Sunday, August 19, 2012

How to Build a Neuron: Step 1

There are many reasons to try to build a neuron, but fully building a model neuron is an extensive process with many steps.  Today we will discuss the very first step in the neuron-building process: determining the activity and  shape of the neuron.

Biocytin filled cortical neurons (source)
To determine the shape of neuron, you have to stain it somehow.  There are several ways to do this, but we will focus on the biocytin filling method.
To determine the activity of a neuron, you have to use electrophysiology to record its electrical activity. The biocytin filling method makes use of the same patch clamp electrode to record the electrical activity of the neuron and to fill it with the biocytin molecule that can be later dyed.  So this method is perfect for building a neuron because with it you can correlate the shape of the neuron directly with its activity patterns. 

Neural activity correlated with neural morphology (source)

A recent Nature Protocols paper by Marx et al. (2012) provides step by step details for how to fill and dye a neuron using the biocytin method. 

The basic biocytin staining protocol is as follows:

1. make brain slices
2. fill the neuron with biocytin while recording its electrical activity
3. fix the brain slice in paraformaldehyde
4. quench the endogenous peroxidase
5. connect the biocytin to avidin (using the vectastain ABC kit)
6. colorize the avidin (using DAB and nickel)
7. mount the slices on gelatin subbed slides
8. dehydrate the slices SLOWLY through very small steps of ethanol concentration
9. clear with xylene and coverslip

Marx et al. provide some excellent specifics in the paper that make the whole process understandable and more importantly, doable. They even have a troubleshooting section which explains what might have gone wrong under several conditions.

Marx et al., 2012 Figure 2
One of their best tips in the paper is to dehydrate the slices very slowly.  They show that when you dehydrate the tissue quickly, you get a cork-screw artifact (A) that is not physiologically meaningful, but when you dehydrate slowly, you get a more accurate morphology. 

So there you have it, Step 1 of neuron building.  Step 2 will be coming soon, can be found here.
And all the "build a neuron" steps will be indexed here.

© TheCellularScale
Marx M, Günter RH, Hucko W, Radnikow G, & Feldmeyer D (2012). Improved biocytin labeling and neuronal 3D reconstruction. Nature protocols, 7 (2), 394-407 PMID: 22301777

No comments:

Post a Comment