Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

1. Preparation of Hypothalamic Brain Slices

1. Obtain, in advance, Institutional Animal Care and Use Committee (IACUC) approval for any procedure involving animals. The animal procedures described here are consistent with the AVMA Guidelines for the Euthanasia of Animals 2013 and were approved in advance by the Oregon Health & Science University IACUC.
2. Prepare 500 ml of the slicing buffer without the MgCl₂ and CaCl₂ (Table 1). Bubble the solution with 95% O₂ and 5% CO₂ at room temperature then add the MgCl₂ and CaCl₂ while continuing to aerate. Adjust the osmolarity to 300 mOsm with sucrose. The osmolarity is measured with a vapor pressure osmometer.
3. Prepare 1 L of the recording media without the MgCl₂ and CaCl₂ (Table 2). Bubble the solution with 95% O₂ and 5% CO₂ then add the MgCl₂ and CaCl₂ while continuing to aerate. Adjust the pH to 7.2-7.4 with NaOH (10 N) and the osmolarity to 300 mOsm with sucrose. The addition of HEPES buffer is optional and was used to further stabilize pH.
4. The slicing and recording solutions may be stored in the refrigerator for several days prior to use.
5. Prepare 3% agarose and pour into a sterile Petri dish (6 mm deep) and refrigerate.
6. Cut three cubes of agarose about 6 mm x 6 mm x 6 mm.
7. Deeply anesthetize a four to eight week old Sprague-Dawley rat with isoflurane. Test for the depth of anesthesia. The depth of anesthesia is deemed appropriate when there is no withdrawal reflex of the hind limb following pinching of the footpad. Once appropriately anesthetized, the rat is euthanized by rapid decapitation. 
8. Rapidly remove the brain being careful to cut the optic nerves first, and place in ice-cold slicing solution saturated with 5% CO₂ and 95% O₂ and allow to cool for 2-3 min.
9. Trim the brain. Place the brain using forceps on a moistened (with slicing solution) circular filter paper set on a glass Petri dish resting on ice.
10. Cut the brain coronally to remove the remaining cerebellum and brain stem, then the rostral cortex and the sides, to leave a block of brain containing the hypothalamus carefully preserving the side containing SCN, optic nerves, and optic chiasm.
11. Spread a thin layer of cyanoacrylic glue large enough to attach the brain and agarose blocks in an ice-cold but dry microtome-slicing chamber.
12. Transfer the ice-cold brain block caudal side down onto the glue with the side containing the SCN positioned for cutting first with the optic nerves forming a "V" pointing up.
13. Place the agarose blocks on the other 3 sides. Fill the chamber with ice-cold slicing buffer continuously bubbled with 5% CO₂ and 95% O₂. The cyanoacrylic glue will solidify with moisture holding the brain and agarose blocks in place.
14. Cut coronal hypothalamic slices (220-250 µm thick) containing the SCN with the vibrating blade microtome. Typically a slice is chosen where the optic chiasm is about 2-4 mm across, appearing like a white band with SCN just dorsal appearing as two small “indentations” in the optic chiasm on either side of the 3^rd ventricle (Figure 1A).
15. Place the slices in the recording chamber (36 °C) mounted on the stage of a Leica DMLFS microscope. The chamber is filled with continuously flowing recording solution (2 ml/min) aerated with 95% O₂ and 5% CO₂ and preheated with an inline heater.
16. Perform whole-cell patch clamp and calcium imaging recordings in SCN neurons 0.5-8 hr after slice preparation.

