Immunohistochemical Visualization of Hippocampal Neuron Activity After Spatial Learning in a Mouse Model of Neurodevelopmental Disorders

The overall goal of the following experiment is to detect changes in hippocampal irk phosphorylation following spatial learning in Grailed two knockout mice as a model of neurodevelopmental disorders. This is achieved by first testing both wild type and in grail two knockout mice in the Morris water maze. As a second step, the mice's brains are removed and slices from wild type and knockout mice are prepared by viome cutting.

Next brain slices from wild type and knockout mice are processed for immunohistochemistry and used to detect phosphorylation changes in the hippocampus. The results show differences in hippocampal RC phosphorylation between wild type and in grail two knockout mice based on light microscopy visualization of the immunohistochemical staining. This method allows to identify specific subsets of hippocampal neurons that are activated following a hippocampus dependent special learning task such as the Maurice water maze.

We first add the idea for this method when we decided to investigate the molecular cascade that underline the molecular deficit in in grade two knockout mice, a mouse model for autism spectrum disorders. To begin train mice from various backgrounds of interest in a spatial learning task such as the Morris Water Mae. Follow the training regimen as described in the accompanying text protocol at the end of the training period, euthanize the mice and then fix and remove their brains.

When ready to continue with the procedure, use a scalpel to remove the cerebellum. Then super glue the rest of the brain upright onto the holder plate of the vibrato. Cut the brain into 20 to 40 micron thick serial sections throughout the dorsal hippocampus.

Transfer each slice into one well of a 24 well plate filled with one milliliter of 0.1 molar PBS transfer three to five dorsal hippocampal sections for each mouse into the wells of a 24 multi-well plate containing 500 microliters of 0.1 molar PBS for each. Well wash the sections contained in each well with 500 microliters of 0.1 molar PBS perform all washes and incubations at room temperature with gentle agitation unless otherwise mentioned. Under a fume hood, incubate the sections in 40%methanol, 1%hydrogen peroxide in 0.1 molar PBS for 20 minutes in order to quench the endogenous peroxidase activity.

Following three more washes in 0.1 molar PBS perme the sections by incubating them in 500 microliters of 0.2%Triton X 100 for five minutes while the sections are incubating, prepare enough blocking buffer for the entire experiment. Then remove the permeable solution and add 100 microliters of the blocking buffer to each. Well incubate the sections for one hour.

Next, remove the blocking buffer and cover the sections with phosphorylated extracellular regulated kinase. Primary antibody diluted one to 500 in blocking buffer. Incubate the sections overnight at four degrees Celsius with gentle agitation the next day after washing the sections three times in 0.1 molar PBS for five minutes.

Each incubate the sections with a biotin conjugated secondary antibody diluted one to 250 in blocking buffer for one hour at room temperature during the incubation. Prepare the A BC reagent at least 30 minutes before use in order to allow enough time for the Aden and biotin complex to form. After washing the sections three more times with 0.1 molar PBS add the A b, C reagent and allow it to incubate with the sample for 45 minutes.

During the next set of three washes in PBS, prepare the DAB working solution in the fume hood according to the manufacturer's specifications, aliquot the DAB working solution in the plate. Then transfer sections into the wells containing the DAB working solution and slowly swirl the plate to allow maximum exposure of the sections to the solution. Monitor the DAB reaction on a microscope until adequate signal develops.

In about two to five minutes, stop the reaction at the desired color intensity by washing the tissue in 0.1 molar PBS. After three more washes in PBS, mount the sections on gelatin coated slides and dry them at room temperature for at least three to four hours. Once air dried transfer slides in the fume hood and dehydrate the sections by sequentially placing them integrated ethanol solutions for two minutes each, then clear the slides in 100%xylene for five minutes.

Cover, slip the slides using mounting medium and leave them in the fume hood overnight to dry transfer the slides to a bright field microscope, equipped with a camera and 20 x objective lens. Acquire multiple brightfield images from each section at 200 x magnification covering the dorsal hippocampus image. Three to five sections from each animal.

Shown here are sections from the dorsal hippo campi regions of a wild type mouse and an Grailed two knockout mouse stained for phosphorylated. Extracellular regulated a reliable molecular readout of learning dependent neuronal activation. Both these mice had undergone a classical hippocampal dependent spatial learning task regimen, the Morris water maze prior to sacrifice.

On the final day of testing, the Grailed two knockout mice displayed a significant spatial learning deficit compared to the wild type mice. To analyze the brain slices from the mice, the total number of positive cells were counted throughout the various regions of the hippocampus. In the CA three parameter layer, wild type mice had significantly more phosphorylated RK positive cells per area than in the knockout mice.

Opposite results were found in the hili and granule cell layer or GCL. After watching this video, you should have a good understanding of how to investigate phos formulation on brain slices from the hippocampus of both genetic and pharmacological mouse models of neurodevelopmental disorders characterized by cognitive deficits. This technique can also be used to study irk force formulation changes in other brain areas, as well as following cognitive behavioral tasks different than Maurice Water maze.

Don't forget that working with DIA benzine can be extremely deserters and per precautions, such as wearing appropriate protections should always be taken while performing this procedure.