(A-F) Loss of TrkB results in reduced localization of GAD67 (green; B) and vGAT (green; E) at GABAergic synapses in the IGL of P21 Wnt1∷Cre; TrkB^fl/fl mice compared to control (A, D). (C, F) Quantification of the area ratio of GAD67:vGluT1 expression (C) and vGAT:vGluT1 expression (F) from control and Wnt1∷Cre; TrkB^fl/fl mice. (G-L) Loss of TrkB results in reduced localization of gephyrin (red; H), but not Neuroligin-2 (NL-2) (red; K) in the IGL of Wnt1∷Cre; TrkB^fl/fl mice compared to control (G, J). (I, L) Quantification of the area ratio of gephyrin:vGluT1 expression (I) and Neuroligin-2:vGluT1 expression (L) from control and Wnt1∷Cre; TrkB^fl/fl mice. The area and intensity of vGluT1 expression (blue) were not perturbed in mice lacking TrkB (Fig. S1; Table S1B), so vGluT1 expression was used to normalize the number and area of glomeruli per section. Scale bar=10 μm. (M) Immunoblot analysis of the expression level of synaptic proteins in cerebellar lysates from two P21 Wnt1∷Cre; TrkB^fl/fl mice compared to control. (N-Q) Quantification of the intensity of individual bands for GAD65, GAD67 and two gephyrin isoforms (98 and 95 kDa) from control and Wnt1∷Cre; TrkB^fl/fl mice. (N) The level of GAD65 was reduced in Wnt1∷Cre; TrkB^fl/fl mice (0.23±0.02) compared to control (0.37±0.02; P<0.01). (O) The level of GAD67, however, was not reduced in Wnt1∷Cre; TrkB^fl/fl mice (0.23±0.02) compared to control (0.21±0.01; P=0.460). (P, Q) Similarly, the level of both gephyrin isoforms was comparable between Wnt1∷Cre; TrkB^fl/fl mice (98 kDa: 0.56±0.05; 95 kDa: 0.81±0.07) and control (98 kDa: 0.48±0.05, P=0.222; 95 kDa: 0.76±0.03, P=0.841). Duplicates from 5 animals were analyzed for each condition.

(A, B) Loss of TrkB did not influence the number of excitatory synapses (black arrows) or granule cell plaques (yellow arrows) per glomerulus in P41 Wnt1∷Cre; TrkB^fl/fl mice (B) compared to control (A) (Ai/Aii and Bi/Bii correspond to dotted box in A and B). However, loss of TrkB resulted in reduced number of inhibitory synapses per glomerulus and per Golgi cell ending in Wnt1∷Cre; TrkB^fl/fl mice (red arrows; B) compared to control (A). (C-F) Quantification of the number of excitatory synapses (e.s.; C; Control: 6.37±0.46, Wnt1∷Cre; TrkB^fl/fl: 6.66±0.39, P=0.863), granule cell plaques (D; C.: 2.56±0.15, W.: 2.33±0.17, P=0.386), and inhibitory synapse per glomerulus (i.s.; E; C.: 4.22±0.39, W.: 2.33±0.33, P<0.01) and per Golgi cell ending (F; C.: 2.10±0.14, W.: 1.20±0.11, P<0.001). (G, H) Loss of TrkB in granule cells did not influence the number of excitatory synapses (black arrows) or granule cell plaques (yellow arrows) per glomerulus in mα6∷Cre; TrkB^fl/fl mice (H) compared to control (G) (Gi/Gii and Hi/Hii correspond to dotted box in G and H). However, loss of TrkB resulted in reduced number of inhibitory synapses per glomerulus and per Golgi cell ending in mα6∷Cre; TrkB^fl/fl mice (red arrows; H) compared to control (G). (I-L) Quantification of the number of excitatory synapses (e.s.; I; C.: 6.77±0.51, W.: 6.96±0.49; P=0.666), granule cell plaques (J; C.: 2.37±0.15, W.: 2.59±0.16; P=0.436), and inhibitory synapses per glomerulus (i.e.; K; C.: 4.48±0.38, W.: 2.56±0.49; P<0.01) and per Golgi cell ending (L; C.: 1.83±0.17, W.: 0.93±0.16; P<0.001). Asterisk denotes Golgi cell ending. Scale bar=2 μm.

