Materials and Reagents

Wistar rats (3 weeks old) (Charles River Laboratories International)
Ketamine-xylasine mixture
Pronase (Sigma-Aldrich, catalog number: 9036-06-0 )
Thermolysin (Sigma-Aldrich, catalog number: 9073-78-3 )
Bovine Serum Albumin (BSA) (Life Technologies, catalog number: 15561020 )
Laminin (Sigma-Aldrich, catalog number: 114956-81-9 )
Ruthenium red
KCl (Sigma-Aldrich, catalogue number: 7447-40-7 )
CaCl₂ (Sigma-Aldrich, catalogue number: 10043-52-4 )
MgCl₂ (Sigma-Aldrich, catalogue number: 7786-30-3 )
NaHCO₃ (Sigma-Aldrich, catalogue number: 144-55-8 )
NaH₂PO₄ (Sigma-Aldrich, catalogue number: 7558-80-7 )
NaCl (Sigma-Aldrich, catalogue number: 7647-14-5 )
Sucrose (Sigma-Aldrich, catalogue number: 57-50-1 )
HEPES (Sigma-Aldrich, catalogue number: 7365-45-9 )
D-Glucose (Sigma-Aldrich, catalogue number: 50-99-7 )
Dissection solution (see Recipes)
Artificial Cerebrospinal Fluid (aCSF) (see Recipes)

Equipment

Bath
Vibratome
0.45 μm sterilize filter
350 μm Sagittal slice containing MnPO
Needle
Pasteur pipettes
Centrifuge
37 °C, 95% O₂-5% CO₂ Cell culture incubator
6 hole petri dish (Thermo Fisher Scientific, catalog number: 50-341-84 )
Micro-cover glasses (Thermo Fisher Scientific, catalogue number: NC9216450 )
15 ml Falcon tubes
Oxygen and Carbogen tank
Binocular

Procedure

Micro-cover glass is coated with laminin (5 μg/ml) and incubated at 37 °C, 95% O₂-5% CO₂ Cell culture incubator, 3 h before culture preparation.
Wistar rats is deeply anesthetized by intraperitoneal injection of a ketamine-xylasine mixture (87.5 and 12.5 ml/kg, respectively) and decapitated.
The brain is removed from the skull and immersed in oxygenated (95% O₂-5% CO₂) ice-cold (2 °C) dissection solution. Oxygenation system consist on a 95% O₂-5% CO₂tank connected to a bath with a line transmitting a gentle bubbling. Then the brain is fixed with glue on a plate which will be place in the Vibratome tub containing oxygenated dissection solution.
A sagittal slice of 350 μm containing the MnPO is prepared using Vibratome (Figure 1).

Figure 1. Use Vibratome to do brain slices
Using binocular, the ventral region of the MnPO is punched (about 3mm in diameter) out with a curve needle (Figure 2), and placed in 1 ml aCSF solution (in falcon) containing 0.1 mg/ml of pronase for 10 min at 37 °C and oxygenated.
Note: Curve needle is homemade from an aluminum needle tip 3 mm diameter, gently curved at 2/3 of length with a soldering and fixed to a 10 ml syringe.

Figure 2. Use Binocular to realize MnPO micropunch
Micropunch is transferred to a new falcon tube with 1 ml aCSF solution containing 2 mg/ml of BSA for 15 min at 37 °C and oxygenated.
Micropunch is transferred to a new falcon tube with 1 ml aCSF solution containing 0.1 mg/ml of thermolysin for 10 min at 37 °C and oxygenated.
Transfer solution containing tissue in a 1.5 ml Eppendorf tube.
The tissue is then mechanically dissociated by successive trituration with glass Pasteur pipettes (Figure 3).
Note: Use 4 Pasteur pipettes whose diameter has previously been gradually reduced with flame from no change to 50 μm. This step is done at room temperature. The come and go flow should be strong enough to separate cells, but also enough gentle to not break cell membrane. The size of micropunch should reduce from pipette to another, and completely disappear using the last pipette.

Figure 3. Mechanic trituration with Pasteur pipette
Transfer solution containing cells in a 1.5 ml Eppendorf tube.
Solution containing cell is centrifuged at 1500 x g during 2 min at room temperature.
Note: Sometime pellet is really small and difficult to see, but there are cells on the wall of the tube.
The supernatant is removed and cells are re-suspended in 50 μl aCSF.
Note: Slow agitation for re-suspension to not break the membranes.
The aCSF solution containing cells (50 μl) is directly platted on micro cover glasses beforehand treated with laminine, then disposed in 6 holes petri dish, and incubated 1 h at 37 °C, 95% O₂-5% CO₂, before patch-clamp recording.
Notes:
1. There is no culture media, cells remains during 1 h in aCSF solution. Do not exceed 1 h of incubation. Density of cell is relatively low, about 100 cells on 1 micro cover glass.
2. Be sure that the solution stays on the micro-cover glass in bead, if not preparation will be dry and unusable.

Recipes

Dissection solution
200 mM sucrose
10 mM D-Glucose
2 mM KCl
1 mM CaCl₂
3 mM MgCl₂
26 mM NaHCO₃
1.25 mM NaH₂PO₄, pH 7.4
Adjusted with CO₂ bubbling and verified with Ruthenium red
Filter sterilize (0.45 μm)
Stored at 4 °C
Artificial cerebrospinal fluid/aCSF
140 mM NaCl
3.1 mM KCl
2.4 mM CaCl₂
1.3 mM MgCl₂
10 mM HEPES
10 mM D-Glucose, pH 7.4 adjusted with KOH, osmolarity 300 MOsm
Filter sterilize (0.45 μm)
Stored at 4 °C

Acknowledgments

This protocol was adapted from: Tremblay et al. (2011). This work was supported by Canadian Institutes of Health Research Grant MOP-178002.

References

Berret, E., Nehme, B., Henry, M., Toth, K., Drolet, G. and Mouginot, D. (2013). Regulation of central Na⁺ detection requires the cooperative action of the NaX channel and α1 Isoform of Na⁺/K⁺-ATPase in the Na⁺-sensor neuronal population. J Neurosci 33(7): 3067-3078.
Tremblay, C., Berret, E., Henry, M., Nehme, B., Nadeau, L. and Mouginot, D. (2011). Neuronal sodium leak channel is responsible for the detection of sodium in the rat median preoptic nucleus. J Neurophysiol 105(2): 650-660.