2010kolluru_saroja.doc

Sai Saroja KolluruDanbury High School

The Quantities of Glutamate Receptors in the Normal Human Brain


Abstract

The purpose of this project is to determine the amount of glutamate (mgu1) receptors in a normal human brain, then predicting the amount of mGlu1 receptors in a Multiple Sclerosis (MS) brain. Glutamate receptors, one of the most abundant amino acids in the human brain, are the causing agents of many diseases such as depression, anxiety and in the case of this experiment, multiple sclerosis. The methodology that was used involved three major steps: preparing a C-11 element, the cutting of different brain sections and finally using quantitative receptor autoradiography to determine the results. The cerebellum section had the highest amount of mGlu1 receptors where the cortex had the least amount. The cerebellum is one of the most important sections in the human brain that is greatly impacted by Multiple Sclerosis as well as other psychological disorders allowing it to have more glutamate receptors than the other sections in the brain. Whereas the cortex as well as the other parts are not as severely affected by MS; therefore, these sections contain less glutamate receptors. This project was conducted at Columbia University and further studies will include the same procedure on a post mortem MS brain.


Introduction

What are the possible quantities of Glutamate-1 receptor (mGlu1) concentrations, in a human postmortem brain section of multiple sclerosis (MS) when compared to a normal human brain?

Neuroscientists, neurosurgeons, universities and pharmaceutical companies spend millions of dollars and hours every year trying to find cures to diseases such as anxiety, depression and multiple sclerosis. This project focuses on multiple sclerosis, an autoimmune condition in which the immune system attacks the nervous system. MS affects the ability of nerve cells in the brain and spinal cord to communicate with each other. Glutamate receptors are causing agents of many disorders. If scientists found a way to reduce the amount of glutamate receptors or try to find which part of the brain has the most amount, then perhaps they can save a lot of time and money creating medications because they play such a vital role in psychological disorders. This entire project takes about 18 months to conduct. The first part of this project was done in the course of five months. This part includes determining the amount of glutamate receptors in the normal (no disease) human brain. The four sections of the brain that were tested include cerebellum, cortex, hippocampus and caudate pautamen. After determining the amount of receptors in a normal human brain using qualitative data, the possible quantities of glutamate receptors is then going to be predicted in a multiple sclerosis brain.

Glutamate is the most abundant Excitatory Amino Acid (EAA) in the mammalian brain and is involved in several pathological and physiological conditions (Ratey and Hagerman, 2008). The functions of glutamate are mainly attributed to two classes of receptors, ion tropic glutamate and metabotropic glutamate (mGlu1) receptors. Ion tropic receptors are coupled with ion channels and mediate fast synaptic transmission required for neuronal plasticity (Dennis, Clark, 2004). Metabotropic receptors are G-protein coupled receptors (GPCR) involved with pain perception and are potential therapeutic targets for multiple diseases due to their ability to modulate the excitatory glutamate transmission and postsynaptic signaling (Turkington, 1996). Among the eight subtypes of glutamate receptors, mGlu1 are the most important and are implicated in neuron protection, pain, multiple sclerosis, motor dysfunction, epilepsy, cerebral ischemia and cerebella long term depression (LTD). There are currently about 250,000 to 350,000 people in the United States who have been diagnosed with multiple sclerosis. However, the physiology of this disease is not well-studied. Therefore, obtaining data on the amount of glutamate receptors in MS brain is significant towards understanding the changes of the receptor concentration in disease and normal state for accurate diagnosis and development of novel target specific medications. The up regulation of glutamate receptors in MS tissue may protect the neurons from chronic toxic injury.

The proposed hypothesis is that the normal brain will have less glutamate receptor concentrations than an infected multiple sclerosis brain. In the human brain mGlu1 receptors are distributed heterogeneously. Since the cerebellum is the one of the most important sections of the brain it will probably be the most concentrated with glutamate receptors. So even in multiple sclerosis brain sections, most likely the highest amount of glutamate receptors will also be in the cerebellum. Cerebellum is associated with cognitive functions and hence mGlu1 receptors that are abundant in cerebellum have a significant impact on the physiology of cognitive disorders. The high concentration of mGlu1 receptors in the brain would allow its quantification using


radio gland binding studies. The cortex will have the lowest amount of glutamate receptors in a normal and MS brain because it does not play a very large role in physiological disorders.


Materials and Methods

[C-11] ligand; 20 mCi; Developed in Radio gland Laboratories of Columbia University Medical Center, Cost: ($500.00 per production)

Tri buffer: 1L, Cost: ($20, Freshly prepared in the laboratories of New York State Psychiatric Institute)

Brain Sections: No living subjects in the study. Postmortem tissue from MH62185 Conte Center for the Neuroscience of Mental Disorder, Brain Bank Collection at the NYSPI will be used.

