TITLE:  Intracellular recordings and post hoc immunofluorescence
AUTHORS: Rachel M Gwynne, Katerina  Koussoulas, Joel  Bornstein

[DESCRIPTION]
The following protocol was submitted on behalf of the authors from the Bornstein lab by the SPARC project. 

This protocol describes methods for standard intracellular recording from mouse myenteric neurons impaled with intracellular electrodes containing biocytin, followed by processing for immunohistochemistry to map the projections of the neurites of enteric neurons. Methods described in this protocol are adapted from decades of studies on guinea-pig enteric neurons and more recent analyses of mouse duodenal myenteric neurons and colonic submucosal neurons. The immunofluorescence is used to reveal either calretinin or neuronal nitric oxide synthase (nNOS), as well as the enteric neurons in the proximal colon of the mouse. The methods can be generalized to whole mount preparations from any gut region in any species.

Mice were sacrificed by cervical dislocation, a  procedure approved by the University Melbourne Animal Experimentation  Ethics committee. The experimental procedures should incorporate all local requirements for standards of animal experimentation.





[STEPS]
SECTION: Electrophysiology
1. Sacrifice mice by cervical dislocation.
SECTION: Electrophysiology
2. Open the abdominal cavity using a midline incision and remove the colon.

SECTION: Electrophysiology
3. To minimize the contractions of the muscle layers during intracellular recordings place the colon in oxygenated [95% O2, 5% CO2] physiological saline [composition in mM: NaCl 118, KCl 4.6, CaCl2 2.5, MgSO4 1.2, NaH2PO4 1, NaHCO3 25, d-glucose 11] containing nicardipine (1.25 μM) (Sigma Aldrich, NSW, Australia) and hyoscine (1 μM)  (Sigma Aldrich, NSW, Australia).
SECTION: Electrophysiology
4. Open 1cm2 segment of the proximal colon along its mesenteric border. Pin it flat in a petri dish lined with a silicone elastomer (Sylgard 184, Dow Corning, NSW, Australia). Remove mucosa and submucosal plexus (SMP) together in a sheet.
SECTION: Electrophysiology
5. To allow access to the longitudinal muscle layer turn the segment over and repin it. 
SECTION: Electrophysiology
6. Remove the longitudinal muscle layer in fine stripes in the longitudinal direction using ultrafine dissection forceps (Dumont #5 INOX, sourced from Fine Science Tools, Canada).
SECTION: Electrophysiology
7. Transfer the preparation to a recording bath (volume 1-2 mL) and superfuse with warmed physiological saline (35 °C) at 5mL/min.

SECTION: Electrophysiology
8. After 1 hour equilibration period, visualize myenteric ganglia using an inverted microscope (Zeiss Axiovert 200). 
SECTION: Electrophysiology
9. Impale myenteric neurons using glass microelectrodes (100 – 200 MΩ resistance, pulled on a Sutter P-97 microelectrode puller) containing 1M KCl with 2% biocytin (Sigma Aldrich, NSW, Australia).
SECTION: Electrophysiology
10. To obtain and acquire voltage recordings, use an Axoprobe 1A microelectrode amplifier (Axon Insturments, USA), a personal computer connected to a PowerLab/4SP acquisition system, and Chart5 for Windows (ADInstruments, NSW, Australia).
SECTION: Electrophysiology
11. Apply single electrical stimuli or trains of stimuli (3, 5 or 15 P at 20 Hz) to an interganglionic fibre tract entering the impaled ganglion using a unipolar stimulating electrode (100 µM stainless steel insulated with 15 µM Teflon, 0.5 – 3.0 mA, duration 0.5 ms) driven by a Master 8 pulse generator via an ISO-Flex stimulus isolation unit (A.M.P. Instruments, Jerusalem, Israel).

Record fast excitatory postsynaptic potentials (EPSP) at hyperpolarized membrane potentials using hyperpolarizing current injected into the neuron, -90 mV.

Record slow EPSPs and inhibitory postsynaptic potentials (IPSP) at resting membrane potential (RMP) or close to RMP (-50 to -60 mV) using hyperpolarizing current.
SECTION: Electrophysiology
12. Examine the excitability of neurons by injecting depolarizing current pulses (500 ms duration) in 50 pA increments over the range 50 – 350 pA while the membrane potential (MP) is held at - 55 mV. 

