Arcuate NPY/AgRP Neurons Decrease Arterial Pressure and Sympathetic Nerve Activity in part via a projection to the Dorsomedial Hypothalamus in Mice

Using d esigner r eceptors e xclusively a ctivated by d esigner d rugs (DREADD) technology, we recently demonstrated that select activation of arcuate nucleus (ArcN) neurons that express Neuropeptide Y (NPY) and agouti‐related protein (AgRP) rapidly and profoundly decreases splanchnic sympathetic nerve activity (SSNA), mean arterial pressure (MAP), and heart rate (HR) in male mice. However, the downstream targets of ArcN NPY neurons are unknown. The present study tested the hypothesis that this response is mediated in part by a projection to the dorsomedial hypothalamus (DMH). The mutated excitatory muscarinic receptor (hM3Dq) was selectively expressed via bilateral injection of Cre‐recombinase‐dependent adeno‐associated virus (AAV)‐hM3Dq‐mCherry into the ArcN of AgRP‐Ires‐cre mice. After >2 wks recovery, mice were anesthetized with α‐chloralose and the otherwise inert ligand Clozapine‐N‐oxide (CNO; 0.3 mg/kg i.p.; n=5), to activate hM3Dq, or saline (sal; n=5), was injected i.p. After ~30 min, CNO decreased SSNA (−25±2 % control), MAP (−9±1 mmHg), and HR (−62±17 bpm) (P<0.01); however, sal had no effects (ΔSSNA: 3±2 % control; ΔMAP: 0±2 mmHg; and ΔHR: 11±9 bpm). Subsequent bilateral injections (30 nL) of the NPY Y1 receptor antagonist BIBO3304 (Y1x; 0.3 nmol) into the DMH of sal‐treated mice elevated (P<0.05) SSNA to 157±10 % control, MAP by 14±4 mmHg, and HR by 141±29 bpm, indicating that DMH NPY tonically inhibits SSNA. In mice given CNO, DMH Y1x increased (P<0.05) these variables (SSNA to 140±9 % control, and MAP by 14±4 mmHg and HR by 125±33 bpm above baseline) to levels similar as in sal‐treated mice given PVN Y1x, suggesting that in CNO‐treated mice Y1x is blocking the actions of both tonic and DREADDs‐induced NPY release in DMH. Next, because DREADDs can be expressed in terminal fields, in another group of AgRP‐Ires‐cre mice (n=5) that received Cre‐dependent hM3Dq into the ArcN, CNO (30 nL of 10 μM/L) was injected into the DMH. DMH CNO decreased SSNA by 23 ± 3 % control, MAP by 11 ± 2 mmHg, and HR by 40±9 bpm (P<0.05). Finally, a Cre‐dependent synaptically‐targeted mCherry AAV was injected into the ArcN of AgRP‐Ires‐cre mice (n=3) and the retrogradely transported indicator Cholera toxin B (CtB) was injected into the rostral ventrolateral medulla (RVLM). A modest number of CtB‐positive neurons were visible in the DMH. However, while mCherry‐labeled fibers were abundant in the DMH, these fibers only occasionally apposed CtB cell bodies, suggesting that NPY in DMH does not inhibit a population of neurons that directly innervates the RVLM. In conclusion, ArcN NPY/AgRP neurons decrease SSNA, MAP, and HR via a projection to the DMH. The downstream neuropathway remains to be determined. Support or Funding Information NIH HL128181, AHA 12GRNT11550018 and 15POST23040042, ADA 1‐13‐BS‐120