Sniffer Cells Detect Angiotensin II Release in the Median Preoptic Nucleus In Vitro

Abstract

The brain is sensitive to Angiotensin II (ANG II). However, neuropeptide release is difficult to study and it's currently unclear if ANG II is utilized as a neurotransmitter within the brain. To address this question, our laboratory has adopted a relatively new approach to study the brain renin‐angiotensin system – sniffer cells.Chinese Hamster Ovary (CHO) cells were transfected with a commercially available plasmid for the angiotensin type 1a (AT1a) receptor (Origene Tech.) and either GCaMP (Addgene #40754) or R‐GECO (Addgene #32462). All plasmids were neomycin resistant and kill curve experiments were performed and Western blot analysis was used to identify and create stable cell lines. Control experiments were conducted with CHO cells expressing only the calcium sensor plasmid. Sniffer cells were plated on glass cover slips and continually perfused with aCSF.Calcium imaging was performed and fluorescent intensity of sniffer cells was measured in response to bath application of neuropeptides. Sniffer cells were also placed on the median preoptic nucleus (MnPO) in in vitro brain slices (produced using standard slice procedures) from adult male Sprague‐Dawley rats. Some rats were anesthetized with isoflurane (2–3%) and injected in the subfornical organ (SFO) with an AAV containing channel rhodopsin2 (AAV2‐CamKII‐C1V1(E122T/E162T)‐TS‐mCherry or AAV2‐hSyn‐ChR2(E123A)‐eYFP‐WPRE) 2–3 weeks prior to the experiments. Fluorescent intensity of sniffer cells was measured in response to optogenetic stimulation of the SFO.Isolated sniffer cells expressing only the calcium sensors did not demonstrate changes in fluorescence intensity related to exposures to ANG II or related peptides (n = 29 cells). Sniffer cells that expressed both the AT1a receptor and the calcium sensor showed dose dependent responses to ANG II and ANG III (both 10–100 nM; n = 10–29). The increases in fluorescence produced by ANG II was blocked by the AT1aR antagonist losartan (10 μM, n = 39). The sniffer cells did not respond to ANG 1–7 (0.1–100 nM, n = 17–41) or bradykinin (0.1–100 nM, n = 17–36). When sniffer cells were placed on the MnPO in brain slices, some demonstrated spontaneous changes in fluorescence intensity that were not observed in healthy sniffer cells alone. The spontaneous activity persisted at least 4 h following slice preparation. This spontaneous activity was abolished by bath application of lorsartan (10 μM, 13/13 cells) and blocked by TTX (1 μM, 9/10 cells). Additionally, no spontaneous calcium signals were detected when slices were incubated with the renin inhibitor Aliskerin (10 μM, 1 h incubation). Optogenetic (20 Hz, 1 sec, 2 ms pulse, 5 mW; n = 23) or electrical stimulation (100 HZ, 1 s, 100 μA; n = 5) of the SFO increased the fluorescence of sniffer cells located on the MnPO. Optogenetic stimulation produced 71 ± 15% increase in fluorescence over baseline while electrical stimulation increase fluorescence 141 ± 36% of baseline in GCaMP expressing cells. The changes in spontaneous fluorescence that were sensitive losartan were not different from the optogenetic evoked responses.The results demonstrate that sniffer cells can be used to measure changes in extracellular ANG II in brain slices in vitro.Support or Funding InformationP01 HL088052This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.