2. Patch Clamp Recording

1. Prepare the electrode solution (intracellular) solution in half the total final volume of water.
2. Adjust the solution to pH 7.3 with KOH (1 N).
3. Add the remaining water. The final osmolarity of the solution should be in the range of 280-290 mOsm.
4. Pass the electrode solution through a 0.2 µm filter, aliquot (0.5 ml) into microfuge tubes, and store at -20 °C.
5. Prepare the calcium probe bis-fura-2 hexapotassium salt stock solution (10 mM) by mixing 1 mg bis-fura-2 hexapotassium salt with 99.29 µl of 0.2 µm filtered water.
6. Aliquot into separate tubes (2-5 µl each) and freeze (-20 °C).
7. Thaw an aliquot of internal solution and the Ca²⁺ probe shortly before use. Add 2 µl of the bis-fura-2 stock solution to 400 µl of the internal solution for a final 50 µM calcium probe concentration. Briefly triturate to mix the solutions. The calcium probe concentration (10-250 µM) varies depending on experimental design and imaging system. Note that bis-fura-2 is comprised of two linked fura-2 molecules.
8. Pull whole-cell patch electrodes, in two stages to an outside tip diameter of approximately 1 µm and resistances of 7-10 MΩ when filled with the intracellular solution.
9. Backfill the electrode with a few µl of the internal solution containing the calcium probe filtered with a 4 mm syringe filter (0.2 µm) for low volume samples via a Microfil tube.
10. Apply a small amount of positive pressure to the electrode as it is guided into the recording chamber under red light illumination while being viewed on a video monitor. 
11. Place the microelectrode over the SCN using a low power objective (4X). The SCN is identified as the two translucent areas on either side of the third ventricle and dorsal to the optic chiasm (Figure 1A).
12. Switch the microscope to a high power magnification (40X or 63X). Subregions of the SCN and individual SCN neurons can be targeted for recording at the higher magnification.
13. Apply gentle positive pressure to the microelectrode as it is advanced onto the surface of a neuron.
14. Apply gentle negative pressure to the microelectrode to form a seal with resistances of 3-10 GΩ.
15. Adjust the membrane voltage to -60 mV with the amplifier controlled with acquisition software.
16. Apply additional negative pressure to the electrode to rupture the cell membrane to whole cell mode.
17. Upon entering whole cell mode the SCN neuron soma and dendrites fill with fluorescent probe.
18. Membrane voltage is measured in current-clamp mode or current in voltage-clamp mode while the calcium probe image data is being monitored and stored (described below). View acquired images along with a graph of 340 nm/380 nm ratio fluorescence data during the experiment.

3. Measurement of Intracellular Ca²⁺

1. Quantitative Ca²⁺ measurements are obtained by recording a pair of images by rapidly exciting the tissue with UV light at 340 nm and 380 nm via a monochronometer with a 10 nm bandwidth and passed through a UG11 optical filter to restrict harmonic wavelengths above 400 nm, a 400 nm DCLP dichroic and emitted light through a 510±40 nm emission filter.
2. The images are acquired using a cooled CCD camera with acquisition time and binning adjusted to minimize photobleaching and maximize recording speed.
3. Quantitative imaging software is used to acquire and display the images.
4. Select regions of interest from the initial image to be converted to relative fluorescence intensity unit data (i.e. soma, dendrite, etc.). Also select a region for measurement of the background fluorescence (Figure 1B).
5. Raw images and quantified optical data are continually saved during the experiment. After the experiment the saved images are generally reanalyzed when more time can be taken to select more accurately the regions of interest to be quantified.
6. The data can be presented as the estimated intracellular calcium concentration (Est[Ca²⁺]i). The ratio (R) of emitted light following excitation and background subtraction at 340 nm divided by 380 nm along with the maximum and minimum fluorescence at 340 nm and 380 nm is used to calculate the calcium concentration⁴. Background subtraction and other calculations are performed using data analysis software.
7. An in vitro determination of maximum and minimum bis-fura-2 fluorescence can be performed using 10 mM CaCl₂ for Ca²⁺ bound and 10 mM EGTA for Ca²⁺ free conditions. The Ca²⁺ disassociation constant, Kd of the probe is taken to be 370 nM for bis-fura-2 (Invitrogen).