Journal: Molecular Psychiatry

Article Title: Emergence of 5-HT5A signaling in parvalbumin neurons mediates delayed antidepressant action

doi: 10.1038/s41380-019-0379-3

Figure Lengend Snippet: 5-HT5A signaling in PV neurons. a Bar graph summary of cAMP level in N2A cells transiently transfected with mouse 5-HT5A. The cells were incubated with the indicated amounts of forskolin and 5-HT. One representative experiment out of three is shown. Bars represent means of cAMP level ( n = 4 wells per group) as percentage of baseline + SD. One Way ANOVA, * P < 0.05 vs. baseline. b Representative western blot images (top) and bar graph summary (bottom) depicting the levels of Kv3.1β and Ser503 pKv3.1β in transfected HEK293 cells treated as indicated ( n = 4 wells per condition). Bars represent mean densities of the pKv normalized to that of the total Kv. Number and arrowhead indicate protein size in kDa. One-way ANOVA. * P < 0.05, ** P < 0.01. OA okadaic acid, PMA Phorbol 12-myristate 13-acetate. c Representative traces of Kv potassium currents evoked with 10 mV potential steps from −70 to +50 mV in PV DG neurons from Veh- and Flx-treated WT mice. d PMA (200 nM) decreased the amplitude of KV currents in Flx-treated mice ( n = 4, 3) but not Veh-treated WT mice ( n = 3, 3). RM one-way ANOVA, * P < 0.05. e Model for serotonergic signaling in PV hippocampal cells. Left: initial exposure to SSRIs does not alter cAMP levels and PKA activity. PP2A activity remains high and Kv3.1β is dephosphorylated. Right: chronic SSRIs increases 5-HT5A surface levels. 5-HT5AR activation reduces cAMP levels and diminishes PKA and PP2A activities to reduce Ser 503 Kv3.1β dephosphorylation

Article Snippet: Staining and analysis of 5-HT5A and Ser503 phospho Kv3.1β (pKv3.1β) were done for all samples at the same time using commercial antibodies against 5-HT5A (LifeSpan Biosciences, LS-A2119), Ser503 phospho Kv3.1β (Phosphosolutions, p1550-503), and PV (Sigma Aldrich, P0388).

Techniques: Transfection, Incubation, Western Blot, Activity Assay, Activation Assay, De-Phosphorylation Assay

Journal: Scientific Reports

Article Title: The transcriptome of mouse central nervous system myelin

doi: 10.1038/srep25828

Figure Lengend Snippet: ( A ) Scheme illustrating the biochemical purification of a light-weight membrane fraction enriched for myelin by homogenizing mouse brains in 0.32 M sucrose, sequential sucrose density gradient centrifugation, and osmotic shocks. Myelin accumulates at the interface between 0.32 M and 0.85 M sucrose. ( B ) Immunoblot analysis of myelin-enriched fractions and equal amounts of brain lysate to compare the abundance of marker proteins for compact myelin (PLP/DM20, MBP), non-compact myelin (CNP, SIRT2), the oligodendroglial nuclear/cytoplasmic compartment (OLIG2), astrocytes (GFAP), microglia (AIF1/IBA1), neuronal plasma membrane (GPM6A), axonal microtubules (TUBB3/TUJ1), and mitochondria (VDAC). Blot represents three biological replicates (male c57Bl6/N mice, age P75). Note that myelin markers were enriched in purified myelin while markers of other cellular sources were reduced. ( C ) Heatmap displaying reverse CT values from qRT-PCRs for three markers each specific for myelin, migroglia, neurons and astrocytes performed on myelin biochemically purified from the brains of 4 individual mice (M1–4) compared to the respective brain lysates (BL1–4) at six month of age.

Article Snippet: Antibodies were specific for PLP/DM20 (A431 , 1:5000), MBP (Dako A0623, 1:500), CNP (Sigma C 5922, 1:1000), SIRT2 (Abcam ab67299, 1:500), OLIG2 (DF308 , 1:200, kindly provided by J. Alberta and C. Stiles, Boston, MA, USA) GFAP (Novocastra NCL-GFAP-GA5, 1:500), AIF (also termed IBA1; Abcam ab107159, 1:500), GPM6A (#24924 , 1:1000), TUBB3 (also termed TUJ1; Covance MMS-435P, 1:1000), and VDAC (Rockland 600-401-882, 1:2000).

Techniques: Purification, Membrane, Gradient Centrifugation, Western Blot, Marker, Clinical Proteomics