This protocol can answer how various circuits in the brain regulate adult neurogenesis and specifically how stimulating or inhibiting neural circuits affect the proliferation of adult neural stem cells. The advantage of this technique is that it is able to specifically target desired neural circuits, as well as reduce the amount of stress introduced to the animals. This method can provide insight into how different brain circuits regulate adult neurogenesis, in particular, how specific cell types that release certain neurotransmitters regulate adult neural stem cell proliferation.
To begin this procedure, place the tissue sections in PBS and mount five to eight sections on a positively charged slide in serial order from anterior to posterior. Let the tissue sections dry at room temperature for two to five minutes and completely adhere to the slides. Next, prepare citrate buffer in a container.
Heat the citrate buffer in a microwave oven for five minutes until the solution is boiling. In the meantime, place the mounted sections in a glass slide holder. After five minutes, carefully place the slide holder with the sections into a pipette box.
Set the microwave oven power to 50%and the cook time to seven minutes. Start a timer for seven minutes, and monitor the solution. Stop the microwave oven when the solution starts to boil, and continue the microwave after boiling stops.
Stop after the timer runs out, even if the cook time on the microwave has not finished. The goal of this step is to keep the water right below boiling temperature without letting the water overly boil. Too much boiling will remove tissue sections from the slide.
Then, transfer the warm box with citrate buffer and tissue slides to an ice bucket for cooling. Cover the box, and wait for about 30 minutes or until the solution is cool to the touch, before proceeding to thymidine analog staining. To stain the tissue sections with thymidine analog, remove the tissue sections from the citrate buffer.
Let the tissue sections dry and completely adhere to the slides, before drawing a border with a hydrophobic pen. Next, permeabilize the sections with permeabilization buffer for 20 to 30 minutes. Wash the sections twice using TBS-triton for five minutes each time.
Then, prepare an Edu reaction solution. Incubate the sections in Edu reaction solution for 30 minutes to an hour. Subsequently, wash them three times in TBS-triton for five minutes each time.
Cover the slides in aluminum foil, or place them in a light-protected chamber to protect from light after this step. At this stage, check if the Edu reaction works by using a fluorescent microscope. Edu-labeled cells should be observed if the reaction works.
Now, block the mounted tissue sections using blocking buffer raised in the same animal as the secondary antibody for 30 minutes to an hour. Then, wash them twice in TBS-triton for five minutes each time. During the blocking step, prepare primary antibody solution by mixing the primary antibody in blocking buffer.
Subsequently, add 250 microliters of primary antibody solution per slide to ensure the tissue sections are completely submerged, and incubate them overnight at room temperature. The next day, wash the tissue sections three times in TBS-triton for five minutes each to remove excess primary antibody. Then, incubate the tissue sections in fluorophore-conjugated secondary antibody prepared in blocking buffer solution for two hours at room temperature.
Wash the tissue sections three times in TBS-triton for five minutes each to remove excess secondary antibody. Next, apply 300-micromolar DAPI solution at one to 100 in PBS for 15 minutes at room temperature. Afterward, wash the tissue sections three times in PBS for five minutes each to remove excess DAPI, and remove the PAP pen circle around the tissue using a cotton swab.
Let the sections dry, before applying mounting media and covering them with coverslips. Using the imaging software, open the image of each dentate gyrus section as a composite image with the channels merged in distinct colors to easily visualize the colocalization. Then, measure the area of dentate gyrus in each section using the polygon selection tool, and record all the sections of each mouse.
This will be the area of dentate gyrus used to calculate the density. Using the software plugin cell counter found under Plugins, Analyze, Cell Counter, Cell Counter, record the number of cells in the dentate gyrus that have colocalizing primary antibody and thymidine analog Edu from the composite image. Additionally, record the total number of Edu-positive and nestin-positive cells with a radial process.
In the case of nestin, it is very important to pay attention to the cells'morphology. If quantifying neural stem cells, ensure that only cells with a radial process are quantified. Enter the cell counts in a spreadsheet software to compile all the data for analysis later.
For example, to obtain the stem cell density, divide the sum of nestin-positive and Edu-positive cells by the sum of dentate gyrus volume in each animal. Calculate the volume of each section by multiplying the area with total Z-step increments, assuming that each step is one micrometer. In this protocol, total steps should be close to 40 since tissue is sectioned at 40 micrometers.
Subsequently, calculate the overall number of proliferating cells, percent of proliferating neural stem cells, and total proliferating cells after stimulating contralateral mossy cells. By using a thymidine analog Edu and antigen retrieval for the nestin staining, the proliferating neural stem cells were successfully labeled. Additionally, by omitting the antigen retrieval step, Tbr2-positive neural progenitor and neuroblast and DCX-positive neuroblast and immature neurons were labeled.
Here is an example of the area quantified and used to calculate cell density, and here is an example of the mounted tissue on a slide. Both successful and sub-par experiments are shown for comparison. Lastly, there are several different quantifications that can be obtained from a successful experiment.
The quantifications include the density of proliferating neural stem cells, the percent of proliferating neural stem cells, total proliferating cells, and the total stem cell pool. Upon contralateral stimulation of mossy cells, a decrease in neural stem cell proliferation was observed. The most important step in this procedure is to control tissue boiling in the microwave oven.
The tissue sections will be damaged if they are boiling for too long. After following this procedure, one could inject different viral vectors to target the same circuit. For example, if one were stimulating a neural circuit, one could inhibit it using an inhibitory DREADD virus.
The techniques presented in this protocol aren't novel. However, the strength of this protocol is that it comprehensively covers all the steps required for answering questions about how circuit impacts adult neurogenesis.
