Extended Data Fig. 6: Comparison of corticothalamic projection strengths derived from EGFP and SypEGFP tracer experiments.

a–d, Maximum intensity projections from four experiments within 500 μm of each other targeting VISp (same experiment labelled VISp-3 below) using either EGFP or SypEGFP tracers in the Rbp4-Cre_KL100 (L5) or Ntsr1_Cre_GN220 (L6) line as indicated. a′–d′, Coronal STPT images near the centre of the densest terminal zone in LGd show axon and presynaptic terminal labelling in LGd and other thalamic targets, including the ventral lateral geniculate (LGd, LGv), the intergeniculate leaflet (IGL) and the lateral posterior nucleus (LP). The anterior pretectal nucleus (APN) in the midbrain is also indicated. SypEGFP labelling is more punctate and has less fluorescence in axons and fibre tracts. a′′–d′′, Coronal STPT images near the centre of one of the densest terminal zones in the middle of LP. a′′′–d′′′, Coronal STPT images near the centre of the second densest terminal zone in the anterior part of LP. This image also contains a portion of the terminal zone in the lateral dorsal nucleus (LD). e–h, Directed, weighted, connectivity matrices (11 × 44) showing log₁₀-transformed normalized projection volumes for the Cre lines representing CT projections labelled from layers 5 (e, f) or 6 (g, h) with EGFP or SypEGFP tracer as indicated. True negatives (including passing fibres) at the regional level were masked and coloured dark grey. The colour map is the same as in Fig. 4. The matrix shows relative differences for connections originating from L5 versus L6 (L5 − L6/L5 + L6) for EGFP-based measures (i) and SypEGFP-based measures (j). k, Normalized projection strengths for CT targets (n = 484) were significantly correlated from matched cortical locations between EGFP and SypEGFP tracers for both Cre lines (Spearman r = 0.71, 0.73; P < 0.0001). On average, EGFP CT NPVs were ~0.5 log unit larger than SypEGFP for Rbp4 experiments, but were not different for the Ntsr1 line. l, Normalized projection strengths for CT targets (n = 484) contacted by L5 or L6 cortical neurons in matched injection locations were also significantly correlated for both EGFP and SypEGFP tracers (Spearman r = 0.51, 0.60; P < 0.0001), although more weakly than for the same line between viruses. Specific connections with different fibre to terminal ratios are coloured by source module (light blue, from VISp; orange, from SSp; dark blue, from RSPagl). m, Relative differences in projection strength to LP and LGd are plotted from n = 6 VISp injection experiments (VISp-1 to VISp-6 in matrix rows above) for each Cre line and viral tracer. n, Relative difference ratios calculated for L5 to L6 using EGFP are plotted against those obtained using SypEGFP (n = 484 CT connections, n = 278 above threshold). There is a significant correlation (Spearman r = 0.68, P < 0.0001). Specific connections are coloured by source module (from l) and labelled with the target.