Norepinephrine Increases Cytosolic Ca2+ in Neurons of the Paraventricular Nucleus of the Hypothalamus

Abstract

The paraventricular nucleus (PVN), an important nucleus of the hypothalamus, is involved in cardiorespiratory and central autonomic responses to a variety of stimuli or stressors. The PVN receives dense innervation from catecholaminergic neurons in the nucleus tractus solitarii, ventrolateral medulla, and locus coeruleus. Stressors, including hypoxia, activate these projections to the PVN, releasing neurotransmitters and modulators including norepinephrine (NE). Within the PVN, adrenergic receptor activation by NE increases excitatory postsynaptic currents, and induces depolarization and elevated action potential discharge. However, NE's role in modulating cytosolic Ca2+ in PVN neurons remains to be determined. In this study, we determined the effects of NE on PVN neuron cytosolic Ca2+ using fura‐2 ratiometric fluorescent Ca2+ imaging. PVN neurons were dissociated from 3–4 wk old male Sprague‐Dawley rats and used within 12 hours of isolation. Neurons were exposed to aCSF baseline (Bsl, 5 min), followed by vehicle (5 min) or NE (100 mM, 5 min). Compared to aCSF Bsl, PVN neurons showed stable basal Ca2+ during vehicle controls (n=34). In contrast, addition of 100 mM NE elevated basal Ca2+ (n=69). To examine the contribution of Ca2+ from internal sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA) stores in this elevation, neurons were exposed to the SERCA blocker Thapsigargin (TG, 1 mM, 30 min). When NE was added in the presence of TG the increase in basal Ca2+ persisted, although slightly attenuated (n=11). To examine if NE elevates cytosolic Ca2+ via extracellular Ca2+ entry, cells were exposed to aCSF with zero Ca2+. In the absence of extracellular Ca2+, NE did not elevate basal Ca2+ and rather showed a significant decrease (n=63). Spontaneous Ca2+ peaks in PVN neurons were also recorded. While NE did not affect the number of spontaneous peaks or the number of cells that exhibited spontaneous peaks, NE did increase spontaneous peak amplitude (n=69). NE in presence of TG replicated NE's increase in spontaneous peak amplitude (n=11). NE in the absence of extracellular Ca2+ significantly decreased spontaneous peak amplitude (n=63). To study the potential influence of NE on use (voltage)‐dependent Ca2+ channels, cells were repeatedly depolarized with 55 mM K+ (5×, 20 sec, 5 min intervals). NE or vehicle was applied between the 3rd and 4th depolarization and the amplitude of Ca2+ peaks and basal Ca2+ was evaluated. Across the 5 peaks, there was a slight decrease in amplitude over time that was not altered by vehicle (n=34). Application of 100 mM NE induced a small, non‐significant increase in Ca2+ peak amplitude, and a significant increase in basal Ca2+ (n=34). In contrast to the NE response that occurred in neurons studied immediately after isolation, PVN neurons incubated for 2–5 days saw the effects of NE greatly diminished, if not completely abolished, and 24 hour incubation in 10 mM NE did not restore NE sensitivity. These preliminary findings demonstrate NE increases cytosolic Ca2+, likely from an influx of extracellular Ca2+, and support previous work that NE is associated with depolarization of PVN neurons.Support or Funding InformationHL 098602This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.