TITLE: Juxtacellular Recordings in Ventral tegmental area and Locus coeruleus
AUTHORS: Cristian González-Cabrera, Kaushik S. More, Matthias Prigge

[DESCRIPTION]
This protocol describes juxtacellular recordings in the VTA and LC of adult mice, involving anesthesia, precise brain targeting, and neuron labeling. Using specialized electrodes and a dual-channel amplifier, neuronal activity is recorded and modulated with current injections. Following recordings, brains are perfused, fixed, and sectioned for detailed morphological analysis.

[STEPS]
SECTION: Preparation of Adult Mice for juxtacellular recordings
1. Use adult mice weighing 25-30 g.
Anesthetize the animal with urethane at a dosage of 1.25 g/kg.
Place the mouse in the stereotaxic frame and align the head angle according to Bregma and Lambda, with a tolerance of a maximum of 50 microns between them.
Expose the skull by shaving the mouse's head and making a sagittal incision on the skin. 
Find the coordinates and drill the bone. Once the dura is exposed, carefully remove it with the forceps (n°).
  
      VTA: A/P 3.25, L/M 0.5
      LC: A/P 5.34, L/M 1.1

Drill a small hole around Lambda and install the reference screw. 


 


SECTION: Juxtacellular set-up
2. Employ an intracellular recording amplifier (NeuroData Dual channel IR-283), an analog-to-digital converter (CED Micro-1401-3), and the Spike2 software..
Use 5-16 MΩ  (40-45 MΩ for intracellular) glass electrodes with a tip diameter of less than 1 μm. 
Fill the electrodes with a solution containing:
250 mM k-gluconate, 5 mM KCl, 1 mM MgCl2, 2 mM EGTA, 5 mM HEPES, 2 mM MgATP, and Tetramethyl-Rhodamin Biocytin tracer or Neurobiotin (2% w/v); pH 7.2.


SECTION: Juxtacellular/Intracellular Recordings and Labelling
3. After penetrating the brain, ensure the electrode impedance is correct by injecting 0.5 nA pulses and adjusting the bridge/balance module.
Move down the electrode at a constant low speed until reaching the dorsal VTA (z=-4.0). 
Start the exploration by moving down not faster than a micron per second.
Isolate spontaneously active neurons until the extracellular spike amplitude is approximately 0.7 to 1.0 mV (for intracellular penetration about 2.0-2.5 mV.)
After baseline recordings, label neurons using the juxtacellular (Pinault, 1996) or intracellular methods.




SECTION: Juxtacellular/Intracellular Recordings and Labelling
4. For juxtacellular labeling:
Label neurons by injecting a small current (1-4 nA for 5-10 minutes, 250 ms ON-250 ms OFF). Make sure that the neuron is being modulated by the current injection.
SECTION: Juxtacellular/Intracellular Recordings and Labelling
5. For intracellular labeling:
Apply AC pulses to gain intracellular access. If unsuccessful, adjust by 2 micrometers and reapply AC pulses.
Once intracellular access is achieved, stabilize neurons with negative current.
Label neurons by injecting a small current (1-2 nA for 10-15 minutes, 250 ms ON-250 ms OFF).
SECTION: Juxtacellular/Intracellular Recordings and Labelling
6. Wait for at least 2 hours before perfusion.
SECTION: Juxtacellular/Intracellular Recordings and Labelling
7. 
SECTION: Post-Recording and Labeling Process:
8. Perfuse the animals via the ascending aorta with 0.01 M PBS followed by 4% paraformaldehyde in PBS.
Post-fix brains between 6-12 hours in 4% PFA.
Cryoprotect the brain in 30% sucrose until it sinks.
Section the brains at 40 μm using a freezing microtome.