Decreased expression of neuronal nitric oxide synthase in the nucleus tractus solitarii inhibits sympathetically mediated baroreflex responses in rat

J Physiol 590.15 (2012) pp 3545–3559 3545

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Decreased expression of neuronal nitric oxide synthase inthe nucleus tractus solitarii inhibits sympatheticallymediated baroreflex responses in rat

Li-Hsien Lin1, Deidre Nitschke Dragon1, Jingwen Jin1, Xin Tian2, Yi Chu2, Curt Sigmund2

and William T. Talman1,3

1Laboratory of Neurobiology, Department of Neurology, Roy and Lucille Carver College of Medicine, University of Iowa, Iowa City, IA, USA2Department of Internal Medicine and Pharmacology, Roy and Lucille Carver College of Medicine, University of Iowa, Iowa City, IA, USA3Neurology Service, Department of Veterans Affairs Medical Center, Iowa City, IA, USA

Key points

• Arteries send nerve signals to the brain in response to changes in blood pressure.• Nerves carrying these signals release chemical transmitters onto other neurons in the nucleus

tractus solitarii, a nucleus in the brainstem.• One chemical that has been considered such a transmitter is nitric oxide, but there has been

debate over the action of nitric oxide in brainstem influences on responses to changes bloodpressure.

• We show that use of a molecular tool that allows us to prevent the normal production of nitricoxide by nerve cells leads to altered reflex regulation of heart rate in rats.

• Our findings, which clarify the role of neuronally derived nitric oxide in control of the cardio-vascular system by the brain, could lead to better control of blood pressure in individuals witheither high or low blood pressure.

Abstract Despite numerous studies it remains controversial whether nitric oxide (NO·)synthesized by neuronal NOS (nNOS) plays an excitatory or inhibitory role in transmissionof baroreflex signals in the nucleus tractus solitarii (NTS). In the current studies we sought to testthe hypothesis that nNOS is involved in excitation of baroreflex pathways in NTS while excludingpharmacological interventions in assessing the influence of nNOS. We therefore developed,validated and utilized a short hairpin RNA (shRNA) to reduce expression of nNOS in the NTS ofrats whose baroreflex activity was then studied. We demonstrate downregulation of nNOS throughtransduction with adeno-associated virus type 2 (AAV2) carrying shRNA for nNOS. Wheninjected bilaterally into NTS AAV2nNOSshRNA significantly reduced reflex tachycardic responsesto acute hypotension while not affecting reflex bradycardic responses to acute increases of arterialpressure. Control animals treated with intravenous propranolol to block sympathetically mediatedchronotropic responses manifested the same baroreflex responses as animals that had beentreated with AAV2nNOSshRNA. Neither AAV2 eGFP nor AAV2nNOScDNA affected baroreflexresponses. Blocking cardiac vagal influences with atropine similarly reduced baroreflex-mediatedbradycardic responses to increases in arterial pressure both in control animals and in those treatedwith AAV2nNOSshRNA. We conclude that NO· synthesized by nNOS in the NTS is integral toexcitation of baroreflex pathways involved in reflex tachycardia, a largely sympathetically mediatedresponse, but not reflex bradycardia, a largely parasympathetically mediated response. We suggest

C© 2012 The Authors. The Journal of Physiology C© 2012 The Physiological Society DOI: 10.1113/jphysiol.2012.237966

3546 L.-H. Lin and others J Physiol 590.15

that, at the basal state, nNOS is maximally engaged. Thus, its upregulation does not augment thebaroreflex.

(Resubmitted 30 May 2012; accepted after revision 8 June 2012; first published online 11 June 2012)Corresponding author W. T. Talman: Department of Neurology, Roy and Lucille Carver College of Medicine, 200Hawkins Drive, Iowa City, IA 52242, USA. Email: [email protected]

Abbreviations AAV2, adeno-associated virus type 2; AP, arterial pressure; cDNA, complimentary deoxyribonucleicacid; CVLM, caudal ventrolateral medulla; eNOS, endothelial NO·; eGFP, enhanced green fluorescent protein; GFAP,glial fibrillary acidic protein; GluR2, glutamate receptor type 2; HR, heart rate; HEK, human embryonic kidney cell;IgG, immunoglobulin G; IR, immunoreactivity; IV, intravenous; MAP, mean arterial pressure; NA, nucleus ambiguus;NMDAR1, N-methyl-D-aspartate receptor type 1; NF160, neurofilament 160; NG, nodose ganglion; nNOS, neuronalnitric oxide synthase; NO·, nitric oxide; NTS, nucleus tractus solitarii; PGP9.5, protein gene product 9.5; RRX,rhodamine red X; RVLM, rostral ventrolateral medulla; shRNA, short hairpin ribonucleic acid; SQ, subcutaneous;TH, tyrosine hydroxylase; VGluT1, vesicular glutamate transporter type 1; VGluT2, vesicular glutamate transportertype 2.

