Journal: Journal of Molecular and Cellular Cardiology
Article Title: The cardiac sympathetic co-transmitter galanin reduces acetylcholine release and vagal bradycardia: Implications for neural control of cardiac excitability
Figure Lengend Snippet: Expression of galanin in stellate sympathetic neurons and GalR 1 on cardiac cholinergic neurons. (A) Reverse transcription-polymerase chain reaction (RT-PCR) products viewed on an ethidium bromide gel after amplification of freshly dissected stellate ganglia and brain mRNA. NTC, no template control, is a negative control assessing the absence of contamination. Galanin mRNA is present in both right and left stellate ganglia. (B) All stellate neurons from freshly dissected left and right stellate ganglia exhibit immunoreactivity for tyrosine hydroxylase (TH, left panel, 20 μm sections) but only occasionally do TH immunoreactive neurons also exhibit galanin immunoreactivity (< 5%, right panel, calibration bar = 20 μm). (C) Dissociated stellate neurons cultured for 3-days exhibit immunoreactivity for TH (left hand panel) and galanin (right hand panel, calibration bar = 20 μm). (D) RT-PCR bands for the galanin receptors GalR 1 (253 base pairs) and GalR 3 (234 base pairs) from mRNA isolated from cardiac ganglia. Guinea pig brain was used as a positive control, and reaction mix without template was the negative control (NTC). (E) Immunohistochemistry of right atrial slices showing expression of GalR 1 receptor protein in neuronal structures only (see inset picture at higher magnification, calibration bar = 10 μm). GalR 1 , GalR 2 and GalR 3 expression was demonstrated in guinea pig midbrain as a positive control (calibration bar = 20 μm). (F) Immunohistochemistry showing choline acetyltransferase (ChAT) staining with Texas Red in a neuron in right atrial slices also staining positive for the galanin GalR 1 receptor in green (fluorescein). Co-localization was demonstrated by overlap of staining in yellow (calibration bar = 20 μm).
Article Snippet: Fixed right atrial slices (or guinea pig midbrain as a positive control) were incubated with rabbit anti-GalR 1 (Sigma, 1:200), anti-GalR 2 (Affinity Bioreagents, 1:150) or anti-GalR 3 (Sigma, 1:150) primary antibodies followed by biotinylated goat anti-rabbit (Vector, 1:200).
Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Negative Control, Cell Culture, Isolation, Positive Control, Immunohistochemistry, Staining