Journal: Journal of Neurochemistry

Article Title: Splice Type‐Specific Effects of Gαo Subunits on Cerebellar Anatomy and Synapse Formation

doi: 10.1111/jnc.70512

Figure Lengend Snippet: Gα o1 and Gα o2 differentially affect the number of VGLUT2 contacts within the cerebellar cortex. (A) Immunohistochemical analysis of the vesicular glutamate transporter 2 (VGLUT2) expression in the adult wild type cerebellum as shown in a midsagittal section. A strong immune signal was detected in the cerebellar glomeruli of the granule cell layer (gcl), together with a distinct punctuate pattern in the molecular layer (ml) and the deep cerebellar nuclei (DCN, see inset for Dapi staining). (B) Double staining for VGLUT2 and Calbindin to visualize Purkinje cell dendrite morphology. In the molecular layer, the VGLUT2 transporter signal characteristically marks synaptic climbing fiber contacts to the proximal to mid‐segmental Purkinje cell dendrites. (C) Regions of the molecular layer depicting VGLUT2 signals in the culmen of wild type and Gα o1 −/− mice used to quantify the VGLUT2 puncta. (D) Exemplary thresholded images of the regions shown in (C) used for analysis in the respective regions of interest (counting area, shown in transparent blue, see insets for magnification). (E) Quantification of the absolute number of VGLUT2 puncta in the counting area (left panel), the density (middle panel), and size (right panel). See higher magnification confocal imaging for details of VGLUT2 signals. (F) Analysis of the effects of Gα o2 knockout on the VGLUT2 staining pattern in the molecular layer of the cerebellum. Exemplary immunohistochemical visualization of VGLUT2 contacts in the central lobule II of wild type and Gα o2 −/− knockout mice. (G) Exemplary thresholded images of the regions used for quantification within the counting area (transparent blue). (H) Quantification of the absolute number of VGLUT2 puncta in the counting area (left panel), the density (middle panel), and size (right panel). See higher magnification confocal imaging for details of VGLUT2 signals. (I) Analysis of the effects of Gα o1/2 (Gα o ) double knockout on the VGLUT2 staining pattern in the molecular layer of the cerebellum. Exemplary immunohistochemical visualization of VGLUT2 contacts in the declive of wild type and Gα o −/− knockout mice. (J) Exemplary thresholded images of the above depicted regions used for quantification within the counting area (transparent blue). (K) Quantification of the absolute number of VGLUT2 puncta in the counting area (left panel), the density (middle panel), and size (right panel). See higher magnification confocal imaging for details of VGLUT2 signals. Bars show means ± SD (individual animal means are depicted), N = 4 animals each WT and knockout, 6–8 sections per animal and genotype. *** p ≤ 0.001.

Article Snippet: Monoclonal antibodies specifically recognizing Gα o2 were from Synaptic Systems (cat. no. 271011, clone 101.4, Western blot) and 201.2 (kind gift from Karsten Spicher, see Spicher et al. , immunofluorescence).

Techniques: Immunohistochemical staining, Expressing, Staining, Double Staining, Imaging, Knock-Out, Double Knockout

Journal: Journal of Neurochemistry

Article Title: Splice Type‐Specific Effects of Gαo Subunits on Cerebellar Anatomy and Synapse Formation

