Ex vivo calcium imaging of sympathetic neurons in intact mouse stellate ganglia.

Sympathetic hyperactivity is a common feature of cardiovascular diseases including hypertension, and sympathetic neurons are hyperactive after a week of angiotensin II (AngII) hypertension. Nerve firing increases intracellular Ca(2+) and we wanted to develop a Ca(2+) imaging method to quantify activity across many neurons at once. Here we describe a method for ex vivo Ca(2+) imaging in intact mouse stellate ganglia. We imaged ganglia from control and AngII (700 ng/min/Kg) hypertensive mice to determine if we could identify Ca(2+) parameters that reflected hyperactivity. We expressed GCaMP6s in tyrosine hydroxylase (TH)-positive neurons (TH(GCaMP6s)). Male and female TH(GCaMP6s) mice 17-25 weeks old were used. Images were obtained under baseline conditions, after stimulation with nicotine (10 μM), and after stimulation with potassium chloride (KCl, 40 mM) as a positive control for GCaMP6s expression. Differential fluorescence responses were quantified using an open-source MATLAB processing tool. An additional MATLAB script was composed to analyze and characterize features of the fluorescent responses. The peak Ca(2+) response after nicotine treatment trended higher in left but not right stellates from AngII-treated mice, and the distribution of peak responses differed significantly in left ganglia. Decay times were similar in all groups. We compared the peak Ca(2+) response after nicotine treatment vs. KCl and found altered frequency distribution of nicotine: KCl responses in left but not right stellates from AngII mice, raising the possibility of selective modulation of cholinergic responses. Calcium imaging allowed simultaneous analysis of multiple cells within ganglia, but did not recapitulate the bilateral hyperactivity identified by electrophysiology.