PVN‐projecting NTS Neurons is Involved in Sympathetic Long‐Term Facilitation After Acute Intermittent Optogenetic Stimulation

Abstract

Caudal Nucleus Tractus Solitarius (NTS) neurons receive inputs from cardiorespiratory visceral afferents and send projections to the paraventricular nucleus of the hypothalamus (PVN). Acute intermittent optogenetic (AIO) stimulation of caudal NTS induces sympathetic long‐term facilitation (LTF), a progressive increase of sympathetic nerve activity. The contribution of PVN‐projecting NTS neurons to sympathetic LTF is unknown. This study used an intersectional viral approach to test the effects of acute intermittent optogenetic (AIO) stimulation of PVN‐projecting NTS neurons in sympathetic LTF. Adult male Sprague‐Dawley rats were anesthetized with isoflurane and injected rAAV2/Ef1a‐DIO‐hchR2(H134R)‐mCherry in the NTS and a retrograde AAV9.hSyn.HI.eGFP‐cre.WPRE.SV40 in the PVN four week prior to LTF experiments. This method was used to induce the CRE dependent expression of channel rhodopsin in PVN‐projecting NTS neurons. To measure LTF, we recorded splanchnic sympathetic nerve activity (SSNA), arterial pressure, and heart rate (HR) in anesthetized (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) and mechanically ventilated adult rats. The caudal NTS was stimulated to induce AIO with a train of 10 stimuli each consisting of pulse of light of 10‐ms duration delivered at 20 Hz for 1 min every 6 min. Twenty minutes after AIO, SSNA amplitude increased by 25.36 ± 24.16% (n=3), and mean arterial pressure (MAP) increased by 7.43 ± 12.88 mmHg (n=3). During AIO induction, the acute SSNA and MAP varied with some rats demonstrating increases (n=2) or decreases (n=1) of SSNA, MAP, and HR. In other animals (n=3) where AIO decreases SSNA (‐58.96 ± 36.18%) and MAP (‐14.37 ± 8.57 mmHg), we tested the possible contribution of the baroreceptors in these decreases by bilateral muscimol (1–10 pmol/100nL) injections in the Caudal Ventrolateral Medulla (CVLM). AIO stimulation after inhibition of CVLM caused an increase in SSNA (1.293 ± 17.65%) and smaller decrease in MAP (−2.46± 6.2 mmHg). These results suggest that the splanchnic sympathetic LTF induced by acute intermittent stimulation of NTS neurons might be mediated by the PVN‐projecting neurons, although the acute responses may be excitatory or inhibitory. Further study is needed to clarify the mechanisms responsible for the differences in the cardiovascular and SSNA responses to acute, single optogenetic stimulation of PVN‐projecting NTS neurons. Despite the observed differences in the acute responses, the results indicate that sympathetic LTF can be induced by the optogenetic activation of NTS neurons that project to PVN.Support or Funding InformationNIH Grant P01 HL088052