This article describes a method for injecting human-derived interneuron precursor cells into the hippocampus of genetically modified mouse pups. The procedure aims to restore the excitatory-inhibitory balance in the hippocampus, which is disrupted due to reduced inhibitory interneurons.
Secure an anesthetized genetically modified mouse pup in a stereotaxic frame using opposite-direction ear bars to prevent injury.
The pup’s hippocampus has reduced inhibitory interneurons, leading to hyperexcitation of excitatory neurons.
Maintain a flat head position and cover the pup with ice to prolong anesthesia.
Disinfect the skull and identify the lambda suture as a reference point.
Position the injection needle at the target hippocampal coordinates.
Using a bent needle, create a microentry hole while minimizing skull damage.
Lower the needle to the target depth and inject human-derived interneuron precursor cells at a controlled rate, minimizing tissue damage.
Slowly retract the needle to prevent cell reflux.
Remove the pup from the frame, warm it until movement resumes, then return it to the litter in the cage.
The injected precursor cells disperse across the hippocampus, mature, and integrate into the neuronal network. Over time, they enhance inhibitory transmission, which restores excitatory-inhibitory balance.
Position the pup by placing it on the stereotaxic frame, and using the ear bars in the opposite direction. Next, clean the surface of the skin using a soft tissue soaked in ethanol. Identify lambda, and set the coordinates to 0 on the digital display console of the stereotaxic instrument.
After relocating the Hamilton syringe to the desired coordinates, use a 90-degree bent insulin needle to penetrate the skull, creating a tiny hole. Then bring the Hamilton syringe down until the needle crosses the skull, and 0 the dorsoventral coordinate.
Lower the needle until the desired dorsoventral coordinates are achieved. Retract the needle slowly once the stem cells have been injected. End the procedure by warming up the pup with the hands until it starts moving before giving it back to the mother.
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