Laser-guided Neuronal Tracing In Brain Explants

We describe a technique to label neurons and their processes via anterograde or retrograde tracer injections into brain nuclei using an in vitro preparation. We modified an existing method of in vitro tracer electroporation by taking advantage of fluorescently labeled mouse mutants and basic optical equipment in order to increase labeling accuracy.

The overall goal of this method is to label neurons and their axonal projections in order to trace their connections to target or source brain nuclei of interest. This method helps answering key questions in neuroscience such as connectivity patterns between different areas or neuronal subpopulations of the brain, as well as their functional significance for a given neuro circuit. The main advantage of this technique is the increased target accuracy during tracer injections.

This is achieved by using basic optical equipment to visualize fluorescently labeled brain nuclei. Another advantage of this method is that it allows for noninvasive genotyping of mouse pops that express fluorescent proteins under the control of neuronal promoters. When genotyping pups check for the expression of the respective fluorescent markers using a laser pointer with the appropriate excitation wavelength.

At the same time, wear filter goggles that block the excitation wavelength, but pass the emission wavelength. Point the laser at the back of the head or the spinal cord of the pup in question. Be careful to not shine the laser in the P'S eyes and keep the laser exposure short.

The excited fluorescent markers should be visible through the skin. When the mouse is older, individual neurons of harvested tissues can be identified. After anesthetizing the mouse and checking for an absence of reflex, remove the skin over the skull, cut the skin along the back of the head to the midsection of the skull.

In even older mice greater than one month, the fluorescence can be observed through the eyes of the animal. Next, using ice cold PBS trans Cardi perfuse the marker positive animal using a 30 gauge needle, after five to 10 minutes, most of the blood will be removed. Then remove the brain and dissect out the region of interest.

Here, the trapezoid body, which is two prominent bulbs in the ventral region of the brain, contains the region of interest. The ventral nuclei of the trapezoid bodies or VNTB begin with transferring the explanted brain region of interest to a dish of oxygenated dissecting solution.There. Secure the explan with 30 gauge syringe needles so that the area for injection is accessible on top.

For the injection use Bo silicate glass pulled injection pipettes that are manufactured in three or four pulling cycles. Load the pipette with the following solution. Cholera toxin subunit B conjugated to Alexa FLUORA 4 5 55, which is mainly for retrograde transport.

With the laser pointer and injection pipettes, all mounted and ready to use proceed with aiming the beam at the labeled brain region or cells to be injected. Observe the field through the band pass filter goggles to see which neurons to target with the injection pipette. Use the micro manipulator to position the pipette into the area of interest.

Then using a pressure injector set to deliver 50 millisecond pulses at 15 PS, I inject the tracer substance in two to 10 pulses. Allow 10 to 15 seconds to pass between pulses so the injected dye can spread. When injecting tetraethyl rumine electrically stimulate the region with the stimulating electrode delivering TTL pulses.

Be sure the electrode is grounded in the bath solution. Deliver eight TTL pulses at eight volts each 50 milliseconds long with 50 millisecond intervals. Stimulate the region with 10 to 20 pulse repetitions over several minutes.

Brain nuclei vary in size, neuronal density, and cell types. This means that the duration and the number of pressure and or electrical pulses might need to be adjusted to achieve optimal labeling results for a given brain region. Using the laser and goggles check the fluorescent tracer signal.

The dye should be taken up by cells and spread around the injected area. After the injection incubate the brain stems in oxygenated artificial cerebral spinal fluid at room temperature for one to four hours. Maintain the oxygenation during the incubation, then fix the tissue in 4%paraform aldehyde.

In PBS overnight at four degrees Celsius the following day, the tissue can be sliced and mounted an intergrade injection into the ventral nucleus of the trapezoid body or VNTB. Using tetraethyl Rodine dextrin shows that projections from the VNTB extend to the medial nucleus of the trapezoid body, or MNTB. Then the MNTB was injected with chole toin, subunit B, which retro greatly labeled the VNTB.

In greater detail, the somata of the glyc enteric VNTB cells appeared to be labeled next. The MNTB was injected with the retrograde label dextrin tri. Unlike the punctate pattern of CTB labeling, dextrin trit labeling was dense and GOGI like.

After watching this video, you should have a good understanding of how to label neurons and their processes with increased the accuracy. The demonstrated technique helps you identifying brain areas and neuronal subpopulations participating in a given neuro circuit once mastered, the injection procedure can be done in about 30 minutes if performed properly. This procedure can be easily combined with other methods like immunochemistry or brain clearing to answer additional questions regarding the morphology, directionality, or functionality of neuronal projections and other neuronal structures.