Figure 1. Distinct cytoarchitecture of cortical layer 1 and layer 2/3.

A, Representative in vivo images of layer 1 (L1) and layer 2/3 (L2/3). The Ca²⁺ indicator Oregon Green 488 BAPTA-1 (OGB-1, green) stained both neurons and astrocytes while Sulforhodamine 101 (SR101, red) stained astrocytes. In L1, most of the cells were astrocytes, indicated by loading with OGB-1 and SR101 (red–yellow). In L2/3, neurons (SR101-negative) outnumber astrocytes (SR101-positive). Time course of Ca²⁺ transients of the numbered astrocytes (1–6) in L1 image is presented in Figure 2A (for L2/3, see Figure S1). Movies of Ca²⁺ transients for L1 and L2/3 are shown in Video S1 and S2. B, The packing densities of astrocytes and neurons are plotted against the depth from cortical pia. L1 (blue circle) and L2/3 (red triangle) were determined by the physical depth and appearance of astrocyte-neuron ratio. Counts of astrocyte and neuron were 19.8±5.6 and 8.9±4.5 in L1 (n = 38 imaging sites), 12.6±4.8 and 63.0±17.1 in L2/3 (n = 34) per imaged area (∼320×320 µm²) (mean±SD in all figures, otherwise noted). While L1 has more astrocytes than layer 2/3, neurons dominated in L2/3 (***p<0.001, Student's t test). C, Immunohistochemical staining for S100β (astrocyte marker, red) and NeuN (neuron marker, green) of a coronal cortical section. Glial dominance in L1 and neuronal dominance in L2/3 is evident. Scale bar: A, 100 µm; C, 200 µm.