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Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient
Published on: September 17, 2011
Preparation of synaptoneurosomes from mouse cortex using a discontinuous percoll-sucrose density gradient
Pamela R Westmark1, Cara J Westmark, Athavi Jeevananthan
1Department of Pathology and Laboratory Medicine, Waisman Center for Developmental Disabilities, University of Wisconsin, USA.
Journal of Visualized Experiments : Jove
|September 28, 2011
Summary
Synaptoneurosomes (SNs), crucial for studying synaptic transmission, can be effectively isolated using a gentle Percoll-sucrose gradient method. This technique yields active SNs rapidly, avoiding damage common with other isolation procedures.
Area of Science:
- Neuroscience
- Cell Biology
- Biochemistry
Background:
- Synaptoneurosomes (SNs) are isolated nerve terminals crucial for studying synaptic transmission.
- They retain pre- and postsynaptic characteristics, enabling analysis of neurotransmitter uptake, storage, and release.
- Current isolation methods using Ficoll or filtration/centrifugation can be problematic, leading to cytotoxicity or mechanical damage, reducing SN activity.
Purpose of the Study:
- To present an improved method for isolating active synaptoneurosomes.
- To overcome the limitations of existing techniques that compromise SN integrity and function.
- To establish a rapid and gentle protocol for obtaining high yields of translationally active SNs.
Main Methods:
- Homogenization and fractionation of mouse brain cortex.
- Isolation of synaptoneurosomes using discontinuous Percoll-sucrose density gradients.
- Comparison with traditional methods involving Ficoll or filtration/centrifugation.
Main Results:
- The Percoll-sucrose gradient method provides a rapid isolation of synaptoneurosomes.
- This technique yields translationally active SNs with good efficiency.
- The method is gentle, employing isotonic conditions and minimizing mechanical damage during centrifugation.
Conclusions:
- Discontinuous Percoll-sucrose density gradients offer an effective and superior method for isolating active synaptoneurosomes.
- This approach enhances the study of synaptic transmission by providing intact and functional SNs.
- The protocol is advantageous due to its speed, gentleness, and high yield of viable synaptoneurosomes.
