ROLE OF CATECHOLAMINERGIC A2 NEURONS OF NUCLEUS OF THE SOLITARY TRACT (NTS) IN CARDIOVASCULAR AND RESPIRATORY ADAPTATIONS TO CHRONIC INTERMITTENT HYPOXIA (CIH) IN RATS

Purpose: To understand the effect of CIH on the mRNA expression levels of AT1a, AT1b and excitatory amino acid (EAAs) receptor subunits in the A2 neurons of NTS and to assess the effect of tyrosine hydroxylase (TH) knockdown in A2 neurons on the cardiovascular responses to CIH Methods: Adult male Sprague-Dawley rats (250-350g) were injected with adeno-associated viruses, either (AAV-GFP-TH) to label the A2 neurons or (AAV-TH-shRNA) to knockdown TH in A2 neurons, into NTS. The control rats for (AAV-TH-shRNA) were injected with virus having scrambled RNA (AAV-sc). All the (AAV-TH-shRNA) and (AAV-sc) injected rats were implanted with radiotelemetry transmitters, 7 days prior to receiving virus injections, to monitor the changes in mean arterial pressure (MAP), heart rate (HR) and respiratory frequency (RF). 14 days after virus injections 7 of AAV-GFP-TH injected rats, all AAV-TH-shRNA and AAV-sc injected rats were exposed to 7 days of CIH (alternating 6 min periods of 10% O2 and 4 min of 21% O2 from 8am to 4pm; from 4pm to 8am rats were exposed to 21% O2). 7 of AAV-GFP-TH injected rats were used as controls and were exposed to room air. Laser capture microdissection (LCM) was performed to capture 7-10 A2 neurons for RNA extraction. Gene expression for different genes were assessed by quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) and compared between the control and CIH rats using 2-ΔΔct method. Hindbrains collected from AAV-TH-shRNA and AAV-sc injected rats were utilized for immunohistochemistry and western blot analysis Results: qRT-PCR study showed that CIH decreased AT1a (p=0.002; control - 1.08 ± 0.13, n=7; CIH - 0.48 ± 0.07, n= 6) and AMPA receptor subunit GluR2 (p=0.03; control - 1.11 ± 0.24, n=7; CIH- 0.52 ± 0.12, n= 6) and increase transcription factor FosB (p=0.03; control - 1.14 ± 0.25, n=7; CIH- 1.97 ± 0.25, n= 5) mRNA expression levels in the A2 neurons. CIH increased MAP and HR during the day in both the scRNA (n= 14, p<.001 MAP; p<.001HR) and shRNA (n=13, p<.001 MAP; p<.001 HR) groups. During the night, MAP and HR remained elevated in the scrambled rats (p<0.001 MAP; p<0.001 HR) but not in the shRNA group which can be attributed to the significant reduction in TH-immunoreactivity in NTS (shRNA: 28±lcells/section; scRNA: 35±lcells/section) Conclusions: These results suggest that A2 neurons play a role in the cardiovascular responses to CIH and following CIH, NTS neurons may become more calcium permeable, as GluR2 is found to resist calcium permeability