I am creating this page for my own learning about the different stains used by the Smith Lab for Array Tomography. Each is a protein marker that is reflective of structure and function of the local environment, and so understanding the salience of each one is important! PICTURES TO COME.
presynaptic marker “…highly concentrated at presynaptic boutons and could be useful as general markers for synapses.”
Bright stained puncta in the nuclei that correspond to electron dense heterochromatin masses, good for registration!
GABAergic synapse marker “GAD puncta, expected to label inhibitory GABAergic synapses, overlap with a small subset of synapsin puncta, and synapsin levels are generally lower in these synapses.”
(gephyrin?) GABAergic synapse marker
glutamatergic synapse marker
glutamatergic synapse marker “There is great variability in the expression levels, subunit composition, and localization of AMPA and NMDA receptors at synapses, which significantly affects their functional properties (e.g., Craig and Boudin, 2001).”
glutamatergic synapse marker “…apposed with synapsin” “immunofluorescence revealed PSD-95 puncta within spine heads that are closely associated with both synapsin and bassoon puncta…”
presynaptic marker
Exceptions …“the list of possible exceptions to this rule has grown recently and now includes ribbon synapses in the retina (Mandell et al., 1990), reticulogeniculate synapses (Kielland et al., 2006), and some GABAergic and VGluT2-containing synapses in the cerebral cortex (Bragina et al., 2007).
presynaptic marker (synaptophysin?) “…highly concentrated at presynaptic boutons and could be useful as general markers for synapses” This is saying why Synph isn’t as good a synapse marker as synapsin: “synaptophysin immunoreactivity is also fairly often detectable at obviously extrasynaptic sites, e.g., in cell body and dendritic cytoplasm and nuclei (Figure 3A). Synaptophysin puncta moreover tend to be smaller and less continuous than synapsin puncta.”
I think that tubilin is another neuron marker, and is expressed by pyramidal neurons that do not express YPF. “…apical dendrites of pyramidal cells not expressing YFP are evident from tubulin immunostaining of their core micro- tubule bundles.”
GABAergic synapse marker “…has been shown to specifically localize to presynaptic boutons (Chaudhry et al., 1998).”
glutamatergic synapse marker “…known to be present in most cortical glutamatergic synapses” “vesicular glutamate transporters VGluT1 and VGluT2… expression reportedly varies depending on the intracortical or subcortical origins of the synapses.” (see VGlut2, below)
glutamatergic synapse marker (continued from VGluT1) “In particular it is believed that VGluT2 is predominantly expressed in thalamocortical synapses”
My sense is that YPF is a good marker for a neuron. However not all neurons (pyramidal) express YPF, in which case we can use tubulin. “YFP fluorescence in the cortex of line H mouse represents a soluble YFP marker transgenically expressed in a large subset of layer 5 pyramidal neurons” “YFP fluorescence is not necessary for the subsequent single synapse analysis, which can be performed also in a wild-type mouse”
“As can be seen in the correlation matrix, both synapsin and synaptophysin, and to a lesser extent bassoon, colocalize with all other synaptic markers, including those of smaller subsets of synapses that contain VGluT2 or GAD. All synaptic markers are anticorrelated with tubulin, which labels microtubules within dendrites and cell bodies. VGluT1 and VGluT2, found in cortical glutamatergic synapses, do not colocalize with the GABAergic markers. PSD-95 and GluR2, both present at the postsynaptic side of glutamatergic synapses, correlate strongly with each other and more weakly with the presynaptic glutamatergic markers. GAD and VGAT, presynaptic markers for GABAergic synapses, show strong correlation. An interesting distinc- tion can be made between the presyn- aptic markers with respect to their coloc- alization with postsynaptic markers. Presynaptic markers that are associated with synaptic vesicles (e.g., synapsin, synaptophysin, VGluTs) show high coloc- alization among themselves, while their colocalization with postsynaptic markers such as PSD-95 and GluR2 is weaker. On the other hand, the presynaptic marker bassoon, which labels the presynaptic active zone, shows similar colocalization with both pre- and postsynaptic markers. This is due to the fact that the synaptic vesicle cluster is situated far enough from the postsynaptic density to be resolved by AT. On the other hand, the presynaptic active zone is only one synaptic cleft (around 20 nm) away from the postsyn- aptic density which is below the resolution capabilities of AT.”
| Please note that quoted material is from: [[http://www.cell.com/neuron/abstract/S0896-6273(10)00766-X | cite]]. I do not want to plagiarize! |