Cell subclass was characterized by expression of a transgenic reporter (mouse) as well as by morphological properties (mouse and human). Cells were assigned to a target layer during the experiment followed by an annotated cortical layer during morphological analysis.
Double, triple, and quadruple transgenic mice were used to label subclasses of excitatory and inhibitory cells. In triple and quadruple transgenics, two distinct subclasses could be labeled with different reporters (typically TdTomato and EGFP). See the Transgenic Characterization for a complete description of the transgenic drivers and reporters.
|Transgenic mouse line||Cre-driver||FlpO-driver|
|Tlx3-Cre_PL56;Ai193 + Fam84b-FlpO AAV
Fam84b targets Layer 5 ET cells
Biocytin filled cells were imaged at 63x magnification to measure morphological features such as spiny-ness, soma shape, and axon and dendritic length. More than 3000 mouse cells and 700 human cells received morphological analysis.
|NEI (Not Enough Information)||Either no biocytin filled cell or poor health precludes definition of dendrite type|
|Aspiny||No visible spines, non-pyramidal morphology|
|Sparsely spiny||Visible spines, non-pyramidal morphology|
|Spiny||Visible spines, pyramidal or stellate morphology|
|Truncation||Obvious cut axon or retraction bulb near surface of the slice that measured ≦ 100 µm|
|Borderline||Axon measured between 100-200 µm|
|Intact||Traceable axon measured > 200 µm|
|Unclear||Axon untraceable but not obviously truncated, likely due to poor health or biocytin fill|
|Truncation distance||Measured length of axon from origin to end. Only measured in "truncated" or "borderline" axons|
|Truncated||Does not reach Layer 1|
|Intact||Reaches Layer 1|
|Truncation distance||Measured length of largest apical branch from soma to end of dendrite. Only measured in "truncated" dendrites|
During the experiment we assigned a target layer to each cell as well as recorded the relative position of the cell within the slice. These cell positions were then mapped onto the biocytin and DAPI stained slice. When both annotated cortical layers, and cell positions were labeled on a slice these cells automatically received a "cortical layer" based on where the center of a cell was in relation to layer boundaries. We were able to assign cortical layers to more than 9000 mouse cells and 1400 human cells. Cells could be assigned a cortical layer even in the absence of biocytin as long as the position of the cells was recorded during the experiment.
Cortical layers drawn in cyan with cell positions outlined in pink