(A-H) The localization of GAD67 (green; B) and gephyrin (red; F) in the IGL of P21 TrkB^SHC/SHC mice was comparable to control mice (A, E). (C, D, G, H) Quantification of the area ratio of GAD67:vGluT1 (C), gephyrin:vGluT1 expression (G) and the intensity of GAD67 (D), gephyrin expression (H) in control and TrkB^SHC/SHC mice. (I-P) The localization of GAD67 (green; J) and gephyrin (red; N) in the IGL of P21 TrkB^PLC/PLC mice is reduced compared to control (I, M). Wild-type TrkB knock-in mice were used as control (TrkB^WT/WT) (Minichiello et al., 2002). (K, L, O, P) Quantification of the area ratio of GAD67:vGluT1 (K), gephyrin:vGluT1 expression (O) and the intensity of GAD67 (L) and gephyrin expression (P) in control and TrkB^PLC/PLC mice. Scale bar=10 μm.

(A, B) The localization of GAD65 (green) in the IGL is reduced in P18 Contactin-1^−/− mice (B) compared to control (A). (C) Quantification of the area ratio of GAD65:vGluT1 expression in control and Contactin-1^−/− mice. (D, E) Similarly, the localization of GAD67 (green) in the IGL of is reduced in P18 Contactin-1^−/− mice (E) compared to control (D). (F) Quantification of the area ratio of GAD67:vGluT1 expression in control and Contactin-1^−/− mice. (G, H) The localization of gephyrin (Berglund et al.) in the IGL is also reduced in P18 Contactin-1^−/− mice (H) compared to control (G). (I) Quantification of the area ratio of gephyrin:vGluT1 expression in control and Contactin-1^−/− mice. Scale bar=10 μm.

(A) Major neuronal subtypes are organized in different lamina in the cerebellar cortex (+:excitatory input, −:inhibitory inputs). Golgi cells (green; G.c.) provide feed-back inhibition to granule cells (red; gr.c.) in the internal granular layer (IGL) (Eccles et al., 1967). (B) The glomerulus is a complex of synapses in which a mossy fiber ending (blue) forms glutamatergic synapses on the inner surface of the enveloping dendrites of granule cells (Berglund et al.) and the axon termini of Golgi cells (green) form inhibitory synapses on the outer surface of the same dendrites (Jakab and Hamori, 1988). (C-E) vGluT1 expression marks mossy fiber endings at P14 (C), P21 (D), and P90 (E). (F, G) Quantification of the area of vGluT1 expression (F) expressed as sum of pixels per region analyzed, and the intensity of vGluT1 expression (G) expressed as fluorescent units (fl.u.) from P14-P90. (H-J) Gephyrin expression (Berglund et al.), found on granule cell dendrites, surrounds mossy fiber endings (blue) at P14 (H), P21 (I), and P90 (J). (K, L) Quantification of the area (K) and intensity (L) of gephyrin expression in the IGL from P14-P90. (M-O) GAD65 expression (green) marks Golgi axon varicosities which surrounds mossy fiber endings (blue) at P14 (M), P21 (N), and P90 (O). (P, Q) Quantification of the area (P) and intensity (Q) of GAD65 expression. (R-T) Golgi axon varicosities surrounding mossy fiber endings (blue) can also be marked by GAD67 expression (green) at P14 (R), P21 (S), and P90 (T). (U, V) Quantification of the area (U) and intensity (V) of GAD67 expression. Scale bar=10 μm. *Box plots contain lower and upper limits (“whiskers”), 1^stand 3^rdquartile (edges of the box), median (white bar), mean (white dot), and outliers (black dots) of the dataset. From herein, refer to Tables S1-3 for mean values and statistics (Mann-Whitney U Test and Kruskal-Wallist Test).

