By: Ramanjot  KaurMentor: Jana  Veliskova

Albert Einstein  College of Medicine

Glutamate receptors: Two categories• There are two  types of Glutamate Receptors: Ionotropic glutamate  receptors and Metabotropic glutamate receptors.

• Glutamate receptors are neurotransmitters that help communication occur throughout the body

• The basic difference between the two types of glutamate receptors is that the ionotropic receptor directly affects the cell when activated and the metabotropic receptors indirectly influences the cell when it is activated.

• Glutamate receptors are neurotransmitters that help communication occur throughout the body.

• In my lab metabotropic glutamate receptors were being concentrated in the brain. 

Task/GoalThe task that I was given was to perform immunohistochemistry on brain sections from ovariectomized rats with or without estrogen replacement to stain for expression of the mGluR1 subunit of the metabotropic glutamate receptors.

The main goal was to identify differences in the expression of the mGluR1 subunit of the metabotropic glutamate receptors between the estrogen-treated and non-treated control rats in the dentate gyrus of the hippocampus.

Materials Citrate bufferBSA (bovine serum albumin)NGS (normal goat serum)Triton X-100mGluR1 subunit primary antibodyPBS (phosphate buffered saline)Anti-rabbit secondary antibodyDAB (diaminobenzidine)AB complex (avidin-biotin)Well platesXylene

Procedure Day one:The sections were transferred from the freezer to new well plates and washed with PBS three times for 10 minutes each.

Next, sections were incubated for 30 minutes at 80 ﾟC in the citrate buffer and then cooled down at room temperature for 10 minutes.

The sections were then washed three times with PBS for 10 minutes.

Next the blocking solution is added to the sections (consists of BSA, NGS, triton X-100 in PBS) for 90 minutes.

Finally the sections were placed in the primary antibody diluted in the blocking solution and left on a shaker at 40C for two days.

Procedure (continued)Day Two:Sections were washed with PBS three times for ten minutes.

Then the sections were incubated with the secondary antibody diluted in the blocking solution.

Next, three washes in PBS for 10 minutes.Incubation in the AB complex for one hour.Washing with PBS three times for 10 minutes.Using DAB to visualized the staining for 6 minutes.

Washing with PBS three times for 10 minutesFinally the sections are mounted on gelatinized slides and examined under the microscope for staining.

Difficulties The sections were very delicate and easily damaged, so it was necessary to be very careful.

It is very important to keep the sections in a solution at all times because the sections dry out in a matter of half a minute. If the sections are dry then they will not have positive staining.

The timing for the incubation with DAB is critical to maintain same conditions for each staining in order to quantify the data.

Wanted ResultsWe wanted for all the sections to be stained and show a distinct difference between the slides from oil-injected controls and estrogen-treated animals.

ResultsIn four animals, the staining showed characteristic pattern of mGluR1 subunit expression, which was identified within the fibers.

There were differences in the expression of the mGluR1 subunit for metabotropic glutamate receptors between the estrogen-replaced and oil-injected ovariectomized rats: Estrogen-treated rats showed a prominent expression of the mGluR1 subunit in the dentate gyrus of the hippocampus, while oil-injected controls had just a weak staining.

DifficultiesThe staining in some animals did not work and I had to repeat the procedure in additional sections from the same animals.

Another problem was a non-specific staining in some slides from 4 animals. This problem needs to be more explored by staining additional sections either from the same animals or to prepare new animals and to use fresh tissue instead of frozen stored sections.

Conclusion The good stained sections are going to be used in my mentor’s project.

More staining needs to be performed for final conclusions regarding the differences in the expression of the mGluR1 subunit for metabotropic glutamate receptors between the estrogen- and oil-injected rats.

Preliminarly we conclude that loss of estrogen by ovariectomy leads to downregulation of the mGluR1 subunit of the metabotropic glutamate receptors in the dentate gyrus of the hippocampus, a structure important for learning and memory.

Future Studies In the future, next year, I want to continue my research in neurology with my mentor in the Bronx.

I also want to continue working with experiments dealing with immunohistochemistry.

References Boeree, C. George. “Neurotransmitters.” 2003,2009. Shippensburg University. August 2009

Devinsky, Orrin. “Epilepsy Patient and Family Guide,” Second Edition. Philadelphia: F. A. Davis Company, 2002.

Johnson, Steven. “Mind Wide Open: Your Brain and The neuroscience of Everyday Life.” New York: Scribner, 2004.

Klein, Bradley. “NEUROTRANSMITTER RECEPTION.” Virginia –Maryland Regional College of Veterinary Medicine. August 2009

Swanson, Chad J., Mark Bures, Michael P. Johnson, Anni-Maija Linden, James A. Monn & Darryle D. Schoepp. “Metabotropic glutamate receptors as novel targets for anxiety and stress disorders” Nature Reviews Drug Discovery. Feburary 2005.

Turkington, Carol. “The Encyclopedia of The Brain and Brain Disorders,” Second Edition. New York: Facts on File, inc., 2002.

Velísková, J. and Velísek, L. “ß-Estradiol Increases Dentate Gyrus Inhibition in Female Rats via Augmentation of Hilar Neuropeptide Y.” (The Journal of Neuroscience, May 30, 2007)

AcknowledgementsDr. Sat BhattacharyaDr. Jana VeliskovaDr. Libor VelisekMs. Zunju HuFriends and FamilyHarlem Children SocietyThank you to all of you