Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain

The goal of the following experiment is to visualize the development of neural circuits in the embryonic mouse brain using three distinct cell labeling systems by using a trans synaptic tracer that travels bidirectionally, a second tracer that travels only in a retrograde fashion and a third label that does not travel from the target cell. It is possible to identify the target neuron upstream neurons and downstream neurons. The results demonstrate that this experimental protocol can be used to analyze the formation of neural circuits in the embryonic female mouse brain at a single cell resolution.

This method can help answer key question in the field of developmental neuroscience, such as when a specific neural circuits are formed and how are they organized during embryonic development. This method can also be used to trace neural circuits in adult mice. Specifically, it can be utilized to analyze synaptic activity between genetically identified neurons, and it can be used to analyze how different experimental conditions may modulate a neural circuit over time.

Begin with a just euthanized pregnant mouse. Clean the ventral side of her abdomen with 70%ethanol, and then make a vertical incision to expose the embryos. Remove the chain of embryos and transfer them to a dish of ice cold PBS in the dish.

Remove the individual embryos using blunted forceps. Separate the individual embryos in solution using fine scissors and forceps, removing extra tissue in the process when the embryos are fully cleaned off. Take a small tail biopsy from each embryo and put into a tube of lysis buffer to collect DNA for gender identification and genotyping.

Then transfer the embryo to freshly made ice cold, 4%PFA on ice. After processing each embryo, let them incubate in the PFA with agitation for at least 1.5 hours, depending on their size. When the incubation is complete.

Wash the fixed tissues three times with ice cold PBS for at least five minutes per wash. Then transfer the embryos to a 30%sucrose solution, kept at four degrees Celsius. When the tissues are fully sunk in the solution, proceed with cryosectioning.

First, begin by labeling a cryo mold using permanent alcohol resistant marker. Next, prepare a slurry of crushed dry ice in 100%Ethanol in an ice bucket using the slurry cool down a glass beaker of isop pentane for about 10 minutes. While waiting, remove the tissues from the sucrose solution and wipe off the excess sucrose with a standard laboratory wipe.

Then transfer the tissue to a pre-labeled cryo mold. With enough OCT to cover the tissue. Do not form any air bubbles in the OCT, especially around the tissue.

Next, orient the tissue using feather weight entomology forceps to cause minimal damage. Freeze the cryo molds in the cooled iso pentane bath without causing any splashes if needed. The frozen molds can be wrapped and stored at minus 80 degrees celsius to section the molds.

Cut them into 14 micron thick serial sections in series of five. Collect each five slice series on a super frost plus glass slide. When preparing solution for this protocol, be sure to add the tween 20 slowly to the TNT buffer, which must be prepared fresh.

Then flush the solution up and down slowly to mix. In preparing the TNB buffer, add the blocking reagent slowly in small increments while stirring. Then heat the solution to 55 degrees Celsius using a water bath.

The solution will become milky. Now, to start work on the tissues first use a scalpel to remove the excess OCT surrounding the tissue sections. Then mark the boundaries of each section using a pap pen.

Let the slides come to room temperature. Then wash them in room temperature one XPBS buffer, three times each wash should be five minutes long. Next, quench the endogenous peroxidase activity by incubating the slides for half an hour in ice cold, 0.3%hydrogen peroxide in methanol.

Follow this with three washes in PBS at room temperature for five minutes per wash. Then incubate the slides for 10 minutes in PBST to perme the tissues. This time, wash the slides three times with TNT.

Now in a humidified chamber, block the tissue in enough TNB to cover each slide. Allow the block to go for half an hour at room temperature. Do not let the slides dry out.

After draining the blocking buffer completely cover the slide with the primary antibody in TNB to bind the tracer. Incubate for two hours at RT or overnight at four degrees Celsius. To remove the primary antibody, wash the slide with TNT three times at room temperature for five minutes per wash.

During these washes, use some gentle agitation. Next, incubate the sections in the secondary antibody solution for an hour. At room temperature later, use three TNT washes to remove the unbound secondary antibody.

Then in a humidified chamber, incubate the tissues in one to 100 stripped havein conjugated horse radish peroxidase in TNB for 30 minutes at room temperature while washing the slides in TNT. After the incubation, make a fresh dilution of TSA solution. Now apply the freshly made TSA dilution to the tissues for exactly 10 minutes at room temperature.

The precise timing of TSA incubation is critical. Therefore, do not try to process too many slides at a time in our hand. Up to 10 slides can be handled in one step.

Additional slides can wait in TNT wash buffer until they're processed Following another round of washes. Incubate the tissues with one to 500 Alexa floor 5 46 stripped. Have it in conjugate in TNB for 30 minutes.

At room temperature, remove the stripped habitant conjugate with three more TNT baths. Then for nuclear staining, apply 5%BIS benza solution in PBS at room temperature for 10 minutes, remove the BIS benzo stain with three TNT washes. Then mount the slides with fluoro Mount G and proceed with their analysis.

Using the described protocol, the maturation of the neural circuits regulating the reproductive axis was analyzed in mice carrying the R 26 BIZ and the R 26 GTT alleles. Expression of the transgenes was activated by creed dependent recombination in embryonic kisspeptin neurons in the arcuate nucleus using a Kisspeptin specific creed driver line double immunofluorescence for kisspeptin in green and BL in red from a kiss. IC R 26 BIZ.

Mouse shows activation of BL in the KISS IC R 26 GTT Mouse double immunofluorescence shows KISSPEPTIN in green and GTT activated by the Cree driver in red. These and other diagnostics demonstrated that the transgenes were functional in E 18.5 experimental embryos. His pepin neurons in the arcuate nucleus were found already communicating with GNRH.

Neurons lack Z was confined to the CRE expressing kisspeptin neurons while BL was trans synaptically transferred to upstream and downstream neurons. Some, but not all. GNRH neurons contained bl.

Thus only a subset of embryonic GNRH neurons is synaptically connected to arcuate nucleus kisspeptin neurons. GNRH neurons did not contain the retrograde tracer. GTT demonstrating that these neurons are downstream of kisspeptin neurons.

Once mastered, this technique can be performed in one day following this procedure. Other methods like dual immunofluorescence can be utilized to identify the biochemical nature of connected neurons. This technique should pave the way for researchers in the field of neuroscience and developmental biology to explore neural connectivity with a single cell resolution both during development and in adult animals.

After watching this video, you should have good understanding of how to visualize the formation and maturation of neural circuits in mice.