Neurological bundle tracking experiment

Summary

Source : Practical Laboratory Techniques in Neurobiology

Operation method

basic program

Principle

Nerve bundle pathway tracing techniques are the most commonly used methods for studying fiber connections between neurons, including tracing using the principles of routing of nerve fibers after injury and neuronal axoplasmic transport, which has obvious advantages in various aspects. Commonly used tracing agents include horseradish peroxidase (HRP), PHA-L, biotinylated-dextranamine (BOA), cholera toxin B (choleratoxin, CT), and fluorescein tracing agents (e.g., FG, Oil, etc.). The specific principles of each tracer can be found in Neuroanatomy, edited by Yunqing Li.

Materials and Instruments

Laboratory Animals
10% BOA
5µL microsampler (Hamilton Company) Mouse brain stereotaxis Power drill

Move

1. After mice were anesthetized by intraperitoneal injection of 1% pentobarbital sodium, the head was fixed with a stereotaxic apparatus.

2. The skin on the top of the head was routinely sterilized, the scalp was incised in a median-sagittal shape, the mouse was positioned according to the coordinates of the atlas, the skull was drilled, and 0.5µL of 10% BDA solution (dissolved in saline) was slowly injected into the striatum with the microplate injector, the injection was completed within 5min, and the needle was left in place for l0min and then slowly withdrawn.

3. After the animals survived for 7d, perfusion fixation was performed and the brain tissues were removed.

4. After dehydration with 25% sucrose, continuous coronal cryosections were performed, with a slice thickness of 14 µm, and the slices were pasted on gelatin-coated slides.

5 The sections containing nigral parts were rinsed into 0.1 MPBS respectively, and then into the horseradish peroxidase-streptavidin complex containing horseradish peroxidase-streptavidin (0.1M PBS 1:500 dilution), and incubated for 2h for DAB color development.

6. Dehydrate, clear and DPX seal. Step 5 can also be carried out by adding anti-biotin fluorescent secondary antibody for incubation, and then sealing the film and observing it with fluorescence microscope.BDA can be used for both paracrine and retrograde tracing, but the effect of paracrine tracing is a little bit better, and it can be clearly observed that in the nigral part of the midbrain the brown color-positive DAB fibers and the green fluorescence-positive fibers can be seen, and it is evident that the morphology of the endings of the nerve fibers traced by the BDA is more clear, and the endings of the nerve fibers from the striatum to the nigral part can be observed. to the projection from the striatum to the substantia nigra.

Caveat

1. There are cis and trans tracking agents, choose the appropriate tracking agent according to the purpose of the experiment.

2. The number of days the animal survives after injection of the tracking agent is determined by the length of the tracked nerve pathway.

Common Problems

1. In the process of stereotactic injection, the microfeeder should be injected slowly into the tracing agent, and the needle should be left in place after injection.

2. The accuracy of the injection site and the amount of injection dose directly determines the effect of injecting the tracer, and novices need to practice more in order to achieve the desired effect.

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