doi: 10.1111/jnc.70512

Figure Lengend Snippet: Gα o1 and Gα o2 differentially affect the number of VGLUT1 contacts within the cerebellar cortex. Immunohistochemical analysis of the vesicular glutamate transporter 1 (VGLUT1) expression in the adult wild type cerebellum as shown in a midsagittal section. A very dense immune signal was detected in the molecular layer. In addition, the cerebellar glomeruli of the granule cell layer (gcl), were marked (see inset for Dapi staining). (B) Double staining for VGLUT1 and Calbindin. In the molecular layer, the VGLUT1 transporter signal forms a dense synaptic carpet sparing the main Purkinje cell dendrite branches. (C) Expression of VGLUT1 in the individual folia. Cerebellar wild type and Gα o1 −/− sections were incubated with a VGLUT1 antibody and protein expression was quantified for the molecular layer of the individual lobules and expressed as brightness values. See representative images. Scale bar 50 μm. Expression levels of VGLUT1 averaged over all folia are given in the bar chart. (D) The same analysis was performed for wild type and Gα o2 −/− cerebella. See representative images. Scale bar 50 μm. Expression levels of VGLUT1 averaged over all folia are given in the bar chart. (E) Expression of VGLUT1 in the individual folia of wild type and Gα o −/− cerebella. See representative images. Scale bar 50 μm. Expression levels of VGLUT1 averaged over all folia are given in the bar chart. (F) Analysis of the effects of Gα o1 knockout on the VGLUT1 staining pattern in the central lobule II of wild type and Gα o1 −/− mice used to quantify individual VGLUT1 puncta. Exemplary thresholded images are shown used for analysis in the respective regions of interest (counting area, shown in transparent blue). (G) Analysis of the effects of Gα o2 knockout on the VGLUT1 staining pattern in the uvula of wild type and Gα o2 −/− mice as shown in (F). (H) Analysis of the effects of Gα o knockout on the VGLUT1 staining pattern in the declive of wild type and Gα o −/− mice as shown in (F) and (G). (B, F–H) Confocal imaging. Data show means ± SD with individual animal means or individual sections depicted in bar charts, N = 3 animals each WT and knockout. 5–6 sections per animal and genotype. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: Monoclonal antibodies specifically recognizing Gα o2 were from Synaptic Systems (cat. no. 271011, clone 101.4, Western blot) and 201.2 (kind gift from Karsten Spicher, see Spicher et al. , immunofluorescence).

Techniques: Immunohistochemical staining, Expressing, Staining, Double Staining, Incubation, Knock-Out, Imaging

Journal: Journal of Neurochemistry

Article Title: Splice Type‐Specific Effects of Gαo Subunits on Cerebellar Anatomy and Synapse Formation

doi: 10.1111/jnc.70512

Figure Lengend Snippet: Gα o1 and Gα o2 differentially affect the cerebellar size. (A) Immunohistochemical analysis of Gα o1 expression in the cerebellum of wild type and Gα o1 −/− mice. Midsagittal cerebellum sections of adult mice of either strain were stained by an antibody preferentially recognizing Gα o1 . In the wild type, Gα o1 showed a very strong reaction predominantly in the molecular layer. Immunostaining against Gα o1 was absent in the knockout. gcl, granule cell layer; ml, molecular layer; pcl, Purkinje cell layer. (B) Analysis of Gα o2 expression. Midsagittal cerebellum sections of adult mice were stained by an antibody recognizing Gα o2 . In the wild type, Gα o2 showed a strong reaction predominantly in the molecular layer. Immunostaining against Gα o2 was absent in the knockout. (C) Analysis of Gα o1/2 (Gα o ) expression. Midsagittal cerebellum sections of adult mice of either strain were stained by an antibody recognizing both Gα o1 and Gα o2 . In the wild type, Gα o showed a very strong reaction predominantly in the molecular layer. Immunostaining against Gα o was absent in the knockout. (D) An exemplary midsagittal DAPI‐stained section of wild type cerebellum with the individual folia labeled. Cranial orientation is to the left. CENT, central lobule; CUL, culmen; DEC, declive; FOTU, fotulum‐tuber‐vermis; NOD, nodulus; PYR, pyramis; UVU, uvula (lobule numbers are given in latin). (E) Comparison of wild type (as shown in D) and Gα o1 −/− cerebellum size. (F) Comparison of wild type and Gα o2 −/− cerebellum size. (G) Comparison of wild type and Gα o −/− cerebellum size. (H) Quantification of overall cerebellar size (given in mm 2 , upper left panel) along with the cranio‐caudal (length) and ventro‐dorsal (height) extension (lower left panel). In addition, the cortical area was calculated for the molecular and granule cell layer (lower right panel). See effects exemplified for CENTIII by Calbindin staining. (I) The same analysis was performed for Gα o2 . See effects exemplified for CENTII by Calbindin staining, (J) Analysis for Gα o . See effects exemplified for CENTII by Calbindin staining. Bars show means ± SD and individual values, N = 4 animals each WT and knockout (except for 3 animals in Gα o2 WT total ML and GCL area analysis), 6–8 sections per animal and genotype. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Article Snippet: Monoclonal antibodies specifically recognizing Gα o2 were from Synaptic Systems (cat. no. 271011, clone 101.4, Western blot) and 201.2 (kind gift from Karsten Spicher, see Spicher et al. , immunofluorescence).

