Acute Intermittent Optogenetic Stimulation of Median Preoptic Nucleus (MnPO) Induces Sympathetic Long‐term Facilitation (sLTF)

Abstract

Acute intermittent hypoxia in the rodent model can produce a ramp increase in sympathetic nerve activity (SNA) that progressively increases and is maintained for up to an hour after stimulation. The induction of this increase, termed sympathetic long-term facilitation (sLTF), involves circulating Angiotensin II acting on the subfornical organ (SFO) and the paraventricular nucleus of the hypothalamus (PVN). The median preoptic nucleus (MnPO) receives inputs from the SFO and projects to the PVN and might mediate the induction of sLTF. This study used an intersectional viral approach to test the ability of acute intermittent optogenetic stimulation of PVN-projecting MnPO neurons to induce sLTF. Male Sprague-Dawley rats (250 g) anesthetized with isoflurane were stereotaxically microinjected with CRE-containing retrograde AAV (AAV9.hSyn.HI.eGFP-cre.WPRE.SV40) in PVN and with either Channel rhodopsin 2 (ChR2)-containing AAV (rAAV2/Ef1a-DIO-hchR2(H134R)-mCherry) or control virus (rAAV2/Ef1a-DIO-mCherry) in MnPO. This resulted in CRE-dependent expression of either ChR2 or mCherry in PVN-projecting MnPO neurons. Following a 4-wk recovery, rats underwent acute intermittent optogenetic (AIO) experiments where splanchnic and or renal SNA, blood pressure (BP), and heart rate (HR) were recorded. Each rat was anesthetized with urethane (800mg/kg) and α-chloralose (80 mg/kg) cocktail delivered by intraperitoneal injection, paralyzed with gallamine triethiodide (20 mg/ml, 0.25 ml/h, iv), bilaterally vagotomized, and mechanically ventilated. Noise levels was determined after a bolus injection of hexamethonium (30 mg/kg iv). MnPO terminals in the PVN were stimulated to induce AIO with a train of 10 stimuli, once per 6 minutes. Each stimuli consisted of a duty cycle of 20-ms light pulse delivered at 5 Hz for 1 min. A single optogenetic duty cycle did not alter splanchnic SNA (P=0.216, ChR2 n = 5 vs control n =2), renal SNA (P=0.234, ChR2 n =3 vs control n =2), blood pressure (P=0.928, ChR2 n = 6 vs control n =4), nor heart rate (P=0.174, ChR2 n = 6 vs control n =4). AIO significantly increased splanchnic SNA (P=0.0163) in rats with ChR2 as compared to rats injected with the control vector 1h post-AIO (ChR2 percent baseline: 160.51±20.08%, n= 5 vs control percent baseline: 77.03±11.76%, n = 2). There were no differences in renal SNA (P=0.302, ChR2 n =3 vs control n =2), BP (P=0.339, ChR2 n = 6 vs control n =4), and HR (P=0.731, ChR2 n =3 vs control n =2) between rats injected with ChR2 as compared to those in injected with the control vector 1h post-AIO. These results suggest that intermittent activation of PVN-projecting MnPO neurons may induce sLTF in the splanchnic SNA but not in renal SNA. Further study is needed to clarify the mechanisms responsible for the differential effects of MnPO-PVN AIO.