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, For synaptic studies, rAAV1/2-HA-Ast-3-mCherry was injected into the NDG using the optimized injection protocol, in preparation for patch clamp studies to examine Ast release with high frequency stimulation. Two weeks later Lv-PRSx8-AstR or Lv-PRSx8-GFP were injected into the cNTS as described above. Brainstem slices were prepared from SD rats 40 ± 1 days after the NTS injection (n = 8; 498 ± 13 g on recording day). Rats were deeply anaesthetised with 5% isoflurane and the medulla removed, blocked and rapidly cooled in artificial cerebrospinal fluid (aCSF, 2 C, containing, Patch clamp recordings of transduced NTS neurons in slices For patch clamp studies, to increase the number of cells available for recordings, 50 nL Lv injections were made into the cNTS at the following 18 sites for a total of 900 nL per SD rat, 1200.
, The slices were continuously perfused in aCSF solution, bubbled with 95% O 2, 5% CO 2, at 32 C. Recording pipettes (2.6 -6.5 MU) were filled with an internal solution containing, Leica Microsystems)
, Signals were sampled at 20 kHz and filtered at 10 kHz. Liquid junction potentials were not corrected (À6.2 mV at 32 C) until post hoc analysis. Ast (5 nM) and HA-Ast (100 nM) was superfused onto the slice in aCSF at 2.5 mL/min. For synaptic studies, all neurons were first classified as receiving vagal afferent input, or not, in voltage clamp mode, Cl À68.8 mV)
, USA) that was placed on the visible ST at a distance of 2-3 mm from recorded neurons. Trains of 5 shocks, FHC
, NTS neurons were classed as a second order neuron, directly receiving primary afferent input, if ST-EPSCs exhibited a synaptic jitter (standard deviation of EPSC latency) of less than 200 ms and exhibited frequency-dependent depression. Otherwise neurons were classified as higher order. High frequency stimulation of the ST (ST-HFS) was carried out at 50 Hz (100 ms shocks) for 2 or 30 s in current clamp mode (I = 0 pA). Likewise input-frequency curves were recorded in current clamp at I = 0 pA, prior to and after ST-HSF in aCSF; prior to and after ST-HSF in ionotropic receptor blockade
, One rat was excluded from the SHR Control cohort as it was delivered at an older age with a higher bodyweight and BP, leaving a cohort of n = 8. In all other regards the animals were treated identically. In the first surgery, rAAV1/2-HA-Ast-3-mCherry was microinjected into the NDG bilaterally. The SHR were allowed to recover in their home cage for 2 weeks, with daily measurement of bodyweight between 8:00 and 10:00. In the second surgery, 14 ± 4 days after the NDG injections, BP telemeters were implanted into the abdominal aorta of SHR anesthetized as described above. Following surgery, the rats were returned to their home cages for recovery for 2 weeks before commencement of control BP recordings. Telemetry devices for recording blood pressure (BP; TA11PA-C40 telemeters; Data sciences International, USA) were implanted following the manufacturer's protocol. Briefly, the abdominal cavity was opened in the midline and the pressure sensing catheter inserted into the abdominal aorta distal to the renal arteries. Vetbond was applied to seal the vascular opening and ensure attachment to the abdominal aorta. The transmitter probe was placed in the abdominal cavity and sutured to the abdominal muscle layer, mM; Tocris, USA) to confirm action potential block. Expression of rAAV1/2-HA-Ast-3-mCherry in nodose ganglion and Lv-PRSx8-AstR or Lv-PRSx8-GFP in NTS of blood pressure telemetered SHR Male SHR (n = 18; $250 g) were purchased in 3 separate cohorts of 6, 2, and 10 animals. Within each cohort, animals were assigned at the start of the protocol to SHR Experimental -those that would eventually receive injections of Lv-PRSx8-AstR into the NTS (n = 9) -and SHR Control -those that would receive injections of Lv-PRSx8-GFP into the NTS (n = 9, 1998.
