Quantitative Autoradiographic Method for Determination of Regional Rates of Cerebral Protein Synthesis In Vivo

R. Michelle Sar&#233;; Anita Torossian; Michael Rosenheck; Tianjian Huang; Carolyn Beebe Smith

doi:10.3791/58503

Quantitative Autoradiographic Method for Determination of Regional Rates of Cerebral Protein Synt...

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Neuroscience

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JoVE Journal Neuroscience

Quantitative Autoradiographic Method for Determination of Regional Rates of Cerebral Protein Synthesis In Vivo

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11:01 min

June 28, 2019

DOI: 10.3791/58503-v

R. Michelle Saré¹, Anita Torossian¹, Michael Rosenheck¹, Tianjian Huang¹, Carolyn Beebe Smith¹

¹Section on Neuroadaptation and Protein Metabolism, National Institute of Mental Health,National Institutes of Health

Overview

This study presents a quantitative autoradiographic method for measuring regional rates of protein synthesis in the brain using L-[1-14C]-leucine. The technique is applied in awake, behaving animals to investigate the brain's adaptive responses during development and neuroplasticity. This approach allows for simultaneous measurements across various brain regions.

Key Study Components

Area of Science

Neuroscience
Protein Synthesis
Methodological Advancements

Background

Protein synthesis is vital for cellular functions and adaptations.
Quantitative measurement in vivo is crucial for understanding brain plasticity.
Existing methods may not provide fully quantitative data.
This study addresses the need for precise measurement techniques.

Purpose of Study

To establish a robust method for determining protein synthesis rates in the brain.
To explore how these rates reflect neuroplastic changes in response to various stimuli.
To facilitate studies on the brain's adaptations during ongoing physiological processes.

Methods Used

The main platform involves quantitative autoradiography and the use of L-[1-14C]-leucine.
The study employs awake, behaving animals for realistic contextual measurements.
It involves surgical procedures to introduce catheters for tracer administration.
Blood samples are collected at various time points to quantify tracer dynamics.
Brain sections are prepared for autoradiography to assess regional synthesis rates.

Main Results

The methodological improvements allow for fully quantitative assessments of protein synthesis.
Initial tests confirm the feasibility of monitoring brain regions simultaneously.
Responses to long-term changes in behavior and development can be accurately tracked.
The findings enable deeper insights into the molecular basis of neuroplasticity.

Conclusions

This study establishes a precise quantitative method for assessing cerebral protein synthesis in vivo.
The approach enhances our understanding of neuroplastic mechanisms and brain adaptation.
Future applications may include fundamental investigations into various neurological conditions.

Frequently Asked Questions

What are the advantages of the quantitative autoradiographic method?

This method allows for fully quantitative measurements in vivo, providing insights into protein synthesis rates across different brain regions simultaneously.

How is the tracer administered in the study?

The tracer is administered intravenously using a Y-connector setup with syringes for both the tracer and saline to ensure accurate delivery and flushing.

What biological outcomes can be derived from this method?

The method provides data on regional protein synthesis rates, which can reflect adaptive changes in the brain associated with development and neuroplasticity.

Are there any limitations to the method?

While the method is robust, it requires careful surgical procedures and handling to minimize stress and ensure accurate measurements during the study.

How can this technique be applied to other studies?

The quantitative autoradiographic method can be adapted for studies investigating various neurological conditions where understanding protein synthesis is vital for exploring disease mechanisms.

What type of data collection is involved in this study?

Timed arterial blood samples are collected at multiple intervals post-tracer administration to quantify tracer clearance and calculate synthesis rates.

Protein synthesis is a critical biological process for cells. In brain, it is required for adaptive changes. Measurement of rates of protein synthesis in the intact brain requires careful methodological considerations. Here we present the L-[1-¹⁴C]-leucine quantitative autoradiographic method for determination of regional rates of cerebral protein synthesis in vivo.

Measurement of regional rates of brain protein synthesis can trace the response of the brain to long term changes such that occur during development and neuroplasticity. Our method has the advantages that measurements are fully quantitative, and they are made in the awake behaving animal. The quantitative autoradiographic technique permits measurements in all brain regions simultaneously.

Demonstrating the procedure will be Anita Torossian, a post-baccalaureate fellow in my laboratory, and Tianjian Huang, our animal surgeon. Begin this procedure with preparation for surgery as detailed in the text protocol. Once on the surgery stage, use surgical scissors to make a one centimeter incision from the upper medial portion of the left thigh rostrally towards the midline revealing the femoral artery and vein.

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