Techniques: Immunohistochemical staining, Expressing, Staining, Immunostaining, Knock-Out, Labeling, Comparison

Journal: Journal of Neurochemistry

Article Title: Splice Type‐Specific Effects of Gαo Subunits on Cerebellar Anatomy and Synapse Formation

doi: 10.1111/jnc.70512

Figure Lengend Snippet: Folia‐specific Gαo subunit expression and determination of Gα o1 and Gα o2 knockout effects on VGLUT2‐positive synaptic contacts within the cerebellar cortex. Displayed are the effects of Gα o1 , Gα o2 , and Gα o knockout on the VGLUT2‐positive synaptic contacts in the molecular layer of the individual folia of the vermis. The same analysis as summarized in Figure for the total molecular layer was conducted for the molecular layer at the level of individual folia. (A) Folia‐specific Gα o1 knockout effects were observed for the absolute number (left panel), density (middle panel) and size of VGLUT2 puncta (right panel). (B) Folia‐specific Gα o2 knockout effects on the same parameters of VGLUT2 contacts as shown in (A). (C) Folia‐specific Gα o2 knockout effects on the same parameters of VGLUT2 contacts as shown in (A). (D) Expression of Gα o1 in the individual folia. Cerebellar wild type sections were incubated with the Gα o1 antibody and protein expression was quantified for the molecular layer of the individual lobules. The knockout is shown for control. See representative images. (E) The same analysis was performed for the expression of Gα o2 . See representative images. (F) Analysis of the combined expression of both isoforms by the Gα o antibody confirmed the results observed with the subtype‐specific antibodies. See representative images. Data show means ± SD, N = 3–4 animals each WT and knockout, 6–8 sections per animal and genotype. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Article Snippet: Monoclonal antibodies specifically recognizing Gα o2 were from Synaptic Systems (cat. no. 271011, clone 101.4, Western blot) and 201.2 (kind gift from Karsten Spicher, see Spicher et al. , immunofluorescence).

Techniques: Expressing, Knock-Out, Incubation, Control

Journal: Journal of Neurochemistry

Article Title: Splice Type‐Specific Effects of Gαo Subunits on Cerebellar Anatomy and Synapse Formation

doi: 10.1111/jnc.70512

Figure Lengend Snippet: Differential effects of single Gα o1 , Gα o2 , and double Gα o1 /Gα o2 knockout on the number and size of VGAT‐positive synaptic contacts in the molecular layer. (A) Immunohistochemical analysis of the vesicular GABA transporter (VGAT) expression in the adult wild type cerebellum as shown in a midsagittal section. A strong immune signal was detected at the Purkinje cell somata, in the cerebellar glomeruli of the granule cell layer (gcl), together with a punctuate pattern in the molecular layer (ml) and the deep cerebellar nuclei (DCN, see inset for Dapi staining). (B) Double staining for Calbindin to visualize Purkinje cell dendrite morphology and VGAT. In the molecular layer, the VGAT signal is detected on all segments of the Purkinje cell dendrites. (C) Analysis of the effects of Gα o1 knockout on the VGAT staining pattern in the molecular layer of the cerebellum. Regions of the molecular layer depicting VGAT signals in the central lobule II of wild type and Gα o1 −/− mice were used to quantify the VGAT puncta. (D) Exemplary thresholded images of the regions are shown used for analysis in the respective regions of interest (counting area, shown in transparent blue, see insets for magnification). (E) Quantification of VGAT puncta in the counting area (left panel), their density (middle panel), and size (right panel). See higher magnification confocal imaging for details of VGAT signals. (F) Analysis of the effects of Gα o2 knockout on the VGAT staining pattern in the molecular layer of the cerebellum. Regions of the molecular layer depicting VGAT signals in the central lobule III of wild type and Gα o2 −/− mice were used to quantify the VGAT puncta. (G) Exemplary thresholded images of the regions are shown used for analysis in the respective regions of interest (counting area, shown in transparent blue, see insets for magnification). (H) Quantification of VGAT puncta in the counting area (left panel), density (middle panel), and size (right panel). See higher magnification confocal imaging for details of VGAT signals. (I) Analysis of the effects of Gα o1 and Gα o2 double knockout on the VGAT staining pattern in the molecular layer of the cerebellum. Regions of the molecular layer depicting VGAT signals in the culmen of wild type and Gα o −/− mice were used to quantify the VGAT puncta. (J) Exemplary thresholded images of the regions are shown used for analysis in the respective regions of interest (counting area, shown in transparent blue, see insets for magnification). (K) Quantification of VGAT puncta in the counting area (left panel), their density (middle panel), and size (right panel). See higher magnification confocal imaging for details of VGAT signals. Bars show means ± SD with individual animal means depicted, N = 3 animals each WT/Gα o1 KO as well as WT/Gα o2 KO, N = 4 animals each WT/Gα o2 KO; 6–8 sections per animal and genotype. *** p ≤ 0.001.

