Supplementary Figure 7: RE⁺ and CB⁺ neurons segregate into two distinct cell populations based on their intrinsic electrophysiological and morphological properties tested in vitro.

(a) Selected electrophysiological properties were compared. Means ± s.e.m. and t-test based p-values are shown for normally distributed data. Medians ± interquartile range and Mann-Whitney-U test based p-values are shown for non-normally distributed data (indicated by black asterisks). All p-values were corrected by the Holm-Bonferroni method. Please note differences in physiological properties when comparing in vitro (this table) and in vivo (Supplementary Table 1) data: RE⁺ neurons measured in vivo had a significantly more depolarized resting membrane potential (t₍₃₃₎ = 5.67, P < 0.0001) and action potential threshold (t(22) = 6.59, P < 0.0001), a smaller AP amplitude (t₍₁₈₎ = -5.75, P = 0.0001) and maximal firing frequency (t₍₄₉₎ = -10.66, P < 0.0001) and saturate at lower injected currents (U = 6, P < 0.0001). Similarly, GABAergic LII neurons were more depolarized (t₍₆₁₎ = -10.78, P < 0.0001), had a lower maximal firing frequency (t_(59.6) = 3.7, P = 0.003) and saturated earlier (t_(54.2) = 10.2, P < 0.0001) in vivo (Supplementary Table 1) than in vitro (n = 49 GAD-EGFP⁺ neurons patched in vitro in LII in 7 mice). (b,c) Representative reconstructions with corresponding in vitro firing patterns of RE⁺ (b) and CB⁺ (c) neurons. Scale bars, 20 mV, 200 ms (firing pattern), 200 μm (reconstruction). (d) k-means clustering suggests that RE⁺ and CB⁺ neurons form two mostly non-overlapping cell clusters. Four selected parameters are plotted. Blue and red dots indicate cell cluster 1 and 2 respectively that correspond largely to CB⁺ und RE⁺ neurons respectively. Calculated centroids are indicated as black crosses. Green arrows indicate cells that were assigned to a cluster that would not have been predicted based on CB/RE expression: 5 CB⁺ neurons and 1 RE⁺ neuron were unexpectedly assigned to cluster 2 (5 red dots with arrow) and cluster 1 (1 blue dot with arrow) respectively. Abbreviations: AP, action potential; AHP, after hyperpolarization.