Fig. 4: Molecular synaptic signatures of two populations of layer 5 neurons.

a Relative protein enrichment in L5 IT and PT neurons. Significant proteins (two-sided, unpaired t-test with permutation-based FDR correction at 5%, s0 > 0.1) are labelled according to Cre line (93 proteins in Tlx3-Cre + /IT and 42 proteins in Sim1-Cre + /PT neurons). b Biological Process gene ontology analysis of differentially expressed proteins. Gene ontology analysis was performed using Fisher’s one-tailed test with FDR multiple-comparison correction. c CSPs are annotated to represent the ‘synapse organization’ module on the volcano plot from panel 4a (two-sided, unpaired t-test with permutation-based FDR correction at 5%, s0 > 0.1). d Ion channels and neurotransmitter receptors are annotated to represent the ‘postsynaptic neurotransmitter receptor activity’ module on the volcano plot from panel 4a (two-sided, unpaired t-test with permutation-based FDR correction at 5%, s0 > 0.1). e, g smFISH analysis of candidate genes in L5 IT and PT neurons labelled by Cre-dependent GFP expression in Tlx3-Cre and Sim1-Cre mice, respectively. Cells that have been quantified are indicated by dotted circles. Scale bars 10 μm. f, h Quantification of RNA expression (average number of RNA puncta per cell). N = 5 mice for Flrt3, Kcna1, Kcna6; n = 6 mice for every other mRNA per Cre line. For each mouse, 8 cells are quantified and averaged as one replicate. Boxplots show distribution of single cells, empty dots display the average of each biological replicate (N). The line across the box describes the mean. The lower and upper hinges correspond to the first and third quartiles. The upper whisker extends from the hinge to the largest value no further than 1.5 * inter-quartile range. The lower whisker extends from the hinge to the smallest value at most 1.5 * inter-quartile range of the hinge. Data beyond the end of the whiskers (outliers) are plotted individually as a small, solid dot. Statistical analysis is performed as an unpaired t-test on the replicates, alpha = 0.05, p-value is reported under transcript name. Source data are provided as a Source Data file. IT intratelencephalic, PT pyramidal tract, PN pyramidal neuron, L5 layer 5.