Determining a Test Growth Hormone Effect on Kisspeptin Neurons using a Whole-Cell Patch Clamp
Transcrição
Secure a transgenic mouse hypothalamic slice in a recording chamber perfused with aCSF.
The slice contains fluorescent fusion protein-expressing kisspeptin neurons, crucial for reproductive system regulation.
Assemble a micropipette comprising a recording electrode in an internal solution.
Microscopically locate a fluorescent kisspeptin neuron.
Apply positive pressure to the pipette and advance it toward the target neuron. Upon contact, the pressure creates an indentation on the neuronal membrane.
Apply brief negative pressure, forming a high-resistance seal between the micropipette and the membrane.
Set the holding voltage at the neuron's physiological resting potential for accurate measurements.
Apply brief negative pressure pulses to break the membrane, establishing a whole-cell configuration.
Confirm the neuron's viability by monitoring the parameters.
Introduce a test growth hormone, triggering signaling pathways and inducing changes in the neuron's membrane potential.
Measure the changes in the membrane potential to determine the growth hormone's inhibitory effect on neuronal activity.