, This approach was applied to the first SH rat cohort (n = 3 per group). However, when observing real-time recordings, we noticed spurious, movement related fluctuations in BP that were clearly non-physiological -BP readings in excess of 300 mmHg. These were relatively random, occurred to different degrees among animals during the same recording period and resulted in increased signal variability that did not reflect a biological signal, Commonly in BP telemeter studies, investigators obtain average readings of BP over defined periods
, All points in excess of 300 mmHg were removed and replaced with the average of the preceding 1 s of data. We compared the resultant data based on this method with the automatic average data and determined that such interference results in random shifts, both up and down, in average BP readings that can be of a magnitude of 10-15 mmHg over a 2 h recording period. As a result, the data included in the manuscript is from a 2h recording period, excluding the 30 min immediately after handling to turn on the BP telemeter, and is only based upon those SH rats in the second and
, The measurements were normalized to the control period for each animal and then presented as mean ± 95% confidence intervals for the groups, with each individual animal also shown. For completeness of the dataset, all raw BP data from all SH rats
, Analysis of the data which included all animals shows the same overall response, SHR Control (n = 8)) are shown (Figure S2B)
, The standard deviation of BP and IBI were derived and plotted for each animal. The baroreceptor sensitivity (BRS) was determined with the sequence method (Kuusela, 2013), and defined as the relationship between a change in the IBI following a unitary change in BP (ms/ mmHg). Individual points for systolic BP and IBI were examined in 10-20 min sections during quiet, inactive periods between 11:00 h and 13:00 h. The data were analyzed using a custom-made MATLAB script that identified periods in which systolic BP and IBI rise or fall monotonically in the same direction for at least three beats, exceeding or meeting a defined threshold of a change of 0.5 mmHg and 1 ms respectively (Figure S3A). The IBI series was advanced by one beat to adjust for the delay between BP and corresponding changes in IBI. A minimum correlation co-efficient (> 0.8) between systolic BP and IBI had to be achieved for the script to consider an actual BRS sequence. Finally, the BRS of each sequence was calculated by the slope of the regression line of systolic BP versus IBI, and the BRS of an animal was the average slope calculated from all the sequences found. Mean slopes of BRS for sequences where systolic BP and IBI increased (called UP-UP, HR) were derived from the pulsatile arterial BP trace. The systolic BP was used as a surrogate measure of the cardiac R-wave and the time between successive pressure maxima as the interbeat interval (IBI)
, After 2 weeks, Lv injections were made into the cNTS at a total of 7 different sites, with 50 nL at each site and a total volume of 350 nL per rat, as described for the SHR above (Table 2). The rats were returned to home cages, re-housed with littermates and weighed daily for the next 31 days. Two additional control SD groups were also generated with microinjections into the cNTS, as described above. The first extra control cohort received just Lv-PRSx8-AstR (n = 10) or just Lv-PRSx8-GFP in the cNTS (n = 10). The second extra control cohort (n = 10) received a unilateral injection of rAAV1/2-HA-Ast-3-mCherry in the NDG, followed 2 weeks later by injection of Lv-PRSx8-AstR in the cNTS, as described previously (Table 2). Following completion of the surgeries the rats were re-housed and bodyweight measured for 14 days, The number of UP-UP and DOWN-DOWN sequences were quantified. Values for all parameters are expressed as individual animals and mean ± 95% confidence intervals for the groups. Expression of rAAV1/2-HA-Ast-3-mCherry in nodose ganglion and Lv-PRSx8-AstR in NTS of SD rats Twenty male SD rats were assigned at the start of the protocol to SD. Experimental -those that would eventually receive injections of Lv-PRSx8-AstR into the NTS (n = 10), and SD
, by inhalation of 4%-5% isoflurane in a sealed container, followed by injection of a mixture of ketamine (60 mg/kg) and medetomidine (250 mg/kg) i.m., and killed by intracardiac perfusion using a peristaltic pump (Cole-Parmer Masterflex L/S Drive System, USA) delivering 0.1 M phosphate-buffered saline (PBS), followed by 4% paraformaldehyde in 0.1 M PBS at a rate of $30 mL/min. The brains were removed and post-fixed in 4% paraformaldehyde in 0.1 M PBS for 6 h at RT, followed by immersion in 20% sucrose at 4 C until processing for immunohistochemistry. The thorax and head were post-fixed in 4% paraformaldehyde in 0.1 M PBS overnight and then washed several times, 2020.
, The NDG were dissected out along with the distal vagal trunk and the superior laryngeal nerve, to its entry to the posterior lacerated foramen