The Electrophysiological Studies of Extrasynaptic Receptors

Prepared by: 1.Muhammad Neqmat Siddiq b. Thal’hah Supervised by: 1.Dr. Tarmizi Che Has INTRODUCTION

The study of electrical current in neuron cell or tissue.

The measurement of electrical Electrophysiology current through the opening and closing of ion channel.

Involves the voltage-calmp machine OBJECTIVES

• To comprehend :

The role of NMDA in Long Term Potentiation (LTP)

To express

The GABA(A)Receptor Epsilon subunit through molecular technique LIGAND-GATED ION CHANNEL

AMPA GLUTAMATE NMDA N-methyl-D-aspartate

Ionotropic receptor /D- Glutamate Highly permeable to Ca2+

Helps in Long Term Potentiation Glutamate Extrasynaptic current Glycine –tonic current /D-serine Agonist – Glutamate & Glycine/ D-serine Negative modulator - Conantokin Blocker- AP5 Positive modulator- CDK5 GABA (A) RECEPTOR

• Gamma-aminobutyric acid (GABA) • Two types of GABA receptors : GABA(A) and GABA(B) • Inhibitory receptor • 19 Subunits • Pentameric • Selectively permeable to CI • Rich in pharmacology GABA (A) Receptor Epsilon (ɛ) Subunit

• Epsilon • Insensitive to benzodiazepines • Produce smaller GABA current amplitude • Restricted to amygdala, thalamus, and subthalamic • The latest GABA(A) subunit to be discovered • Is believed to play a role in learning and memory as well as lymbic system. Methodology -NMDA study

Brain Slice 1) Preparation Solutions / drugs

Electrodes

2) Giga Seal Pick the best pyramidal cell

3)Current analysis Methodology of DNA & RNA Preparation

Lysogeny Broth (LB)

1)Preparation Terrific Broth (TB)

Agar plate- Ampicillin(Antibiotic)

2)Cloning of bacteria

3)Single Colony

4)Overnight Culture Methods of DNA & RNA Preparation

Resuspension Buffer

Lysis Buffer 5)Miniprep Kit Neutralization Buffer

Wash Buffer

6)Linearized plasmid of SmaI /RT/Overnight DNA

Mastermix : Restriction Enzyme(SmaI) = 2µl Cutsmart Buffer = 1µl DNA(Epsilon) = 7µl Total =10µl Methodology of DNA & RNA Preparation

DNA Wash Buffer

7)Clean up Kit DNA Elution Buffer

RNA Transcription 8)RNA Preparation

RNA Precipitation Result - NMDA study

Microelectrode

Glass micropipette under high magnification Result NMDA Study Cont.

Current Produced

Response Result ( GABAA Rs ) Study Colony of bacteria DNA(GABAA R ɛ subunit)

Nanodrop machine Gel Electrophoresis Result (Linearized Plasmid)

Gel Electrophoresis RNA(GABAA R ɛ subunit) Discussion

Supernatant produce better colony • Before spreading the bacteria on the agar, Ms. Fatin demonstrated the step for us by using supernatant instead of the pellet. • After incubating the bacteria overnight, the supernatant produce better and clearer colony of bacteria than the pellet ones. • Because of that, we repeated the experiment to prove the new theory of supernatant, which is the lower the concentration, the better the colony. • So, we decreased the volume of bacteria (-50%) used to spread on agar. Instead of 50µl, we used 25µl of bacteria. • We compare the colony of bacteria produced by using supernatant and it was better from previous ones. Hypothesis accepted! Safety Precautions

GABAA study NMDA study • Sterile () • Shrinking of the neuron cell • Pipetting techniques • Choosing the best cell • Heat shock (time) • The condition of the rats (5- • RT PCR (temperature) 7 weeks old male) • Labelling • No air bubbles in • Adjust the speed of microelectrode Centrifuge according to • Careful when handling the protocol controller • Talk when necessary • To achieve the best Giga Seal • Spread the bacteria under • The condition of the the flame machine • Time constraint( 5-6 hrs) What I have gained

Science is a wide I had increased field which Don’t limit science to doctors and my soft skills in encompasses engineers only, be open-minded as the study of science creates job that doesn’t exist yet. terms of self- nature and living confidence etc things.

Life is full of challenges, I know what I want to be in the I must choose sometimes what we plan will future, find the job that suits the right career me well path not happen, so be ready for everything

Follow the If the experiment is failed, I don’t To put passion in rules and give up easily. Do everything will everything I did protocols all my best. Acknowledgement

• I would express my highest gratitude to: • 1. Prof. Dato’ Dr. Jafri Malin Abdullah • 2. Dr. Tarmizi Che Has (My supervisor) • 3.Dr. Farizan Ahmad • To my supervisor specifically, Dr. Tarmizi Che Has, for teaching and introducing electrophysiology to me, sharing his own experience as a lecturer, providing a convenient environment for me to study, finding lecturers to give talks to the VRP students and above all, to accept me as his student. • To all the lecturers who motivate me in their talks: encouraging to study overseas, to choose the right job in the future which suits me well. • To all the staffs (Ms. Nurul, Mrs. Aza, Mrs. Mazira, who help me adapting myself here, sharing their experience and help me in choosing my career. • To Ms. Fatin, the graduate tutor, for guiding me while doing research, showing me the right technique when using laboratory's apparatus. To the future VRP students

• Grab the chance • Submit your resume to join this wonderful programme to explore the world of science. • Come with the right intention • Have the right mind set which is to gain knowledge from all the talks and research activities. • Get to know with everyone • Make new friends to gain a lot of experience.(Including staffs, lecturers and students) • Enjoy doing research • Put your passion while conducting your project. Feel the heat! • Apply the knowledge in daily life