Journal: Frontiers in Pharmacology

Article Title: CyPPA, a Positive SK3/SK2 Modulator, Reduces Activity of Dopaminergic Neurons, Inhibits Dopamine Release, and Counteracts Hyperdopaminergic Behaviors Induced by Methylphenidate ¹

doi: 10.3389/fphar.2012.00011

Figure Lengend Snippet: CyPPA inhibits spontaneous and evoked release of dopamine form TH/SK3 positive cultured rat midbrain neurons . (A) Western blot detection of SK3 in rat midbrain cultures. Extracts of HEK293 cells transiently transfected with rSK1, rSK2, and rSK3 and adult rat brain membranes (RBM) as well as lysates from cultured midbrain neurons were separated on 4–15% SDS-PAGE and immunoblotted with anti-SK3 antibody. A band of approximately 70 kDa was detected in midbrain cultures, RBM lysates, and HEK293 cells transfected with rSK3. This band was absent in lanes loaded with HEK293 cells expressing rSK1 and rSK2. (B) Immunodetection of SK3 subunits in rat midbrain cultures. Cultured midbrain neurons (8 days in vitro ) were double-labeled for SK3 and tyrosine hydroxylase (TH). In TH-positive dopaminergic neurons, the SK3 subunit displayed a primarily somatic–dendritic localization whereas little immunoreactivity was associated with axons (arrow). Scale bar 20 μm. (C) Basal release and stimulated release with 25 mM KCl or inhibited release with 30 μM Cd 2+ and with 1 μM TTX. (D) Basal release and stimulated release with 3 μM haloperidol and inhibited release with 10 μM quinpirole. Blue bars represent inhibited release with increasing concentrations of CyPPA (* P < 0.05, ** P < 0.01). The data presented are representative of three independent experiments.

Article Snippet: Neurobasal media and B-27 were obtained from Invitrogen (Nærum, Denmark) and 3 H-DA was acquired from PerkinElmer Life and Analytical Sciences (Boston, MA, USA) The primary antibodies used were a rabbit polyclonal anti-SK3 antibody (1:100, APC-025) from Alomone Labs (Jerusalem, Israel) and a monoclonal mouse anti-tyrosine hydroxylase antibody (1:150, clone LNC1) from Millipore (Copenhagen, Denmark).

Techniques: Cell Culture, Western Blot, Transfection, SDS Page, Expressing, Immunodetection, In Vitro, Labeling

Journal: Journal of Neuroscience

Article Title: Early Onset of Ataxia in Moonwalker Mice Is Accompanied by Complete Ablation of Type II Unipolar Brush Cells and Purkinje Cell Dysfunction

doi: 10.1523/jneurosci.2294-13.2013

Figure Lengend Snippet: Figure 1. Immunolocalization of TRPC3 in cerebellar paraffin sections from wild-type and TRPC3 / mice. A, Enrichment of TRPC3-immunoreactive UBCs in nodulus (X) and ventral uvula (folia IXb and IXc) in a wild-type mouse. Other lobules (IXa, VIII) contain few TRPC3- positive UBCs. PCs show only moderate immunostaining. B, Immunostained cells in nodulus show typical UBC morphology: mid-sized soma (arrow) and intensely immunoreactive brush (arrowhead). C–H, Confocal images from a wild-type mouse illustrate colocalization of TRPC3 with mGluR1 in type II UBCs (C–E). TRPC3 is absent from CR-positive type I UBCs (F–H). Arrowsandarrowheadsindicatesomataanddendriticbrushes,respectively(white,typeIIUBC; blue, type I UBCs). I, J, TRPC3 immunostaining is abolished in the cerebellar sections from a TRPC3/mouse(I);however,theimmunostainingofmGluR1intypeIIUBCs(arrowheads) and PCs (labeling in ml) appears unaffected (J). Asterisks indicate PC somata. ml, Molecular layer; gcl granule cell layer.

Article Snippet: PC immunoreactivity was apparent in cryosections labeled with the Alomone Labs TRPC3 antibody, whereas UBCs were barely labeled (data not shown).

Techniques: Immunostaining, Labeling

Journal: Journal of Neuroscience

Article Title: Early Onset of Ataxia in Moonwalker Mice Is Accompanied by Complete Ablation of Type II Unipolar Brush Cells and Purkinje Cell Dysfunction

doi: 10.1523/jneurosci.2294-13.2013

Figure Lengend Snippet: Figure 2. TRPC3 channels expressed in type II UBCs are functional. A, Time course of the current (at 70 mV) in control conditions and in the presence of DHPG and DHPGPyr3 in type II UBCs obtained from Tg(Grp-EGFP)DV197Gsat (control, black symbols,n 10)andTRPC3 /(redsymbols,n 5)mice.Currentswerenormalizedtothecontrolcurrent(averagedbetween 240and300s).Atotalof50MDHPGincreasedthecurrentupto2.5foldinUBCsfromcontrolmice.SubsequentapplicationofPyr3 (30 M), a TRPC3 antagonist, decreased the DHPG-sensitive currents by 54.5 10.6% (p 0.05, ANOVA with Newman–Keuls post hoc test). In type II UBCs of TRPC3 / mice, DHPG only induced a detectable current in three of five cells, and it was not blockedbyPyr3.B,VoltagerampusedtomeasurethePyr3-sensitivecurrentshowninCandD.C,D,RepresentativePyr3-sensitive currentsobtainedbydigitalsubtractionoftheaveragecurrentinDHPGandPyr3fromtheaveragecurrentinDHPG(markedascand b in A, respectively) in control mice (C) and TRPC3 / mice (D).

Article Snippet: PC immunoreactivity was apparent in cryosections labeled with the Alomone Labs TRPC3 antibody, whereas UBCs were barely labeled (data not shown).

Techniques: Functional Assay, Control