Article Snippet: Monoclonal antibodies specifically recognizing Gα o2 were from Synaptic Systems (cat. no. 271011, clone 101.4, Western blot) and 201.2 (kind gift from Karsten Spicher, see Spicher et al. , immunofluorescence).

Techniques: Knock-Out, Immunohistochemical staining, Expressing, Staining, Double Staining, Imaging, Double Knockout

Journal: Journal of Neurochemistry

Article Title: Splice Type‐Specific Effects of Gαo Subunits on Cerebellar Anatomy and Synapse Formation

doi: 10.1111/jnc.70512

Figure Lengend Snippet: Folia‐specific determination of Gα o1 and Gα o2 knockout effects on VGAT‐positive synaptic contacts within the cerebellar cortex. Displayed are the effects of Gα o1 , Gα o2 , and Gα o knockout on the VGAT‐positive synaptic contacts in the molecular layer of the individual folia of the vermis. (A) Significant reductions appearing in the Gα o1 knockout regarding the absolute number (left panel), density (middle panel), and the size of VGAT puncta (right panel) are indicated. (B) The same analysis was performed for the Gα o2 knockout. Mainly positive significant effects are indicated. (C) Folia‐specific analysis as in (A) and (B) for the Gα o knockout. Significant reductions are indicated. (D) Number of Parvalbumin‐positive cells within the molecular layer of the lobules CENTIII and uvula in wild type and Gα o1 knockout animals. (E) Number of Parvalbumin‐positive cells within the molecular layer of the lobules CENTIII and pyramis in wild type and Gα o2 knockout animals. (F) Number of Parvalbumin‐positive cells within the molecular layer of the lobules CENTIII and culmen in wild type and Gα o knockout animals. See representative images for wild type and Gα o2 −/− cerebella stained with an antibody recognizing Parvalbumin. Asterisks depict cell somata. Data show means ± SD, N = 3 animals each WT/Gα o1 KO as well as WT/Gα o2 KO, N = 4 animals each WT/Gα o2 KO (VGAT analysis). Parvalbumin counting N = 3 animals each condition. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Article Snippet: Monoclonal antibodies specifically recognizing Gα o2 were from Synaptic Systems (cat. no. 271011, clone 101.4, Western blot) and 201.2 (kind gift from Karsten Spicher, see Spicher et al. , immunofluorescence).

Techniques: Knock-Out, Staining

Journal: Journal of Neurochemistry

Article Title: Splice Type‐Specific Effects of Gαo Subunits on Cerebellar Anatomy and Synapse Formation

doi: 10.1111/jnc.70512

Figure Lengend Snippet: Alterations of Purkinje cell morphology by the knockout of Gα o1 , Gα o2 , and the double knockout. (A) Morphometrical analysis of Purkinje cell primary dendrite width in the CENTIII lobule of wild type and Gα o1 −/− mice. Gα o1 knockout effects are displayed as relative cumulative frequency and overall means. White bars in images represent 3 μm. (B) Analysis as shown in (A) performed for primary dendrites in the culmen. Knockout effects are displayed as above. White bars in images represent 3 μm. (C) Analysis of dendrite outgrowth. Regions of interest (ROI) of 180 × 100 μm within the molecular layer of wild type and Gα o1 −/− cerebella were analyzed and the total Calbindin stained area as well as the proportion of the ROI occupied by the staining were quantified. Thresholded images. (D) Analysis as shown above for the knockout of Gα o2 in the CENTIII lobule. White bars in images represent 3 μm. (E) Analysis of effects in the uvula. White bars in images represent 3 μm. (F) Analysis of dendrite outgrowth as performed in (C). Thresholded images. (G) Analysis performed for primary dendrites in the CENTIII lobule of wild type and Gα o knockout mice. (H) Analysis of knockout effects in the declive. (I) Analysis of dendrite outgrowth as performed in (C) and (F). Thresholded images. Data show means ± SD with individual sections depicted in bar charts, N = 3 animals each WT and knockout. 5–6 sections per animal and genotype. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: Monoclonal antibodies specifically recognizing Gα o2 were from Synaptic Systems (cat. no. 271011, clone 101.4, Western blot) and 201.2 (kind gift from Karsten Spicher, see Spicher et al. , immunofluorescence).

Techniques: Knock-Out, Double Knockout, Staining