Massive ablation of M/T cells in adult mice induces plasticity of apical dendrite.
A. Schematic representation of the transgenic strategy to ablate M/T cells in adult animals. The Tbx21 promoter is specific for M/T cells. The Tbx-cre transgene controls the expression of the Cre recombinase in M/T cells, enzyme, thus regulating the selective expression in M/T cells of the receptor (iDTR) for diphtheria toxin (DT), from a CAG:loxp stop loxP-iDTR reporter mouse. Upon systemic injection of DT into the bloodstream, DT gets transported into M/T cells expressing iDTR, and selectively kills them.
B. Two possible scenarios are expected after ablating most of the M/T cells in adult animals. (top) M/T cells density reduction does not induce changes in the apical dendrite structure. (bottom) The absence of peer M/T cells induces structural plasticity in remaining M/T cells, whose apical dendrites extend into nearby glomeruli.
C. Quantification of the number of remaining M/T cells (tdTomato (tdT) positive cells) in the OB of adult mice (P120) in different cell ablation experiments. 437.6 ± 119.2 M/T cells remained in Tbx::DTA::Ai9 mice (∼1% of the full set of M/T counted in wt mice; n=8; light red). 1,158 ± 99 M/T cells remained in Tbx::iDTR::Ai9 mice (∼3% compared to wt mice; n=4; light green). 9,819 ± 1544 M/T cells remained in Tbx::iDTR::Ai9 mice when half of the DT concentration (10 μg/Kg of body weight) was injected (∼25% compared to wt; n=3; dark green). Wild type (Tbx::Ai9 ) mice had 38,546.8 ± 2,912.2; n=3; gray). Data are shown as average ± standard deviation.
D-H. Confocal images of M/T cells after sparse labeling using AAV-PHP.eB-DiO-GFP. (C) Tbx (wild type) mice at P120. (D) Tbx::DTA mice at P120. (E) Tbx::iDTR mice at P120 (cell ablation induced at P60). (F) Tbx::iDTR mice at P240 (cell ablation induced at P180). (G) Tbx::iDTR mice at P120 (cell ablation induced at P60 using half of the DT dose). Blue = DAPI staining. Scale bar in H is 50 µm and applies to D-H.
I. Percentage of M/T cells with an apical dendrite innervating one, two, three or more glomeruli for experimental conditions in D-H. In Tbx::DTA ∼35% of M/T cells had an apical dendrite innervating more than one glomerulus, while in control mice (Tbx ) ∼99% M/T cells had an apical dendrite innervating a single glomerulus(Tbx (n=158) and Tbx::DTA (n=149); p-value<0.0001; χ2 test). In Tbx::iDTR mice, when ablation was induced at P60, 48% of M/T cells had an apical dendrite innervating more than one glomerulus (n=188; p-value=<0.0001, compared to Tbx ), but no significant differences were observed when compared to Tbx::DTA (p-value=0.05). No significant differences were observed in Tbx::iDTR mice when comparing cell ablation performed at P60 versus P180 (n=216; p-value=0.15) or P60 versus M/T cells ablation using half-dose of DT at P60 (n=236; p-value=0.05).