/dryad.66t1g1kbt
Description of the data and file structure
Slice whole cell recording using pClamp of hb9-GFP cells. Each experiments start with the cell name ( letter followed by date code). An Excel with metadata info has been made (SummaryEphysData_hb9). Only non-leaky cells were kept to continue the experiment. Only the cells with a good access resistance were kept for the analysis.
Files and variables
File: A210921_0000.abf
Description: Membrane test protocol to evaluate access resistance, input resistance, and capacitance.
File: A210921_0002.abf
Description: Current clamp protocol to evaluate spiking property
File: A210921_0003.abf
Description: Current clamp protocol to evaluate spiking property- test
File: A220509_0002.abf
Description: Voltage clamp protocol-test
File: A210921_0007.abf
Description: Voltage clamp protocol to evaluate T current
File: A210921_0004.abf
Description: Current clamp protocol to evaluate spiking property
File: A210921_0001.abf
Description: I=0 protocol to evaluate the resting membrane potential of the cell
File: A220509_0004.abf
Description: membrane test protocol to evaluate access resistance, input resistance, and capacitance.
File: A220509_0003.abf
Description: Voltage clamp protocol to evaluate T current
File: A220509_0006.abf
Description: Voltage clamp protocol to evaluate T current
File: A220926_0000.abf
Description: membrane test protocol to evaluate access resistance, input resistance, and capacitance.
File: A220509_0005.abf
Description: Current clamp protocol to evaluate spiking property
File: A220926_0001.abf
Description: Current clamp protocol to evaluate spiking property
File: C221011_0005.abf
Description: membrane test protocol to evaluate access resistance, input resistance, and capacitance.
File: A221007_0000.abf
Description: membrane test protocol to evaluate access resistance, input resistance, and capacitance.
File: A220509_0000.abf
Description: membrane test protocol to evaluate access resistance, input resistance, and capacitance.
File: A221007_0001.abf
Description: I=0 protocol to evaluate the resting membrane potential of the cell
File: A221007_0004.abf
Description: Voltage clamp protocol to evaluate T current
File: A220509_0008.abf
Description: Voltage clamp protocol to evaluate T current
File: A210921_0005.abf
Description: Voltage clamp protocol to evaluate T current
File: A220509_0007.abf
Description: Voltage clamp protocol to evaluate T current
File: A221007_0003.abf
Description: Current clamp protocol to evaluate spiking property
File: C221011_0004.abf
Description: Voltage clamp protocol to evaluate T current
File: A210921_0006.abf
Description: Voltage clamp protocol to evaluate T current
File: A221007_0005.abf
Description: Voltage clamp protocol to evaluate T current test
File: A221007_0002.abf
Description: Current clamp protocol to evaluate spiking property
File: C221011_0002.abf
Description: Current clamp protocol to evaluate spiking property
File: A221007_0006.abf
Description:
File: C221011_0000.abf
Description: membrane test protocol to evaluate access resistance, input resistance, and capacitance.
File: C221011_0006.abf
Description: Current clamp protocol to evaluate spiking property
File: A220509_0001.abf
Description: Current clamp protocol to evaluate spiking property
File: C221011_0003.abf
Description: Voltage clamp protocol to evaluate T current
File: A220509_0009.abf
Description: membrane test protocol to evaluate access resistance, input resistance, and capacitance.
File: C221011_0001.abf
Description: I=0 protocol to evaluate the resting membrane potential of the cell
File: SummaryEphysData_hb9.csv
Description: This dataset contains intrinsic electrophysiological properties from whole-cell patch-clamp recordings of individual neurons. The columns are defined as follows:
- Cell Id, unique identifier assigned to each recorded neuron
- Abf file, name of the raw Axon Binary File (.abf) containing the electrophysiological recording
- Mouse ID, unique identifier of the mouse from which the cell was recorded
- Date of Experiment, date on which the recording was performed
- Internal, type of intracellular (pipette) solution used
- Biocytin (mg/ml), concentration of biocytin included in the internal solution for post hoc morphological analysis
- Rp (MOhm), pipette resistance measured prior to break-in
- Cell type, experimenter-defined neuronal classification
- RMP (mV), resting membrane potential measured under zero current injection
- Ra (MOhm), access resistance in whole-cell configuration
- RmmV), membrane resistance (MΩ)
- Cm (pF), membrane capacitance
- Rheobase (pA), minimum injected current required to evoke an action potential
- 1st AP threshold (mV), voltage at which the first action potential is initiated
- Amplitude (mV), peak amplitude of the first action potential
- Half Width (ms), action potential half-width measured at half-maximal amplitude
- AHP (mV), afterhyperpolarization amplitude following the first spike
- Sag(mV), voltage sag amplitude during hyperpolarizing current injection
- T-current max amplitude, maximum amplitude of T-type calcium current recorded in voltage-clamp mode
- Rebound (mV), rebound depolarization amplitude following hyperpolarization
- Tcurrent Analysis, indicator specifying whether T-type current analysis was performed
- Voltage, holding potential used during voltage-clamp protocols;
- C221011, A220509, and A221007, internal experiment identifiers corresponding to specific recording sessions or protocols.