Introduction

Within the nucleus tractus solitarii (NTS) there is robustexpression of neuronal nitric oxide (NO·) synthase(nNOS) as well as endothelial NOS (eNOS) (Simonian &Herbison, 1996; De Vente et al. 1998; Atkinson et al. 2003;Lin et al. 2007). The former is located primarily in neuronswhile the latter is found predominantly in endothelial cellsand glia (Lin et al. 2007). The two isoforms of NOS lie inclose proximity within their cellular elements in NTS andthus NO· synthesized by either could diffuse (Garthwaite,1995) to local NTS neurons where it could modulate thearterial baroreflex, one of the multiple visceral functionsunder control of NTS neurons (Spyer 1982; Spencer &Talman 1986; Feldman & Ellenberger 1988; Bauman et al.2000). Others have shown that NO· derived from eNOSand released as a result of angiotensin’s action on NTSmicrovessels may act as a paracrine agent to inhibit thebaroreflex (Paton et al. 2007). Our own studies havesuggested that NO· generated from nNOS may augmentbaroreflex signal transmission in NTS. Specifically, wehave found that nNOS and vesicular glutamate trans-porters, markers of glutamatergic excitatory synapses,colocalize in NTS (Lin & Talman 2001; Lin & Talman,2002; Lin et al. 2004; Lin & Talman, 2005). Further, NTSneurons colocalize both nNOS and ionotropic glutamatereceptors (Lin & Talman, 2001; Lin & Talman, 2002), thussupporting the potential for both presynaptic and post-synaptic excitatory actions of NO· generated by neurons.Physiological responses to glutamate receptor activation,which is considered integral to baroreflex transmission(Talman et al. 1980a; Lawrence & Jarrott, 1994), areblocked by selective nNOS inhibitors (Talman & NitschkeDragon, 2004). Furthermore, blockade in NTS of solubleguanylate cyclase, the enzymatic ‘receptor’ for NO· itself(Knowles et al. 1989), also blocks responses to glutamatereceptor activation (Chianca et al. 2004). The same nNOSinhibitors that blocked responses to glutamate receptoractivation also blocked arterial baroreflex responses when

the inhibitors had been applied to the NTS (Talman &Nitschke Dragon, 2004). Despite these findings doubtsremain about a role for NO· in excitatory transmissionof the baroreflex. In part this has been due to skepticismabout selectivity of the nNOS inhibitors used in earlierpharmacological studies. In keeping with an effort to studythe contribution of nNOS to baroreflex transmission,others (Carvalho et al. 2006) have studied nNOS knockoutmice and reported that there was a significant reductionof baroreflex responses when compared with wild-typemice. However, as used, the knockout mouse model didnot allow one to assess nNOS specifically in the NTS orany other select site in the CNS.

Hypothesizing that NO from nNOS acts in an excitatorymanner on baroreflex transmission in NTS we soughtto determine if loss of expression of nNOS in NTSwould attenuate baroreflex function or if upregulationof nNOS would lead to augmented baroreflex responses.Further, in utilizing a novel nNOS shRNA, expressedvia adeno-associated virus vectors (AAV2), we sought tovalidate the efficacy and selectivity of that approach to theremoval of nNOS influences in NTS.

Methods

All studies were performed in anaesthetized adult maleSprague–Dawley rats whose level of anaesthesia was testedevery 15 min as previously described (Talman et al. 1991)by assessing whether graded tail pinch led to changes inblood pressure or withdrawal movements. All methodswere reviewed and approved by the Institutional AnimalCare and use Committee of the University of Iowa andadhered to standards established in the National ResearchCouncil’s Guide for the Care and Use of Laboratory Animals.

Preparation of AAV2 vector with cDNA for nNOS

AAV2nNOScDNA was prepared as described in our earlierpublication (Lin et al. 2011). Briefly, rat nNOS cDNA

C© 2012 The Authors. The Journal of Physiology C© 2012 The Physiological Society

J Physiol 590.15 nNOS and the baroreflex 3547

(a gift from Dr David S. Bredt, Johns Hopkins MedicalSchool) was cloned into a modified rAAV2 packagingplasmid pFBGR (Gene Transfer Vector Core, Universityof Iowa) with a CMV promoter. The AAV2nNOScDNAvectors were then prepared by a triple baculovirusinfection in SF-9 insect cells by The Gene Transfer VectorCore of The University of Iowa according to methodsdescribed previously (Urabe et al. 2002). The titre of theAAV2nNOScDNA vector was 1.12×1013 viral genomes perml. The vectors were stored at −80◦C in 20 mM Tris-HCl(pH 8.0) containing 250 mM NaCl. They were diluted tomatch the titre of AAV2nNOSshRNA (see below) and thendialysed against phosphate buffered saline (PBS, pH 7.4)at 4◦C for 15 min immediately before use.

