Week 9: Methodology And Results

Welcome Back! Last week, I presented on the topic of excitotoxicity and its potential role in eliciting Parkinsonian symptoms. This week, I have made progress on my research paper by finalizing the Methodology and Results sections. 

Here is what I have produced so far:

Methodology

In order to conduct this experiment, I will utilize the patch-clamp process to record the membrane potentials of a living neuron under the effect of 4-Aminopyridine. The machinery I will be using is the Micro Manipulator, Vibratome, and Brain Splice Rig. The Micro Manipulator is used to change the x, y, and z coordinates, and will be used in this experiment to locate a living neuron when the cerebral cortex of the Zebra Finch is tainted with 4-Aminopyridine. The Vibratome is a device that is going to be used to slice the cerebral cortex into splices. Lastly, the brain splice rig will hold the splice into place, allowing me to inject the drug into the neuron. I will monitor any changes in the membrane potentials via a program and note the significance of environmental factors in inducing Parkinson’s disease based on the results. 

First, with the assistance of my professor, we will paralyze the Zebra Finch using isoflurane to extract its brain. To prevent neuron degeneration, the brain will be immersed in the cerebral spinal fluid (CSF). After allowing the brain to rest in the CSF for a suitable period, I will utilize the vibratome to slice the brain and sterilize any bacteria using an autoclave. Subsequently, I will commence the patch-clamp process, meticulously positioning the brain slice on the brain splice rig and puncturing the membrane using microelectrodes. Then, I will use the Micro Manipulator to locate a living neuron; moreover, I will inject the intracellular solution into the cell by using a micropipette to prevent neuron implosion and also pump atmospheric pressure to remove any debris. Once I locate a living neuron, I will first record its action potentials without the drug’s effects. After obtaining a brief recording of its action potentials, I will proceed to inject another living neuron that has not been punctured with 4-Aminopyridine. As the neuron is expected to expire shortly after being injected, I will need to act swiftly to record its membrane potentials. By obtaining recordings of both a neuron unaffected by the drug and a neuron induced with the drug, I will be able to determine the effectiveness of these drugs and environmental factors in inducing excitotoxicity, which can lead to Parkinsonian symptoms.

Results

At the conclusion of the experiment, it was observed that the drug effectively inhibited the majority of the potassium voltage-gated channels, thereby impeding the formation of action potentials and resulting in a brief period of depolarization. A comparative analysis of the first and second images reveals that the absence and presence of the drug had a significant impact on the membrane potentials. Notably, the second image highlights a distinct phase of suppressed depolarization, providing a clear demonstration of the inhibitory effects of such drugs and environmental factors, such as insecticides.  

This image illustrates the action potentials of a living neuron that isn’t under the effect of the drug, 4-Aminopyridine (Figure 1).  

This image illustrates the membrane potentials of the neuron that is under the effect of the drug. The drug temporarily closed most of the potassium-voltage-gated channels, thereby preventing action potentials from forming and allowing for constant depolarization for a short period of time (Figure 2). 

In my next post, I intend to provide the final updates concerning the Discussion/Significance and Bibliography sections of my research paper. See you then!