Combinations of Patch-Clamp and Confocal Calcium Imaging in Acutely Isolated Adult Mouse Amygdala Brain Slices

Materials and Reagents

1. Pipette tips (USA Scientific, catalog numbers: 1112-1820 ; 1110-1800 ; 1111-3840 )
2. Single edge razor blade (Fisher Scientific, catalog number: 12-640 )
3. Patch-clamp grass pipette (Sutter Instrument, catalog number: BF100-58-10 )
4. C57BL/6J male mice, age 9-12 weeks (THE JACKSON LABORATORY, catalog number: 000664 )
   Note: Mice are group housed before and during the experiment.
5. Oregon Green^TM 488 BAPTA-6F, Hexapotassium Salt, cell impermeant (Thermo Fisher Scientific, catalog number: O23990 )
   Note: Aliquots should be stored at -20 °C.
6. Isoflurane (Henry Schein Animal Health, catalog number: 029404 )
7. 95% Oxygen and 5% CO₂ cylinder (Airgas, catalog number: X02OX95C2003102 )
8. Sucrose (Fisher Scientific, catalog number: BP220-1 )
9. Potassium chloride (KCl) (Fisher Scientific, catalog number: P217-500 )
10. Sodium phosphate monobasic (NaH₂PO₄) (Acros Organics, catalog number: 447760010 )
11. Magnesium sulfate (MgSO₄) (Acros Organics, catalog number: 124900010 )
12. Sodium bicarbonate (NaHCO₃) (Fisher Scientific, catalog number: S233-500 )
13. Calcium chloride (CaCl₂) (Fisher Scientific, catalog number: C79-500 )
14. Glucose (Fisher Scientific, catalog number: BP350-1 )
15. Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271-1 )
16. Magnesium chloride (MgCl₂) (Fisher Scientific, catalog number: M33-500 )
17. Potassium methanesulfonate (KSO₃CH₃) (Sigma-Aldrich, catalog number: 83000 )
18. HEPES (Sigma-Aldrich, catalog number: H3375 )
19. Adenosine 5'-triphosphate magnesium salt (MgATP) (Sigma-Aldrich, catalog number: A9187 )
20. Guanosine 5'-triphosphate sodium salt hydrate (Na₃GTP) (Sigma-Aldrich, catalog number: G8877 )
21. Phosphocreatine (Sigma-Aldrich, catalog number: P1937 )
22. Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 417661 )
23. High sucrose dissection solution (see Recipes)
24. Normal artificial cerebrospinal fluid (ACSF) (see Recipes)
25. Patch-clamp pipette solution (see Recipes)
    

Equipment

1. Blunt-ended scissors (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 78702 )
2. Blunt-ended forceps (Fisher Scientific, catalog number: 13-812-39 )
3. Iridectomy scissors (Sklar Surgical Instruments, catalog number: 64-2035 )
4. Point-ended scissors (Fisher Scientific, catalog number: 13-808-2 )
5. Hemostats Rankin forceps (Fisher Scientific, catalog number: 13-812-45 )
6. Metal spatula (Fisher Scientific, catalog number: 14-374 )
7. Vibrating blade microtome (Leica Biosystems, model: Leica VT1000 S )
8. Pipettes, single channel, 0.2 µl-1 ml (Gilson, model: PIPETMAN^® Classic )
9. Confocal microscope (Nikon Instruments, model: Eclipse FN1 )
10. 4 °C refrigerator (Whirlpool, model: WRR56X18FW )
11. MultiClamp 700B Amplifier (Molecular Devices, model: MultiClamp^TM 700B )
12. Digidata 1550B plus HumSilencer (Molecular devices, model: Digidata^® 1550B )
13. P-97 Micropipette Puller (Sutter Instrument, model: P-97 )
    

Software

1. pCLAMP 10 Software Suite (Molecular devices)
2. NIS-Elements Confocal (Nikon instruments)
   

Procedure

Note: Animal care and procedures met National Institutes of Health standards. Local animal care ethical standards must be adhered to. The University of Iowa Animal Care and Use Committee (ACURF #4041016) and the University of Toledo Institutional Animal Care and Use Committee (Protocol #108791) approved all procedures.

1. C57BL/6J mouse
   The 4 weeks old C57BL/6J mice are originally ordered from Jackson laboratory (Stock No. 000664). The mice are housed and bred in the animal facilities at the University of Toledo. Mice are maintained on a standard 12-h light-dark cycle and received standard chow and water ad libitum. Animal care and procedures met National Institutes of Health standards. The University of Iowa Animal Care and Use Committee (ACURF #4041016) and University of Toledo Institutional Animal Care and Use Committee (Protocol #108791) approved all procedures. Experimental groups are male mice matched for age (ranging from 9 to 12 weeks) and assigned randomly to experimental groups.
   
   
2. Whole-cell patch clamp and confocal calcium imaging
   
   1. Anesthetize a 9-12 weeks old mouse with over-dose isoflurane. Pour 5 ml isoflurane into a sealed glass desiccator followed by putting in a mouse. Decapitate after the mouse lacks eyelash and limb reflections.
   2. Dissect the brain and rinse in the pre-oxygenated (5% CO₂ and 95% O₂) ice-cold high sucrose dissection solution (see Recipes) for 5 min. Then glue the brain on the plate of the sample chamber of the Leica Vibrating blade microtome (Figure 1). One brain is sufficient for one-day experiment.
      
      
      Figure 1. Schematic of the procedure of the brain slicing using vibratome. A. The brain is dissected and rinsed in the pre-oxygenated (5% CO₂ and 95% O₂) ice-cold high sucrose dissection solution. B. The cerebellum is removed and the cerebrum will be glued to the vibratome. C. Vibratome slice the brain coronally into 300 µm sections. D. Slices are incubated in normal pre-oxygenated ACSF.
      
