At the cell-type level, bulk labeling of VPM boutons in combination with in vitro-labeled dendrite morphologies was used previously to estimate the number and 1D innervation profile of VPM synapses for L2, L3, L4ss/sp, L5st/tt, and L6 neurons.
Experimentally however, current techniques for bulk labeling of cells in vitro often affect neuronal function creating a barrier for exploring structure-function questions.
We selected relatively larger crystals (up to ≈60 μm) for DiI placements into the cerebral peduncle and ventrobasal complex to achieve bulk labeling.
One possibility is that the layer 5 arborizations are altered in a more subtle manner and that we are unable to detect the effects using bulk labeling techniques.
Specifically, we examined Ca2+ transients in these cells, as assessed by changes in fluorescence in cells bulk labelled with the Ca2+ dye, OGB1.
Bulk labeling of TC axons using cytoplasmic- or bouton-specific GFP shows that VPM and POm projections establish vertical axon bundles in the vibrissal cortex.
Here, the superposition of individual VPM neurons resembled the innervation pattern obtained by bulk labeling (Meyer, Wimmer, Hemberger, et al. 2010).
(4) The ideal labeling method would not only be suited to bulk labeling of populations of neurons, but importantly also provide intense labeling of sparse populations of neurons, or even single neurons.
Furthermore, the previously estimated VPM bouton density in L4 and the rank order of VPM innervation of excitatory cell types was in good agreement to the bulk labeling data (Meyer, Wimmer, Hemberger, et al. 2010).
Perhaps not surprisingly, the spread of the vsd signal after stimulation of a single whisker bears remarkable similarity to the population axonal projection pattern observed after bulk labeling of layer 2/3 neurons (Miller et al. 2001; Petersen et al. 2003).
This is, because to date the most successful reconstructions of complete neuronal morphology require sparse or single-cell labeling since the axons of bulk labeled neurons become indistinguishable in the densely packed neuropil unless resolved with electron microscopy (da Costa and Martin 2013; Helmstaedter 2013).