Phosphor images, Vendor: Packard; Cost per experiment: ($ 1000.00 per study.)

Safety precautions : All experiments were performed in lead shielded caves to avoid radioactivity contamination. Gloves and goggles were used throughout the experiment to avoid accidental spills of chemicals and biological specimens. All operations were followed by the safety guidelines of Columbia University Medical Center and New York State Psychiatric Institute.

Experimental Procedure:Preparation of [C-11] ligand:

1. Dissolve corresponding desmethyl-precursor (0.5-1.0 mg) in 500 L of acetone in a capped 1 mL V-vial. Add sodium hydroxide (10 L, 5 M) for the resultant solution and allow the solution to stand for two minutes.

2. Approximately 5 minutes at room temperature transport high specific activity [C-11] CH3OTf by a stream of argon (20-30 mL/ min).

3. At the end of the trapping, the product mixture, dilute 0.5 ml of acetone nitrate and directly inject the material into a semi preparative RP-HPLC (Phenomenal C18, 10 x 250 mm, 10 ) and elute with a solution of acetone nitrate: water containing 0.1 M (40:60) at a flow rate of 10 mL/min.

4. Collect the product fraction with retention time between 8-9 minutes based on detector and dilute with 100 mL of deionizer water, passed through a classic C-18 Sep-Pak cartridge and washed with 10 mL water.

5. Reconstruct the product 1 mL of absolute ethanol will afford [C-11] ligand.6. Analyze a portion of the ethanol solution by RP-HPLC (Phenomenal, Prodigy ODS (3)

4.6 250 mm, 5; mobile phase: acetic nitrate: 0.1 M AMF solution (40:60), flow rate: 2 mL/min, retention time: 6 min, wavelength: 254 nm) to determine the specific activity and purities. Also analyze a solution of [C-11] ligand in 10% ethanol-saline by RP-HPLC to confirm the purities and specific activity.

7. Transfer the Required amount of [C-11] ligand after the determination of specific activity measurements to autoradiography experiments.

Brain Sections/ Source of Agent: No living subjects are involved in this study.

1. Obtain Human brain tissue at autopsy from MS and age matched control. 2. Assay four sets of hippocampus, frontal cortex, striatum, and cerebellum in every


mm, for mGlu1 (2 slides; total and non-specific binding) by quantitative receptor autoradiography using [C-11] ligand.

Quantitative receptor autoradiography of [C-11] ligand to the mGlu1

Autoradiography of mGlu1 receptors will be performed as follows:

1. Cut brief sections (~ 20m) from the blocks at -15 to -20°C in a cryostat.2. Thaw and mount onto acid-washed gelatin-subbed glass slides, and rapidly dry at (37

°C).3. Store sections for 2 weeks at -70°C before radio gland incubation.4. Dry sections from one matched pair and cover with dental wax and incubate with 5-10

nM of [C-11] ligand in a humidity chamber for 60 minutes.5. Conduct washes in large Plexiglas jars, to ensure the presence of excess buffer to remove nonspecific binding.

6. Determine non-specific binding by using 10 M of a known specific mGlu1 antagonist (all buffers will be isometric to preserve the integrity of the tissue).

7. Optimize washing buffer using 10 M of a known specific mGlu1 antagonist. 8. Dry slides under a stream of cold air and expose to ST-phosphor-imaging screen

(Packard, wrapped in Mylar film) with high- and low-activity [14C] standards (American Radio labeled Chemicals) for 90 minute.

9. Scan screens with a Packard Cyclone phosphor-imaging system and analyze with OptiQuant Acquisition and Analysis software (Packard).


Equipment Used for Experiment

Cyclotron: machine that produces radioactivity [C-11]

The radio labeled MGlu1 ligand is prepared inside this hot cell. It is referred to as “hot” because it is for radioactivity use only.


Preserved Brains

Various Sections


Incubating place: this picture shows the 'lead chamber' where they incubate the slices with radioactivity

Gamma counter: the radioactivity is counted using a gamma counter.

All Pictures were taken by Mrs. Jaya Kumar and Mr. Dileep Kumar, Columbia University.


Results

There were very consistent results throughout the experiment. Three trials were conducted using the multiple sections of the different parts of the brain. There are two different types of labels on the chart. NS stands for non-specific which means that the entire section of that part of the brain was not tested. So the non-specific part stood as the control for this experiment. Total stands for that the entire section was examined when determining the results. As predicted the cerebellum had the highest amount of glutamate receptors and the cortex had the lowest amount of glutamate receptors.


