Involvement of Phox2B Neurons Located in the Commissural NTs with the Maintenance of Hypertension in SH Rats
Authors: Del Rosso de Melo Mariana , Ong Sarah JM , Connelly Angela A , Bassi Jaspreet K , Colombari Eduardo , Allen Andrew M 
Published: June 2021

Inhibition of neurons located in the commissural portion of the nucleus of the solitary tract (cNTS) decreases high blood pressure in different models of hypertension, such as the spontaneously hypertensive rat (SHR). The aim of this study was to identify the phenotype of the neurons responsible for this effect using different chemogenetic techniques. Firstly, AAV‐CBA‐hM4DREADD‐mCherry or AAV‐CBA‐TdTomato were injected into the cNTS of male SHR and Sprague‐Dawley (normotensive control – NT). Recordings of mean arterial pressure (MAP) and heart rate (HR) were made approximately 3 weeks later, in anesthetised rats. Injection of CNO i.v. in SHR expressing hM4Di in cNTS, rapidly decreased both MAP (Δ MAP: −56.6 ± 6.2 vs. saline: −2.2 ± 2.4 mmHg, 10 min after injection) and HR (Δ HR: −70 ± 8.1 vs. saline: −14.3 ± 8 bpm, 20 min after injection). Similar CNO injections in SHR expressing TdTomato also decreased BP. The response was slower in onset, but eventually achieved the same maximal response to that of the SHR expressing hM4Di (Δ MAP: −62.1 ± 7.3 vs. hM4Di: −55.5 ± 6.7 mmHg; Δ HR: −61.9 ± 14.4 vs. hM4Di: −70.4 ± 7.4 bpm; 40 min after the injection). The effective depressor and bradycardic effects of hM4DREADD activation by CNO was obtained by subtracting the values of SHR‐TdTomato from the SHR‐hM4Di. It was found that the maximum reduction in the MAP and HR produced by hM4Di activation occurred at 5 minutes (Δ MAP: −39.9 mmHg and Δ HR: −42.4 bpm). The same experiments were repeated in NT rats and neither MAP nor HR was affected by CNO injection. Using immunohistochemistry and in situ hybridization histochemistry (RNASCOPE) we analyzed the phenotype of hM4Di‐mCherry‐transfected neurons. Of the transfected neurons 38.4 ± 1.9% expressed Phox2B, 17 ± 2% expressed TH, and only 0.4 ± 0.17% expressed HSD2. The transfected neurons expressed mRNA for GAD1 (22 ± 0.75%), and VGlut2 (17.6 ± 1.8 %). To more selectivetly study the role of Phox2B neurons we used a lentivirus with the inhibitory allatostatin receptor (AstR) expressed under the control of the Phox2 receptor promoter PRSx8. The Lv‐PRSx8‐AstR‐GFP or Lv‐PRSx8‐GFP viruses were injected in the cNTS of another group of SH rats. In these animals microinjection of saline into the cNTS did not produce any changes either in MAP or HR in either group (Δ MAP: SHR‐GFP: −0.9 ± 3.2 vs. SHR‐AstR: −5.6 ± 3.3 mmHg, p>0.05) (HR: SHR‐GFP: −8.7 ± 6.6 vs. SHR‐AstR: −6.9 ± 5 bpm, p>0.05). Microinjection of allatostatin into the NTS produced a large decrease in MAP and HR in SHR‐AstR rats that was of similar magnitude to that of CNO in the hM4D‐expressing SHR (Δ MAP: SHR‐GFP: − 1.5 ± 9 vs. SHR‐AstR: −44.1 ± 4.5 mmHg; Δ HR: SHR‐AstR: −8.3 ± 9.3 vs. allatostatin: −59.8 ± 16.8 bpm; considering the peak of the response). We conclude that Phox2‐expressing neurons in cNTS are important for maintaining the high levels of BP in SH rats. Whilst our intial studies with the CBA promoter suggested both GABA and glutamatergic neurons might be involved, the majority of Phox2‐expressing neurons in the medulla are glutamatergic.Support or Funding InformationFAPESP (2016/02091‐1; 2015/23467‐7), CNPq, NHMRCThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.