Mechanical recordings & optical stimulation
A 2.5 mm stainless steel rod was inserted through the lumen of the colon and mounted in an organ bath (120 * 40 * 12 mm; LWH) located on a heated base. Krebs solution (35.5-36°C) superfused the bath (∼5 mL/min). Smooth muscle force was recorded via 4 evenly spaced hooks in the colonic muscularis externa, each linked to an isometric force transducer (Grass FT03C) by suture thread. THIS CORRESPONDS TO DATA IN CHANNELS 1-4 ORIENTED PROXIMAL TO DISTAL. SAMPLING RATE 1kHz; UNITS, g.
Initial base resting tension was set between 0.5 - 1.0 g. Preamplified signals (Biomedical Engineering, Flinders University) were digitized by a PowerLab 16/35 (ADInstruments, Bella Vista, NSW, Australia) and recorded using LabChart 7 software (ADInstruments) on iMac computer. Post hoc analysis of the mechanical recordings was done using LabChart 8 software on PC.
For optical stimulation during mechanical recordings in vitro, two LEDs (emitting 470 nmλ photons; C470DA2432, Cree Inc., NC, USA) were used, driven by a variable power supply. The area of light emission from each LED was 240 μm x 320 μm (0.0768 mm2). To characterise LED function, light power density across a range of currents was measured 5 mm from the LED using a standard photodiode power sensor (S120C; Thorlabs, NJ, USA) and a power meter (Thorlabs, PM100USB). The stimulator panel within LabChart software was used to set parameters and manually trigger LED pulse trains via the 10V analogue output of the PowerLab and an ILD1 opto-isolator. LIGHT STIMULATION PULSES SHOWN IN CHANNEL 5.
Ih channel blocker experiments readme
Intraluminal pellet CMC recordings
To record proximal and distal colon CMCs separately, full length colon was bisected halfway between the caeco-colonic junction and terminal rectum, creating equal length proximal and distal colon preparations. Each preparation was suspended vertically on a stainless-steel holder inside a glass, water jacketed organ bath containing Krebs solution (Fig.6J, K). A 2.7 mm diameter synthetic pellet (polymethyl methacrylate, “Perspex”) was placed inside the gut lumen and linked by stainless steel rod to a force transducer (MLT0420, ADInstruments), allowing measurement of both anterograde and retrograde propulsive forces on the pellet. Signals were amplified by bridge amplifier (FE224, ADInstruments), digitized at 1kHz (PLCF1, ADInstruments) and recorded using LabChart 8 software.
ZD7288 (73777, Sigma-Aldrich) was dissolved in water as stock solution at 10 mM. Caesium Chloride (C4036, Sigma-Aldrich) was dissolved in water as stock solution at 200 mM. Tetrodotoxin citrate (T-550, Alomone Labs) was dissolved in water as stock solution at 3 mM. Control, ZD7288, CsCl and washout periods were at least 30 mins; TTX was applied for at least 10 minutes.
CHANNEL 1 = PROXIMAL; CHANNEL 2 = DISTAL
SAMPLING 1 kHz
UNITS = g
Time and type of drug application noted in corresponding channels
What is an .adicht file?
An .adicht file is a native data file format created by ADInstruments software, primarily used for recording and analyzing physiological signals. These files store waveform details, timing, and annotations from PowerLab systems, often used in scientific research and education. They are opened in LabChart or the free LabChart Reader.
Key Details About .adicht Files:
Usage: Used for storing data such as heart rate, blood pressure, and neural signals recorded on Windows or Mac computers.
Compatibility: They can be opened in LabChart Pro or LabChart Reader.
File Conversion: These files can be exported to other formats, including MATLAB (.mat), text files (.txt), and WAV files.
Version Compatibility: While the extension is the same, older versions of LabChart may not open files created in newer versions.
Read-only: When using LabChart Reader, these files are read-only to ensure data integrity.
