Journal: Scientific Reports

Article Title: Molecular layer interneurons shape the spike activity of cerebellar Purkinje cells

doi: 10.1038/s41598-018-38264-1

Figure Lengend Snippet: Conditional deletion of Vgat in molecular layer interneurons does not lead to gross cerebellar changes in cellular composition, cellular distribution, or layer patterning. ( a–x ) Cerebellar cell types were present and appeared unchanged in location and morphology despite the lack of VGAT in basket cells and stellate cells. Dotted lines indicate the borders of the Purkinje cell layer (PCL) with the molecular layer (ML) and the granular layer (GL). Scale = 20 μm. Control: N = 14, n ≥ 42; basket cell condition: N = 4, n ≥ 12; stellate cell condition: N = 6, n ≥ 18. ( a–c ) CAR8 and IP3R1. ( d–f ) GABAαR6. ( g–i ) NFH. ( j–l ) Neurogranin. ( m–o ) VGLUT1. ( p–r ) VGLUT2. ( s–u ) Calretinin. ( v–x ) CART.

Article Snippet: The CART signal was amplified using a biotinylated secondary antibody (Vectastain Elite ABC method; Vector Labs; Burlingame, CA, USA) and used to visualize climbing fibers mainly in lobules IX and X . Purkinje cells were marked with anti-calbindin (1:1,000; Cat. # 300; Swant, Marly, Switzerland), rabbit polyclonal anti-carbonic anhydrase or CAR8 (CAVIII, 1:1000 l; Cat. # sc-67330, Santa Cruz Biotechnology), goat polyclonal anti-IP3R1 (1:500; Cat. # sc-6093, Santa Cruz Biotechnology, Dallas, TX, USA), goat polyclonal anti-RORα (1:250; Cat. # sc-6062, Santa Cruz Biotechnology, Dallas, TX, USA), and mouse monoclonal anti-ankyrin-G (1:200; Cat. # MABN466, clone N106/36, Millipore Sigma, Burlington, MA, USA).

Techniques:

Journal: bioRxiv

Article Title: Molecular layer interneurons shape the spike activity of cerebellar Purkinje cells

doi: 10.1101/376517

Figure Lengend Snippet: ( A-B ) Example images of sagittal cerebellar sections used for quantification of ML thickness wherein Purkinje cells were stained with either calbindin or CAR8 and all neurons were stained with either nissl or DAPI to facilitate visibility of the Purkinje cell layer (PCL) compared to the molecular layer (ML) and granule layer (GL). Scale = 50μm. ML thickness was unchanged in the stellate silencing condition ( A ) or the basket cell silencing condition ( B ). ( C ) Quantification of ML thickness in all conditions. ML thickness is not significantly changed from control in either basket cell or stellate cell mutant animals (basket cell control mean = 179.9μm ± 3.833, basket cell mutant mean = 181.1μm ± 3.164, P = 0.8200; stellate cell control mean = 159.7μm ± 9.201, stellate cell mutant mean = 157.2μm ± 5.493, P = 0.8288). ( D-G ) TEM images revealed normal synapses in all conditions. Purkinje cells and processes are colorized in magenta and identified basket and stellate synaptic terminals are colorized in green. Scale = 200nm. Inhibitory synapses onto Purkinje cell dendrites in the ML were unchanged from the control ( D ) in stellate cell mutant mice ( E ). Similarly, inhibitory synapses onto Purkinje cell somas were unchanged from the control ( F ) in basket cell mutant mice ( G ). ( H-M ) Gephyrin expression was unchanged in stellate cell mutant mice compared to control. Scale = 20μm. Control mice ( H-J ) have uniform expression of VGAT in the ML ( H ) and similarly uniform expression of gephyrin at inhibitory synapses in the ML ( I ). Triple staining reveals gephyrin is present at inhibitory synapses in the ML. Example triple labeled synapses (arrowhead) are shown in the blowup ( J ). Stellate cell mutant mice do not have uniform expression of VGAT in the ML as a result of the targeted deletion of VGAT ( K ). However, gephyrin appears uniformly expressed (L) suggesting it is present at synapses as normal, despite the depletion of VGAT ( M ). ( N-S ) Postsynaptic structures are also unchanged in basket cell mutant mice. HCN1 staining suggests the region of the basket cell pinceau is unchanged from control ( N ) in basket cell mutant mice ( O ). Scale = 20μm. ( P-S ) The Purkinje cell axon initial segment (stained with ankyrin G and indicated by arrowheads) is obvious in control ( P-Q ) and basket cell mutant mice ( R-S ) throughout the cerebellum with example images show from both anterior and posterior lobules. Purkinje cells are stained with calbindin with their somas indicated by asterisks. Scale = 10μm. ( A-B, H-S ) Dotted lines indicate the borders of the Purkinje cell layer (PCL) with the molecular layer (ML) above and the granule layer (GL) below.

Article Snippet: Purkinje cells were marked with anti-calbindin (1:1,000; Cat. # 300; Swant, Marly, Switzerland), rabbit polyclonal anti-carbonic anhydrase or CAR8 (CAVIII, 1:1000l; Cat. # sc-67330, Santa Cruz Biotechnology), goat polyclonal anti-IP3R1 (1:500; Cat. # sc-6093, Santa Cruz Biotechnology, Dallas, TX, USA), goat polyclonal anti-RORα (1:250; Cat. # sc-6062, Santa Cruz Biotechnology, Dallas, TX, USA), and mouse monoclonal anti-ankyrin-G (1:200; Cat. # MABN466, clone N106/36, Millipore Sigma, Burlington, MA, USA).

Techniques: Staining, Control, Mutagenesis, Expressing, Labeling

Journal: bioRxiv

Article Title: Molecular layer interneurons shape the spike activity of cerebellar Purkinje cells

doi: 10.1101/376517

Figure Lengend Snippet: ( A-X ) Cerebellar cell types were present and appeared unchanged in location and morphology despite the lack of VGAT in basket cells and stellate cells. Dotted lines indicate the borders of the Purkinje cell layer (PCL) with the molecular layer (ML) and the granule layer (GL). Scale = 20μm. CAR8 and IP3R1 staining revealed normal Purkinje cell location and morphology ( A-C ). GABAαR6 showed normal expression in granule cells ( D-F ). NFH expression was unchanged in Purkinje and basket cells ( G-I ). Neurogranin expression in Golgi cells was unchanged in all conditions ( J-L ). In the mutants, VGLUT1 staining in mossy and parallel fibers was unchanged ( M-O ) compared to control conditions and similarly VGLUT2 was present in mossy fiber terminals in the granule cell layer and climbing fibers in the molecular layer ( P-R ). Staining of unipolar brush cells with calretinin was similar between controls and mutants ( S-U ). CART staining of climbing fibers in the mutants was also consistent with controls ( V-X ).

Article Snippet: Purkinje cells were marked with anti-calbindin (1:1,000; Cat. # 300; Swant, Marly, Switzerland), rabbit polyclonal anti-carbonic anhydrase or CAR8 (CAVIII, 1:1000l; Cat. # sc-67330, Santa Cruz Biotechnology), goat polyclonal anti-IP3R1 (1:500; Cat. # sc-6093, Santa Cruz Biotechnology, Dallas, TX, USA), goat polyclonal anti-RORα (1:250; Cat. # sc-6062, Santa Cruz Biotechnology, Dallas, TX, USA), and mouse monoclonal anti-ankyrin-G (1:200; Cat. # MABN466, clone N106/36, Millipore Sigma, Burlington, MA, USA).

Techniques: Staining, Expressing, Control