Examine the number of action potentials (AP) and the duration of AP firing for each current pulse amplitude. Measure the duration of AP firing from the start of the first AP to the end of the last AP triggered by the pulse.

To determine the input resistance (IR) apply hyperpolarizing current pulses (50 pA - 300 pA) and plot against the associated voltage changes.
SECTION: Pharmacology
13. Make up concentrated stock solutions of antagonists in distilled water.
SECTION: Pharmacology
14. On the day of the experiment dilute the stock solutions to working concentrations.

Drugs used in the experiment included:
hyoscine (1µM) - Sigma Aldrich, NSW, Australia
hexamethonium (200µM) - Sigma Aldrich, NSW, Australia
nicardipine (2.5µM) - Sigma Aldrich, NSW, Australia
NOLA (100µM) - Sigma Aldrich, NSW, Australia
TNP-ATP triethylammonium salt (5µM) - Tocris, sourced from In Vitro Technologies Pty Ltd. Vic, Australia)
ondansetron (3µM) - Glaxo Research Group, Greenford, UK
SECTION: Pharmacology
15. Record a minimum of 3 synaptic responses before adding antagonist to the perfusing solution.
SECTION: Pharmacology
16. Record at least 3 responses between 5 and 20 minutes with antagonists present, before washing the drugs from the bath (20-30 minutes).
SECTION: Pharmacology
17. Where possible, record responses after the washout period to determine if any effect seen with an antagonist present is reversible. 
SECTION: Immunofluorescence
18. Following electrophysiological recordings:
SECTION: Immunofluorescence
18.1. Remove the proximal colon from the recording bath.
SECTION: Immunofluorescence
18.2. Repin it into a petri dish lined with silicone elastomer.
SECTION: Immunofluorescence
18.3. Fix it overnight in 4% formaldehyde solution (40% formaldehyde diluted with 0.2M phosphate buffer, pH 7.0). 
SECTION: Immunofluorescence
18.4. Remove fixative with 3 x 10 minutes washes in phosphate buffered saline (PBS).
SECTION: Immunofluorescence
18.5. Soak the proximal colon in 10% CAS block plus 0.1% Triton X-100 for 20 minutes. 
SECTION: Immunofluorescence
19. Apply primary antibodies (AB) against calretinin or nitric oxide synthase (NOS) and incubate for 2-3 nights in humidified containers at 4 °C .

SECTION: Immunofluorescence
19.1. Wash excess primary AB with 3 washes in PBS.
SECTION: Immunofluorescence
20. Add secondary ABs to visualize calretinin or NOS labelling with streptavidin Alexa-Fluor 594 (high binding affinity for biotin injected into neurons during recordings) to allow identification of impaled neurons
SECTION: Immunofluorescence
20.1.  Incubate for 3 hours in dark humid containers. 
SECTION: Immunofluorescence
20.2. Remove secondary ABs by washing 3 x in PBS. 
SECTION: Immunofluorescence
21. Mount the preparations on glass slides  (Livingstone premium grade, thickness 1.0-1.2 mm) in Dako mounting medium (Carpinteria, CA, USA).
SECTION: Confocal Microscopy
22. To visualize impaled neurons and either calretinin or NOS labeling capture Z-stack images (.czi files) using two channels of a confocal laser scanning microscope.  

SECTION: Confocal Microscopy
22.1. Set the step distance so that adjacent planes overlap.
SECTION: Confocal Microscopy
22.2. Take high power (63x or 40x oil) images of neuron cell bodies.
SECTION: Confocal Microscopy
22.3. Trace and image neuron projections using 10 or 20x air objectives ensuring overlap between consecutive image stacks.
SECTION: Confocal Microscopy
22.4. Project Z-stacks into single images (.tif files) using ImageJ.
SECTION: Confocal Microscopy
22.5. To produce detailed black and white images (also .tif files) further process images in Corel Photo-Paint. 
SECTION: Confocal Microscopy
22.6. Stitch neuron projection images (.tif files) together in Corel Draw to produce a "map" for each neuron and export as a jpeg file.
SECTION: Confocal Microscopy
23. Each neuron map is linked into the excel spreadsheet containing the electrophysiological data obtained from that neuron.