      
   3. Vibratome slice brains coronally into 300 µm sections in the pre-oxygenated (5% CO₂ and 95% O₂) ice-cold high sucrose dissection solution.
   4. Incubate slices in normal artificial cerebrospinal fluid (ACSF) at room temperature (22-25 °C) for at least 1 h before recording (see Recipes).
   5. For experiments, transfer individual slices to a submersion-recording chamber and continuously perfuse with the 5% CO₂/95% O₂ ACSF (~3.0 ml/min) at 22-25 °C (Figure 2).
      
      
      Figure 2. Schematic of the Ca²⁺ imaging perfusion and recording system
      
      
   6. Observe the slices under an upright confocal microscope (Nikon Eclipse FN1). The slices containing the amygdala is identified according to The Mouse Brain in Stereotaxic Coordinates: second edition (Paxinos and Franklin, 2003).
   7. Dissolve Oregon Green 488 BAPTA-6F (100 μM) in the patch-clamp pipette solution (see Recipes).
   8. Use the Sutter P-97 micropipette puller to make the patch-clamp pipette. The pipette resistance (measured in the bath solution) is 3-5 MΩ. Before moving into the brain slice, a holding positive pressure (~0.1 ml suction through a 1 ml syringe) is given to the pipette. Once the pipette tip attaches to the cell membrane, the positive pressure is released followed by a transient negative suction through the pipette. High-resistance (> 1 GΩ) seals are formed in voltage-clamp mode.
   9. Then hold the membrane potential at -80 mV. With the high-resistance seals, an additional transient negative suction is given to break the cell membrane. Whole-cell patch-clamp configuration is made from pyramidal neurons in the lateral amygdala.
   10. After obtaining the whole-cell configuration, wait 15-20 min for the intracellular diffusion of the Oregon Green.
   11. Cells are then switched from voltage-clamp mode to current-clamp mode for Ca²⁺ imaging.
   12. Perform imaging using a high-speed confocal laser scanning microscope NIKON Eclipse FN1 with a long-working distance water-immersion objective (25x; NA 1.1). NIS-Elements Confocal software is used to capture signals and data analysis.
   13. Intracellular calcium signals in dendrites near the soma of pyramidal neurons in the lateral amygdala are evoked by electrical stimulation at cortical inputs with a series of frequencies (20 Hz, 50 Hz and 100 Hz for 1 sec) (Figure 3).
       
       
       Figure 3. Postsynaptic [Ca²⁺]_i recording in the amygdala slices. A-B. Schematic for measuring changes in postsynaptic [Ca²⁺]_i. Brain slices are prepared, cortical input stimulation is identified, and lateral amygdala pyramidal neurons are loaded with the fluorescent Ca²⁺ indicator Oregon Green 488 BAPTA-6F (100 µM) via a patch pipet. Changes in postsynaptic [Ca²⁺]_i induced by presynaptic stimulation at 20, 50, and 100 Hz are assayed when the ACSF is saturated with 5% CO₂ (pH 7.35). C. Example of loading of Oregon Green 488 BAPTA-6F via a patch pipet in the amygdala neurons. D. Representatives of changes in [Ca²⁺]_i signal with stimulation of lateral amygdala neurons.
       
       
   14. Detect fluorescence signals at one frame/10-50 msec.
   15. Analyze the real-time calcium imaging data using NIS-Elements Confocal software (Figure 3).
       

Data analysis

Relative changes in fluorescence are calculated and normalized to baseline measurements as ∆F/F0, where F0 is the fluorescence intensity before stimulation and ∆F is the change in fluorescence during presynaptic stimulation.

Recipes

1. High sucrose dissection solution (pH 7.30 at 22-25 °C)
   205 mM sucrose
   5 mM KCl
   1.25 mM NaH₂PO₄
   5 mM MgSO₄
   26 mM NaHCO₃
   1 mM CaCl₂
   25 mM glucose bubbled with 95% O₂/5% CO₂
2. Normal artificial cerebrospinal fluid (ACSF) (pH 7.35 at 22-25 °C )
   115 mM NaCl
   2.5 mM KCl
   2 mM CaCl₂
   1 mM MgCl₂
   1.25 mM NaH₂PO₄
   11 mM glucose
   25 mM NaHCO₃ bubbled with 95% O₂/5% CO₂
3. Patch-clamp pipette solution
   135 mM KSO₃CH₃
   5 mM KCl
   10 mM HEPES
   4 mM MgATP
   0.3 mM Na₃GTP
   10 mM phosphocreatine (mOsm = 290)
   Adjust pH to 7.25 with KOH
   

Acknowledgments

We thank Thomas Moninger, Theresa Mayhew, and Sarah Horgen for assistance. JD is supported by the American Heart Association (15SDG25700054) and the National Institutes of Mental Health (R01MH113986). The authors declare that there are no conflicts of interest or competing interests.

References

1. Cameron, M., Kekesi, O., Morley, J. W., Tapson, J., Breen, P. P., van Schaik, A. and Buskila, Y. (2016). Calcium imaging of AM dyes following prolonged incubation in acute neuronal tissue. PLoS One 11(5): e0155468.
2. Du, J., Price, M. P., Taugher, R. J., Grigsby, D., Ash, J. J., Stark, A. C., Hossain Saad, M. Z., Singh, K., Mandal, J., Wemmie, J. A. and Welsh, M. J. (2017). Transient acidosis while retrieving a fear-related memory enhances its lability. Elife 6: e22564.
3. Paxinos, G. and Franklin, K. B. J. (2003). The mouse brain in stereotaxic coordinates. 2nd edition. Academic Press.