Chart of the different sections of the brain. As shown below, Cerebellum had the highest amount of MGlu1 receptors shown by the green and yellow colors. Whereas the Cortex has the

lowest amount of MGlu1 receptors as shown by the dark purple and blue. The data that was gathered pertains to all three trials.

TRIAL 1

TRIAL 2


TRIAL 3


Conclusions

The question tested in this experiment was: What are the possible quantities of Glutamate-1 receptor (mGlu1) concentrations, in a human postmortem brain section of multiple sclerosis (MS) when compared to a normal human brain? The hypothesis is that in the normal human brain, the cerebellum will have the highest amount of glutamate receptors. Compared to all the sections in the brain, the cerebellum is the most important and is responsible for most of the physiological functions that take place in the brain (Turkington). It has been found that activation of group 1 receptors enhances the excitatory effects of glutamate by modulation of ion channel activity and are positively coupled with phospholipids (Ratey and Hagerman, 2008). This is the main reason why this experiment dealt with only glutamate 1 receptors only. Studies of human postmortem brain samples suggest an up regulation of mGlu1 protein expression in chronic Temporal Lobe Epilepsy (TLE), multiple sclerosis and neuronal tumors patients compared to controls (Murphy, 2005). Glutamate receptors play a big role and are key factors in certain diseases. The cerebellum is also responsible for a lot of diseases that occur such as multiple sclerosis. The data for every trial conducted in this experiment supported the hypothesis. The consistent trend that was seen during all three trials was that the cerebellum had the most glutamate receptors then any of the other sections in the brain. The cortex section of the normal human brain had the lowest amount of glutamate receptors. This data supported the hypothesis.

New York Institute of Psychiatry which is part of Columbia University donated a normal human brain for this project. This brain was then cut into the different sections that were used for this experiment such as: cerebellum, hippocampus, caudate pautamen and cortex. Then these four sections were further cut up into the different slices so they can be used in each of the trials. After a particular part of these sections were then cut for the control part of the experiment. The [C-11] tracer was also built to find the glutamate receptors in the normal human brain and then autoradiography was used to determine the results. This was the methodology that was used during this experiment.

The results produced are very important because they explain which section of the brain contains the most amounts of glutamate receptors. This is very significant, because neuroscientists can find ways to reduce the amount of glutamate receptors in the brain that cause these various diseases instead of spending a lot of money in trying to make medication that cures multiple sclerosis. If there was a way to prevent the growth of these receptors, then perhaps it will be easy for patients suffering from these diseases and doctors/scientists who are trying to stop the growth. Multiple sclerosis and many other diseases are from mGlu1 receptors. These results, to a certain extent will be useful to neurologists, neurosurgeons, pharmaceuticals making the various medications and universities.

The major difficulty during this experiment was determining the control. This particular section of this 18 month project is actually the control experiment because it determined the amount of glutamate receptors in the normal human brain. When the whole project is completed this part will serve as the control to the multiple sclerosis sections of the brain. Another difficulty during this experiment was the use of radioactive materials. There had to be a lot of safety precautions taken into consideration before any of the materials were used. But with the help of mentors and professors at the University, it was very convenient in using the materials.


This project will be completed during the course of another year. The methodology will be used in a multiple sclerosis brain and a study will be conducted on this brain. The amount of glutamate receptors will be determined using a C-11 tracer and this will help patients with MS. Another goal after determining the amount of receptors in an MS brain is to create a tracer that reduces or easily finds glutamate receptors in a diseased brain. Hopefully, by September 2010, this entire project will be completed and a small alternate method of curing or helping with the research of multiple sclerosis as well as glutamate receptors will be found!


Bibliography

Best, & Ben (2009, February 16). Brain Neurotransmitters. Retrieved November 13, 2009,

From http://www.benbest.com/science/anatmind/anatmd10.html

Dennis, & Clark (2008, December 8). Effects of Glutamate Receptors. Retrieved November 1,

2009, from http://stke.sciencemag.org/cgi

Ratey, J. J., & Hagerman, E. (2008). Spark (pp.25-89). New York City : Little Brown Company .

Turkington (2008). The Brain Encyclopedia . New York City : Fact on Files

Wang, S., & Aamodt , S. (2008). Welcome to Your Brain (pp. 54-79). New York City : Bloomsbury USA.

William , & Bunney (2008). Diseases and Glutamate Receptors . Retrieved December 31, 2009,

From http://www.acnp.org/g4/GN401000116/CH114.html

http://www.benbest.com/science/anatmind/anatmd10.html

2010kolluru_saroja.doc

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