Horseradish peroxidase as marker for anatomical em

In order to map synaptic connections in large volumes such as the retina, Anderson et al have argued (see here) that molecular profiling needs to be correlated with electron microscope (EM) images. More on their paper later.

For now, here’s an example of how a molecular profiler can be put to good use. Li et al electroporated expression constructs with genes for horseradish peroxidase targeted to the plasma membrane. Horseradish peroxidase emits amplifiable light at a wavelength of 428 nm. These researchers used it as an anatomical label to correlate spatially with the serial section EM of their tadpole neurons.

One of the advantages of horseradish peroxidase is a uniform plasma membrane distribution including mitochondria / vesicles. It also helps identify long axons / dendrites with small diameters. But on the other hand it has to be electroporated while the animal is still alive to have an effect, unlike some other markers.

Here’s a series of EM images of a distal dendritic branch (blue) that synapses with axon terminals (pink) at white arrow heads:

scale bar = 1 micrometer

You can see how the dendrite recedes as you look to the right in the series of images, which the researchers can reconstruct in their model of the microcircuit.

In order to identify synapses, these researchers used two main criteria:

1) At least two serial sections in which docked and clustered synaptic vesicles oppose the plasma membrane of the next neuron.

2) The distance from the intracellular portion of the presynaptic membrane to the cytosol of the postsynaptic membrane should be significantly longer at synaptic sites than non-synaptic sites. Consistent with this, the presynaptic membrane and the synaptic cleft should be much thicker in electron dense material at synaptic sites than non-synaptic sites.

The following chart quantifies the average membrane thickness at synaptic and non-synaptic sites, although these differences are actually slightly less distinct in neurons expressing horseradish peroxidase:

Reference

Li J, Wang Y, Chiu S and Cline HT (2010) Membrane targeted horseradish peroxidase as a marker for correlative fluorescence and electron microscopy studies. Front. Neural Circuits doi:10.3389/neuro.04.006.2010. Link here.