Cardiovascular neurons of the rat brain express extracellular calcium-sensing receptors

Decrease of calcium in the cerebrospinal fluid (CSF) raises blood pressure whereas increase of calcium lowers it. Although these phenomena have been known for more than 50 years, precise mechanism has not fully been understood. We hypothesized that the extracellular calcium-sensing receptor (CaSR), originally cloned from bovine parathyroid cells, may participate in the blood pressure (BP)-lowering effect of calcium in the CSF. To test the hypothesis, we examined whether the cardiovascular neurons in the medulla oblongata express CaSR. Adult male Sprague–Dawley rats weighing 300–450 g (n=6) were anesthetized with urethane. Femoral artery and vein catheters were inserted for recording BP and drug infusion, respectively. They were randomly assigned to the two groups. In one group, BP was raised with infusion of phenylephrine for 60 min. Physiological saline was infused in the control group. After the end of infusion, 2-h of waiting time was allowed for induction of c-Fos protein, a marker for cellular activation. Then, the animals were transcardially perfused with heparinized phosphate-buffered saline (PBS) followed by 4% paraformaldehyde in PBS. The brains were cut coronally into 40 μm sections using a freezing microtome and collected in PBS. Coronal sections were processed for Fos and CaSR immunohistochemistry. Sections were incubated first in the primary antibody to CaSR for 96 h at 4 °C. After thorough rinsing, sections were processed with the avidin–biotin immunoperoxidase method, and CaSR was visualized as red staining with Nova Red. Thereafter, sections were incubated with antisera to Fos protein for 48 h at 4 °C. In a similar manner, c-fos was visualized as brown-black with nickel-enhanced diaminobenzidine. CaSR was found in the facial nucleus, the nucleus ambiguus, the rostral ventrolateral medulla, and caudal ventrolateral medulla (CVLM) but not in the nucleus tractus solitarius in both the control and BP-raised groups. In the control group, c-fos immunoreactivity was very low. Therefore, double-stained neurons were seldom seen. In the BP-raised group, on the other hand, many neurons in the CVLM showed both of c-fos- and CaSR-like immunoreactivity. These results indicate that an increase of calcium in the CSF may activate depressor neurons in the CVLM through CaSR on them.