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Synaptic Physiology Methods: Cell Classification

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.

 

Transgenic Classification

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
Double Transgenic    
Pvalb-IRES-Cre;Ai14 Pvalb-TdTomato  
Sst-IRES-Cre;Ai14 Sst-TdTomato  
Nr5a1-Cre;Ai14 Nr5a1-TdTomato  
Tlx3-Cre_PL56;Ai14 Tlx3-TdTomato  
Sim1-Cre_KJ18;Ai14 Sim1-TdTomato  
Triple Transgenic    
Sst-IRES-Cre;Pvalb-T2A-FlpO;Ai193-hyg Sst-EGFP Pvalb-TdTomato
Vip-IRES-Cre;Sst-IRES-FlpO;Ai193-hyg Vip-EGFP Sst-TdTomato
Vip-IRES-Cre;Pvalb-T2A-FlpO;Ai193-hyg Vip-EGFP Pvalb-TdTomato
Sim1-Cre_KJ18;Sst-IRES-FlpO;Ai193-hyg Sim1-EGFP Sst-TdTomato
Sim1-Cre_KJ18;Vip-IRES-FlpO;Ai193-hyg Sim1-EGFP Vip-TdTomato
Sim1-Cre_KJ18;Pvalb-T2A-FlpO;Ai193-hyg Sim1-EGFP Pvalb-TdTomato
Quadruple Transgenic    
Sst-IRES-Cre;Ai140;Pvalb-T2A-FlpO;Ai65F Sst-EGFP Pvalb-TdTomato
Vip-IRES-Cre;Ai140;Sst-IRES-FlpO;Ai65F Vip-EGFP Sst-TdTomato
Vip-IRES-Cre;Ai140;Pvalb-T2A-FlpO;Ai65F Vip-EGFP Pvalb-TdTomato
Nr5a1-Cre;Ai140;Pvalb-T2A-FlpO;Ai65F Nr5a1-EGFP Pvalb-TdTomato
Nr5a1-Cre;Ai140;Sst-IRES-FlpO;Ai65F Nr5a1-EGFP Sst-TdTomato
Nr5a1-Cre;Ai140;Vip-IRES-FlpO;Ai65F Nr5a1-EGFP Vip-TdTomato
Pvalb-IRES-Cre;Ai140;Rorb-T2A-tTA2;Ai63 Pvalb-EGFP Rorb-TdTomato
Tlx3-Cre_PL56;Ai140;Pvalb-T2A-FlpO;Ai65F Tlx3-EGFP Pvalb-TdTomato
Tlx3-Cre_PL56;Ai140;Sst-IRES-FlpO;Ai65F Tlx3-EGFP Sst-TdTomato
Tlx3-Cre_PL56;Ai140;Vip-IRES-FlpO;Ai65F Tlx3-EGFP Vip-TdTomato
Sim1-Cre_KJ18;Snap25-GFP;Pvalb-T2A-FlpO;Ai65F Sim1-EGFP Pvalb-TdTomato
Sim1-Cre_KJ18;Ai139;Sst-IRES-FlpO;Ai65F Sim1-EGFP Sst-TdTomato
Sim1-Cre_KJ18;Snap25-GFP;Vip-IRES-FlpO;Ai65F Sim1-EGFP Vip-TdTomato
Ntsr1-Cre_GN220;Ai140;Pvalb-T2A-FlpO;Ai65F Ntsr1-EGFP Pvalb-TdTomato
Ntsr1-Cre_GN220;Ai140;Sst-IRES-FlpO;Ai65F Ntsr1-EGFP Sst-TdTomato
Ntsr1-Cre_GN220;Ai140;Vip-IRES-FlpO;Ai65F Ntsr1-EGFP Vip-TdTomato
Viral tools    
Tlx3-Cre_PL56;Ai193 + Fam84b-FlpO AAV
Fam84b targets Layer 5 ET cells
Tlx3-EGFP Fam84b-TdTomato

 

 

Morphological Classification

Biocytin filled cells were imaged at 63x magnification to measure morphological features such as spiny-ness, soma shape, and axon and dendritic length. 3525 mouse cells and 583 human cells received morphological analysis.

Morphology annotation Description
Dendrite type  
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
Axon  
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
Apical dendrite  
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

 

Layer Annotation

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 5251 mouse cells and 835 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

Cell Number Target Layer Annotated Cortical Layer
1 5 5
2 5 5
3 5 5
4 5 5
5 5 5
6 5 5
7 5 5
8 5 4