Our .adicht files
Unit = grams force
Channel 1 = proximal colon force exerted on an intraluminal synthetic pellet
Channel 2 = distal colon force exerted on an intraluminal synthetic pellet
Comments indicate the timing and type of drug application
20230522_J281_6_colon.adicht, 20230523_J281_0_colon.adicht, 20230524_J287_1_colon.adicht, 20230524_J287_5_colon.adicht, 20230525_J277_2_colon.adicht, 20230526_J279_4_colon.adicht, 20230529_J279_5_colon.adicht, 20230530_J278_6_colon.adicht, 20230531_J285_0_colon.adicht, 20230601_J281_1_colon.adicht, 20230605_J286A_4_colon.adicht, 20230607_J278_7_colon.adicht, 20230607_J286_6_colon.adicht, 20230608_J287_0_colon.adicht, 20230608_J287_2_colon.adicht, 20230609_J281_2_colon.adicht, CS__20231212_01.adicht, CS__20231212_02.adicht, CS__20231212_03.adicht, CS__20231212_04.adicht, CS__20231212_05.adicht, CS_20231205_01.adicht, CS_20231205_02.adicht, CS_20231206_03.adicht, CS_20231206_04.adicht, CS_20231207_05.adicht, CS_20231207_06.adicht, ZD___Cs_Prep_specs.csv, ZD__20231207_01.adicht, ZD__20231207_02.adicht, ZD__20231208_03.adicht, ZD__20231208_04.adicht, ZD__20231211_05.adicht, ZD__20231211_06.adicht, ZD__20231211_07.adicht, ZD__20231211_08.adicht, ZD_20231205_01.adicht, ZD_20231205_02.adicht, ZD_20231206_03.adicht, ZD_20231206_04.adicht, ZD_20231206_05.adicht, ZD_20231206_06.adicht
File: ZD_Cs_Prep_specs.csv
Column Descriptions for CSV file
- Filename: Unique identifier for each physiology recording file. The prefix encodes the drug condition (CS = CsCl, ZD = ZD7288, + = with TTX), followed by the date (YYYYMMDD) and a sequential number.
- Sex: Biological sex of the mouse (F = female, M = male).
- Weight_g: Body weight of the mouse in grams at the time of the experiment.
- Age_weeks: Age of the mouse in weeks at the time of the experiment.
- CsCl_2mM: Whether 2 mM cesium chloride (CsCl) was applied during the recording (Yes/No). CsCl is a non-selective blocker of hyperpolarization-activated cation (HCN/Ih) channels.
- ZD7288_5-10uM: Whether 5–10 µM ZD7288 was applied during the recording (Yes/No). ZD7288 is a selective HCN/Ih channel blocker.
- TTX: Whether tetrodotoxin (TTX) was applied during the recording (Yes/No). TTX blocks voltage-gated sodium channels to isolate non-neural responses.
- Experiment_Group: The experimental drug condition group, derived from the filename prefix: CsCl (CsCl alone), ZD7288 (ZD7288 alone), ZD7288+TTX (ZD7288 with TTX), or CsCl+TTX (CsCl with TTX).
Code/software
Need to use Clampfit to open the data, can also use Python with the pyabf package Opto readme
Electrophysiological recordings:
Whole cell patch clamp electrophysiological recordings of Hb9:GFP+ neurons were performed according to Osorio & Delmas (14), with modifications for recording from the distal colon. The protocol is described in brief below.
Tissue dissection and preparation:
Mice aged 8–10 weeks were culled by CO2 and cervical dislocation. The colon was removed and flushed with ice cold oxygenated Krebs solution [118 mM NaCl, 4.8 mM KCl, 1 mM NaH2PO4, 25 mM NaHCO3, 1.2 mM MgCl2, 2.5 mM CaCl2 and 11 mM glucose, supplemented with scopolamine (2 M) and nicardipine (6 μM)], then placed in a Sylgard-lined Petri dish with ice cold oxygenated Krebs solution for further dissection. Krebs' solution was changed out for a fresh oxygenated solution every 5 minutes. Under a dissection microscope, the distal colon was pinned, and the mucosa was peeled away using fine forceps, leaving a few millimeters of mucosa along the edges of the tissue for pinning stability. The muscularis was then flipped over and re-pinned, serosa side up, and the longitudinal muscle was carefully peeled away. The tissue was transferred to a custom 3D-printed recording chamber lined with a thin layer of clear Sylgard and re-pinned under light tension, with the myenteric plexus facing up. The tissue was kept at 32°C and was continuously perfused with oxygenated Krebs solution. Hb9:GFP+ neurons were visually identified within a ganglion under epifluorescence illumination with a 455nM LED (Thorlabs, M455L2) and a 470 (excitation)/525 (emission) nm wavelength filter set. A local perfusion of protease XIV (0.2% in Krebs) (Sigma, P5417) was applied on top of the targeted cell to digest any muscle fiber residue. A 1–2MΩ pipet with a trimmed arm hair glued to the tip was used to brush and clean the surface of the ganglion. Further cleaning with 1mg/mL collagenase (Worthington, CLS-4) and 4mg/mL dispase (Sigma, D4693) in Krebs solution was also performed to expose the GFP neuron for patching.
