The scope of our research is to study metabolic disorder in relation to hypothalamic dysfunction. We're interested in studying the biochemical and cellular mechanism of targeted primary mammalian hypothalamic neuron, especially the one expressing leptin receptor. The hypothalamus is a complex brain region involving the maintenance of feeding, sleeping, motivation, and metabolism.
And this imply a very diverse neural community. Only few of this population have been studied thoroughly and most of the other needs to further investigated and their cellular mechanism further explained. Hypothalamic neurons and their function are investigated mainly with electrophysiology, chemogenetics, and optogenetics.
And these techniques are very informative in relation to understand animal behavior and brain circuitry. However, very little is known about the cellular and molecular mechanism that drive this behavior. And only by looking deeper at the cellular level we can address these gaps.
Start by fire polishing three Pasteur pipettes using a Bunsen burner, creating decreasing diameters. Next, sterilize the abdomen of a euthanized pregnant mouse down with 70%ethanol. Cut open the abdominal cavity from the pubic symphysis to the xiphoid process of the ribcage.
Now extract the uterine horn from the abdominal cavity and place it in a 100-millimeter plate filled with ice cold HBSS solution. Extract and separate all embryos from the washed uterus using fine forceps. Quickly decapitate the extracted mice embryos and place the heads in a 60-millimeter Petri dish filled with HBSS.
Place a pair of fine forceps in the eye cavity to hold the brain. With another pair, peel the skin and skull until the brain is visible. Scoop the brain out of the olfactory bulbs from the skull and flip it upside down.
Observe the cortex on the ventral side and the hypothalamus on the dorsal side. Use curved forceps to remove the layer of meninges and blood vessels until the brain appears white and clear, and separate the hypothalamic area from the rest of the brain. Cut the hypothalamus into three to four small fine pieces and use a pipette to transfer the pieces into a 15-milliliter tube.
Next, fill the tube with six milliliters of HBSS. Then add enzyme mix one once the tissue has settled and the supernatant removed. Incubate the tube in a 37 degrees Celsius water bath for 15 minutes, agitating every five minutes to resuspend the tissue.
Now add 30 microliters of enzyme mix two and dissociate the tissue by pipetting up and down 10 times with a large diameter past your pipette. After an additional 10 minute incubation, add the remaining 15 microliters of enzyme mix two. Utilizing two fire polished pipettes of decreasing diameter, pipette the tissue 10 times.
Immediately add 10 milliliters of HBSS with 0.5%BSA to the tube, then centrifuge the tube at 300 G for 10 minutes at room temperature. Aspirate the supernatant and resuspend the cell pellet in one milliliter of HBSS with 0.5%BSA. Begin by centrifuging the isolated brain cell suspension at 300 G for eight minutes.
Resuspend the pellet in 80 microliters of HBSS with 0.5%BSA solution. Add 20 microliters of non-neuronal cell biotin antibody cocktail and incubate. Wash the cells with two milliliters of HBSS with 0.5%BSA, then resuspend the centrifuge pellet in 80 microliters of HBSS with 0.5%BSA.
Now add 20 microliters of anti-biotin microbeads and pipette to mix thoroughly. After cold incubation, add 0.5 milliliters of HBSS/BSA solution. Set up the stand before rinsing the column with 0.5 milliliters of HBSS with 0.5%BSA.
Next, place a 15-milliliter tube under the column and pass 0.5 milliliters of the cell suspension through it. Perform three washes with 0.5 milliliters of HBSS with 0.5%BSA solution to capture residual neuronal cells. Remove the column from the magnet and place it inside a new 15-milliliter tube.
Add one milliliter of HBSS with 0.5%BSA and use the plunger to collect magnetically labeled non-neuronal cells. After cell centrifugation, resuspend the cells in one milliliter of HBSS with 0.5%BSA solution. Count the cells on a Neubauer counting chamber under a brightfield microscope.
LepR positive neurons began to form neurites after 48 hours. At DIV4, axonal extensions showed progress, while dendritic processes began to appear. At DIV6, the neurons were sufficiently developed.
Immunofluorescence studies showed that no glial cells or other non-neuronal cells were observed. The neuronal nature of the cells was confirmed by microtubule associated protein-2 staining. At DIV10, 30%of LepR positive cells expressed POMC.
Synaptic connectivity and functionality were observed in general cultures containing heterogeneous hypothalamic neuronal populations. Begin by centrifuging the murine pure neuronal cell suspension at 300 G for eight minutes. Gently aspirate the supernatant and resuspend the pellet in 80 microliters of HBSS with 0.5 BSA solution.
After adding the specific antibody, incubate the tubes at four degrees Celsius for 10 minutes. Wash the excess antibody with HBSS/BSA solution and centrifuge. Add 20 microliters of antibiotic and microbeads to the pellet resuspended in HBSS/BSA.
Incubate the tube at four degrees Celsius for 10 minutes. Then, add 0.5 milliliters of HBSS with 0.5%BSA solution for every 10 to the seven cells. Rinse the column with 0.5 milliliters of HBSS with 0.5%BSA solution.
Once the dripping stops, place a 15-milliliter tube beneath the column and pass 0.5 milliliters of cell suspension through it. Collect the eluent containing nonspecific neuronal cells, then clean the column with three washes of 0.5 milliliters of HBSS with 0.5%BSA. Now, remove the column from the magnet and place it in a new 15-milliliter tube.
Add one milliliter of HBSS with 0.5%BSA and use the plunger to flush out the targeted cells. After centrifugation and supernatant aspiration, resuspend the pellet in 0.5 milliliters of plating medium. Count the cells in a Neubauer counting chamber under a brightfield microscope.
Plate the targeted cells as the positive control and non-specific cells as the negative control in the prepared 24-well plate. Incubate at 37 degrees Celsius for 12 hours. LepR positive neurons began to form neurites after 48 hours.
At DIV4, axonal extensions showed progress, while dendritic processes began to appear. At DIV6, the neurons were sufficiently developed. Immunofluorescent studies showed that no glial cells or other non-neuronal cells were observed.
The neuronal nature of the cells was confirmed by microtubule associated protein-2 staining. At DIV 10, 30%of LepR positive cells expressed POMC. Synaptic connectivity and functionality were observed in general cultures containing heterogeneous hypothalamic neuronal populations.
