Neurons in the Nucleus Tractus Solitarius show no discernable change in intracellular calcium during acute application of 17β-estradiol

Purpose: Neurons in the Nucleus Tractus Solitarius (NTS) are important regulators of cardiovascular and hormonal responses to stress and a potential site for estrogen modulation of stress responses. Our goal was to elucidate whether acute estrogen receptor activation is associated changes in intracellular calcium in NTS neurons in male and female rats. Methods: Adult male and female Sprague-Dawley rats were anesthetized with isoflurane and the hindbrain removed and cut into 300um thick sections. The NTS slices were then incubated for 50min with 10μM of Fura-2AM, 0.1% F-127 at 40°C and then washed for 20min in aCSF gassed with 95%O2+5%CO2. A single slice was transferred to the recording chamber on an upright epi-fluorescent microscope. The slice was held in place with a nylon mesh and superfused with normal aCSF at a rate of 2.5 ml/min. Application of 100 nM 17β-estradiol or 63 mM KCl dissolved in aCSF solution was done using multi barrel patch pipette positioned close to the neuron so that injection (volume) occurred in the same direction as the flow of aCSF being perfused. Fluorescence of Fura-2AM was excited by epi-illumination with light provided by a 75 W Xenon lamp band-pass filtered alternatively at 340 or 380 nm. Emission light pass through a barrier filter (510 nm). Pairs of 340 and 380 nm images were acquired at intervals of 5s and analyzed off-line with NIS-Elements AR 3.2 software. All images were captured with a charge-coupled device (CCD) camera. Results: A total of 19 sections were examined from 4 female rats and an average of 7 cells/slice were analyzed in each section. In response to superfusion of the slice with 100 nM 17β-estradiol no discernable change in the 340/380 ratio was observed in any cell. In 58 neurons from 10 slices in an additional 2 rats, a higher concentration of 1mM 17β-estradiol still failed to alter the 340/380 ratio. Cell viability was confirmed by application of 63 mM KCl which induced a 14 ± 5.4% increase in the 340/380 ratio relative to baseline in the cells studied. Conclusions: Acute application of 17β-estradiol did not alter intracellular calcium in NTS neurons. This could reflect a true lack of coupling between estrogen receptors and calcium signaling mechanisms in NTS neurons. However, if estrogen receptors are on a subset of NTS neurons, perhaps the fura-2 did not label the relevant population of NTS neurons. Further, perhaps estrogen does not have an acute (non-genomic, membrane bound receptor) effect in NTS and a more prolonged application of 17β-estradiol.