Patching and recording
Patch pipettes (4–6 MΩ) pulled from borosilicate glass were filled with internal solution containing in mM: 144 K-gluconate, 3 MgCl2, 0.5 EGTA, 10 HEPES, pH 7.2 (285/295 mOsm), and 2% biocytin (Millipore Sigma, B4261–100MG). Patch clamp recordings were collected with a Multiclamp 700A (Molecular Devices) amplifier, a Digidata 1440 digitizer, and pClamp10.7 (Molecular Devices). Recordings were sampled and filtered at 10kHz. Passive properties analysis was performed using pClamp10.7. Analysis of action potentials was performed using a custom MATLAB (MathWorks) software. All recordings were performed at 32°C. Membrane potentials were not corrected for liquid junction potential. Immediately after whole cell configuration, the cell was maintained at −70 mV, and a short voltage clamp membrane test protocol consisting of 20 times 600ms, 10mV depolarization steps was performed to assess cell health and recording conditions. Recordings were performed in Hb9:GFP+ colonic neurons with an access resistance less than 30MΩ (16.69 ± 2.63 MΩ). Next, the current clamp mode was used to measure resting membrane potential (RMP), input resistance (Rin), and APs stimulated. Membrane potential was not adjusted from resting potential, and cells were depolarized by 1-second current pulses in 10pA increments until APs were triggered (rheobase). Finally, if the seal was still stable, a voltage clamp steady-state inactivation of T-current protocol was performed as previously described (44). In brief, a sequence of depolarization from −90 to −45mV for 500 ms, quickly followed by a depolarization to −40mV for 200ms. Tissues were then fixed and immunostained according to Wholemount preparations and Immunohistochemistry sections above.
Mechanical recordings & optical stimulation
Optogenetic stimulation experiments were performed as previously described (29). A 2.5 mm stainless steel rod was inserted through the lumen of the colon and mounted in an organ bath (120 * 40 * 12 mm; LWH) located on a heated base. Krebs solution (35.5–36°C) was superfused into the bath (~5 mL/min). Smooth muscle force was recorded via 4 evenly spaced hooks in the colonic muscularis externa, each linked to an isometric force transducer (Grass FT03C) by suture thread. Initial base resting tension was set between 0.5 – 1.0 g. Preamplified signals (Biomedical Engineering, Flinders University) were digitized by a PowerLab 16/35 (ADInstruments, Bella Vista, NSW, Australia) and recorded using LabChart 7 software (ADInstruments) on an iMac computer. Post hoc analysis of the mechanical recordings was done using LabChart 8 software on a PC.
For optical stimulation during mechanical recordings in vitro, two LEDs (emitting 470 nmλ photons; C470DA2432, Cree Inc., NC, USA) were used, driven by a variable power supply. The area of light emission from each LED was 240 μm × 320 μm (0.0768 mm2). To characterise LED function, light power density across a range of currents was measured 5 mm from the LED using a standard photodiode power sensor (S120C; Thorlabs, NJ, USA) and a power meter (Thorlabs, PM100USB). The stimulator panel within LabChart software was used to set parameters and manually trigger LED pulse trains via the 10V analogue output of the PowerLab and an ILD1 opto-isolator.
Intraluminal pellet CMC recordings
To record proximal and distal colon CMCs separately (45, 46), the full-length colon was bisected halfway between the caeco-colonic junction and terminal rectum, creating equal-length proximal and distal colon preparations. Each preparation was suspended vertically on a stainless-steel holder inside a glass, water jacketed organ bath containing Krebs solution (Fig.6J, K). A 2.7 mm diameter synthetic pellet (polymethyl methacrylate, “Perspex”) was placed inside the gut lumen and linked by a stainless steel rod to a force transducer (MLT0420, ADInstruments), allowing measurement of both anterograde and retrograde propulsive forces on the pellet. Signals were amplified by a bridge amplifier (FE224, ADInstruments), digitized at 1kHz (PLCF1, ADInstruments), and recorded using LabChart 8 software.
ZD7288 (73777, Sigma-Aldrich) was dissolved in water as a stock solution at 10 mM. Caesium Chloride (C4036, Sigma-Aldrich) was dissolved in water as a stock solution at 200 mM. Tetrodotoxin citrate (T-550, Alomone Labs) was dissolved in water as a stock solution at 3 mM. Control, ZD7288, CsCl, and washout periods were at least 30 mins; TTX was applied for at least 10 minutes.