This article describes a method for microinjecting tetrodotoxin into the hypothalamus of awake female rats to study its effects on neuronal firing and hormone secretion. The procedure involves careful preparation of microsyringes and microinjectors to ensure accurate delivery of the toxin.
Take microsyringes filled with distilled water and connect them to tubings linked to microinjectors.
Press the microsyringe plungers until a water droplet appears at the microinjector tips, then retract the plungers slightly to create an air pocket in the tubings.
Place pads soaked with a toxin-inactivating substance and paraffin films above them.
Pipette tetrodotoxin onto the parafilm and aspirate it into the microinjectors.
Mount the microsyringes onto the microinjection pump and advance the plungers until a drop appears at the microinjector tips. Discard the drops.
Hold an awake female rat with guide cannulas implanted in its hypothalamus.
Insert the microinjectors into the guide cannulas.
Perform the microinjection.
Tetrodotoxin blocks the sodium channels in hypothalamic neurons, preventing neuronal firing.
This inhibition suppresses neurons secreting gonadotropin-releasing hormone, thereby inhibiting ovulation.
Leave the microinjectors in place to prevent backflow. Then, remove them and replace the caps on the guide cannulas.
Configure the microinjection pump with the infusion rate and total time of injection. Then fill 2 10-microliter Hamilton syringes with sterile distilled water and insert a piece of PTFE tubing into the tubing connector of each micro injector, ensuring that the length of the tubing does not constrain the movement of the rat.
Connect the tubing to the Hamilton syringes and press the plunger until a drop of water is visible at the tip of the micro injector. Retract the plunger to create a 2-centimeters air pocket in the tubing. Place a pad that is soaked with the inactivating substance and a 2 by 2 centimeters square of paraffin film above it. Pipette a sufficient amount of TTX to fill the needle of the injector and 1 centimeter of the tubing above the film.
Absorb the TTX drop by gently retracting the plunger. Mount the syringes in the micro injection pump and use its controls to manipulate the plunger until a drop of TTX can be seen at the tip of each micro injector. Use only rats that showed at least three consecutive cycles after the surgery, taking into consideration their stage of the cycle and the time of the day.
Hold the rat with a firm grip. Remove the cap and obturators from the cannulas. Insert the micro injectors into the guide cannulas and return the animal to the cage. Turn on the pump and wait until the microinjection finishes. Leave the micro injectors in place for two additional minutes to avoid reflux of the solution, then remove them. Replace the obturators. Put on the cap and return the animal to the colony room.
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