The dynamic activity of C1 neurons determines the level of blood pressure during hemorrhage in freely behaving rats

Abstract

Background Adrenergic neurons in the rostral ventrolateral medulla (RVLM), called C1 neurons, regulate vasomotor sympathetic nerve activity (SNA) and blood pressure (BP), and are thought to contribute to BP homeostasis during hemorrhage. In unanesthetized rats, hemorrhage has two sequential phases: 1) compensated, when SNA is markedly elevated and BP is maintained at nearly normal levels and 2) decompensated, when SNA is suppressed, and BP falls dramatically. We hypothesize that dynamic changes in C1 neuron activity determines the level of SNA and BP during hemorrhage. Aim To measure C1 neuron activity during hypotensive hemorrhage in non-anesthetized freely behaving rats. Methods The real-time activity of C1 neurons was monitored using a genetically encoded calcium indicator, jGCaMP7s, with in vivo fiber photometry in non-anesthetized rats. A virus carrying a Cre-dependent GCaMP7 transgene (pGP-AAV1-syn-FLEX-jGCaMP7s-WPRE) was injected in the RVLM of adult female and male Th-Cre rats and an optic fiber was implanted at the site of injection. Four weeks later, rats were instrumented for BP measurements and vascular access for blood withdrawal. The next day, the activity of C1 neurons was measured while the rats were subjected to a controlled hypotensive hemorrhage (18 ml/kg of blood withdrawal over 20 minutes, rate 0.3-0.5 ml/min). Isosbestic-corrected fluorescence (DF/F0) is expressed as a Z-score based on a baseline recording period expressed as mean ± SD. Tests include Student's t-test or repeated measures one-way ANOVA. Differences were considered significant at p<0.05. Results Four weeks after the injection, GCaMP7 was expressed selectively in C1 neurons (76 ± 12 % of GCaMP7-positive neurons expressed tyrosine hydroxylase and PNMT). Transient hypotension induced by intravenous sodium nitroprusside (SNP) activated C1 neurons (from -0.3 ± 0.6 to 11.3 ± 4 A.U., p=0.0002, n=8), whereas elevations in BP induced by intravenous phenylephrine (PE) did not consistently reduce C1 neuron activity (from 0.1 ± 0.3 to -0.9 ± 2.3 A.U., p=0.343, n=7). During hemorrhage, the activity of C1 neurons increases during the compensated phase (when BP is stable despite the blood loss) in relation to the pre-hemorrhage period (from 0.17 ± 0.8 to 5.2 ± 2.1 A.U., p=0.0006, n=8). At the onset of the decompensated phase, the activity of C1 neurons drops dramatically in relation to the compensated phase (from 5.2 ± 2.1 to 2.9 ± 2.8 A.U., p=0.0017, n=8). Conclusions Our results indicate that C1 neurons have low resting activity and are very sensitive to hypotension in quite awake freely behaving rats. And the activation of C1 neurons is critical for BP stability during compensated hemorrhage and their inhibition contributes to the reduction in SNA and hypotension during decompensated hemorrhage.