(A-H) Loss of TrkB in presynaptic Golgi cells resulted in reduced GAD67 localization (green; B), but did not alter gephyrin localization (red; F) in the IGL of P21 mGluR2∷Cre; TrkB^fl/fl mice compared to control (A, E). (C, D, G, H) Quantification of the area ratio of GAD67:vGluT1 (C), gephyrin:vGluT1 expression (G) and the intensity of GAD67 (D), gephyrin expression (H) in control and mGluR2∷Cre; TrkB^fl/fl mice. (I-P) Loss of TrkB in postsynaptic granule cells did not influence GAD67 localization (green; J), but resulted in reduced gephyrin localization (red; N) in the IGL of mα6∷Cre; TrkB^fl/fl mice compared to control (I, M). (K, L, O, P) Quantification of the area ratio of GAD67:vGluT1 (K), gephyrin:vGluT1 expression (O) and the intensity of GAD67 (K), gephyrin expression (P) in control and mα6∷Cre; TrkB^fl/fl mice. Scale bar=10 μm.

(A-I) Homozygous mice carrying TrkB^F616A allele were treated with water or 1NMPP1 from P0 to P28 and analyzed at P28. The localization of GAD65 (green; B), GAD67 (green; E) and gephyrin (red; H) in the IGL is reduced in TrkB^F616A mice treated with 1NMPP1 compared to TrkB^F616A mice treated with water (A, D, G). (C, F, I) Quantification of the area ratio of GAD65:vGluT1 (C), GAD67:vGluT1 (F) and gephyrin:vGluT1 expression (I) in control and 1NMPP1-treated mice. (J-R) Homozygous mice carrying TrkB^F616A allele were treated with water or 1NMPP1 from P30 to P50 and analyzed at P50. The localization of GAD65 (green; K), GAD67 (green; N) and gephyrin (red; Q) is reduced in TrkB^F616A mice treated with 1NMPP1 compared to control (J, M, P). (L, O, R) Quantification of the area ratio of GAD65:vGluT1 (L), GAD67:vGluT1 (O) and gephyrin:vGluT1 expression (R) in control and 1NMPP1-treated mice. Scale bar=10 μm.

(A, B) Loss of TrkB results in reduced localization of Contactin-1 in the IGL of P21 Wnt1∷Cre; TrkB^fl/fl mice (B) compared to control (A). (C, D) Quantification of the area ratio of Contactin-1:vGluT1 (C) and the intensity of Contactin-1 expression (D) expression in control and Wnt1∷Cre; TrkB^fl/fl mice. For (E-T), yellow represents only colocalized Contactin-1 on individual markers in blue (see Materials and Methods). (E-L) Loss of TrkB results in reduced Contactin-1 localization (yellow) on Neurogranin⁺ Golgi cell axons (blue) and YFP⁺ granule cell dendrites (blue) in Wnt1∷Cre; TrkB^fl/fl; Thy1∷YFP mice (F, J) compared to control (E, I). (G, H, K, L) Quantification of the area ratio of colocalized Contactin-1:Neurogranin (colocal.:NG; G), Contactin-1:YFP (colocal.:YFP; K) and the intensity of colocalized Contactin-1 expression (K, L). (M-T) The colocalization of Contactin-1 (yellow) on vGluT⁺ mossy fibers (blue) and on GFAP⁺ astroglia is not perturbed in P21 Wnt1∷Cre; TrkB^fl/fl mice (N, R) compared to control (M, Q). (O, P, S, T) Quantification of the area ratio of colocalized Contactin-1:vGluT1 (colocal.:vGluT1; O), Contactin-1:GFAP (colocal.:GFAP; S) and the intensity of colocalized Contactin-1 expression (P, T). Scale bar=10 μm. (U) Immunoblot analysis shows that the expression of Contactin-1 was not perturbed in lysates from four P21 Wnt1∷Cre; TrkB^fl/fl mice compared to control. (V) Quantification of the intensity of the band for Contactin-1 (135 kDa) normalized to the intensity of tubulin in control (0.94±0.04) and Wnt1∷Cre; TrkB^fl/fl lysates (0.90±0.07; P=0.885).