Preparation of AAV2 vector encoding shRNA for nNOS

The sequence used for generating shRNA for nNOS wasa double-stranded DNA of 21 nucleotides from 2281to 2301 region of nNOS DNA. The first nucleotide ofthe target sequence started at ‘G’, which is required bythe RNA polymerase III promoter. We added a poly Ttermination signal for antisense oligonucleotide and anEcoRI restriction enzyme cutting site for cloning of theDNA insert. With the loop sequence of TATCGC added,the DNA sequences for shRNA of nNOS therefore wereGAGCTATCGGCTTTAAGAAATtatcgcATTTCTTAAAGCCGATAGCTCTTTTTgaattcc.

A mouse U6 promoter was also cloned into themodified pFBGR plasmid (Gene Transfer Vector Core,University of Iowa) with downstream PmeI and EcoRIrestriction sites. The sense (modified at 5′ phosphate) andthe antisense oligonucleotides for the shRNA constructwere synthesized as two complementary DNA sequencesby Integrated DNA Technologies (Coralville, IA, USA),annealed in equimolar amounts, cut by EcoRI, ligatedbetween PmeI and EcoRI site and then cloned to theplasmid. The vector also contained humanized renillagreen fluorescent protein (hrGFP) with a CMV promoter.The AAV2nNOSshRNA vectors were then prepared by atriple baculovirus infection in SF-9 insect cells by The GeneTransfer Vector Core, The University of Iowa as describedabove. The titre of AAV2nNOSshRNA was 5.9×1012 viralgenomes per ml.

Cell culture

Human embryonic kidney (HEK293) cells were used toexamine the efficiency of recombinant AAV2 plasmidthat contained nNOS shRNA (AAVp-nNOSshRNA).HEK293 cells were purchased from ATCF (AmericanType Culture Collection, Manassas, VA, USA) andgrown in six-well plates in Dulbecco’s modified Eagle’smedium and supplements with heat-inactivated fetalbovine serum and antibiotics. Because HEK does not

naturally express nNOS the cells were incubated withAAVp-nNOScDNA (to induce nNOS expression) in theabsence or presence of plasmids containing shRNAfor nNOS (AAVp-nNOSshRNA) for 48 h before theywere harvested for real time polymerase chain reaction(RT-PCR) and Western blot analysis.

NTS tissue preparation for protein and RNA analysis

For RT-PCR and Western blot analysis of nNOS expressionin vivo, rats were anaesthetized with isoflurane (5%induction and 2% maintenance) delivered by nasalcone in 100% O2, and were bilaterally injected withAAV2nNOSshRNA (individual increments of 25–50 nl toa combined total of 200 nl) into the NTS (0.4 mm rostralto the calamus scriptorius, 0.5 mm from the midline, and0.5 mm below the surface of the brainstem at the level ofthe area postrema) (Nayate et al. 2008) and were killed withan overdose of pentobarbital (150 mg kg−1) 2 weeks later.The NTS from each rat was dissected with stainless steeltubing (inner diameter of 0.96 mm) from six consecutive150 μm frozen medullary transverse sections that hadbeen cut with a cryostat. The tissue punches were storedat −20◦C until they were used for Western blot analysis orplaced in cold RNAlater (Qiagen Inc., Valencia, CA, USA)overnight and then stored at −20◦C for real time RT-PCR.In some animals injections of AAV2nNOSshRNA or PBSwere made unilaterally for subsequent immunofluorescentanalysis of nNOS.

Real time RT-PCR

Real time reverse transcriptase quantitative polymerasechain reaction (RT-PCR) was used to assess nNOS mRNAin rat NTS after transduction with AAV2nNOSshRNAas described in our earlier publication (Lin et al. 2011).Following extraction with TRIzol reagent (Invitrogen,Carlsbad, CA, USA), RNA of NTS was prepared usingthe RNeasy Mini kit (Qiagen). RNA concentrationswere determined using a NanoDrop spectrophotometer(Thermo Scientific, Wilmington, MA, USA). Reversetranscription using 300 ng RNA for each sample wasperformed according to methods described in an earlierpublication (Chu et al. 2002). We performed real timeRT-PCR in a 96-well plate with identical amountsof reverse transcription product using nNOS TaqManExpression Assays and eNOS TaqMan Expression Assayspurchased from Applied Biosystems (Carlsbad, CA, USA).These kits contain probes and primers for real timeRT-PCR for nNOS and eNOS. RT-PCR for β actin (RatACTB Endogenous Control, Applied Biosystems) was usedas an endogenous reference control and was performed inthe same well as nNOS or eNOS. FAM fluorphore was usedfor nNOS or eNOS, VIC fluorphore was used for β actin.Expression levels of nNOS or eNOS were first normalized



by β actin level, and relative expression levels were thenobtained using the ��Ct method (Wakisaka et al. 2010).

Western blot

Procedures were similar to those used in our previouspublication (Lin et al. 2011). Briefly, we homogenizedHEK293 cells or NTS tissue in buffer containing 2%sodium dodecyl sulphate (SDS). After centrifugation,protein concentration of the supernate was determinedusing Bio-Rad DC Protein Assay (Bio-Rad Laboratories,Hercules, CA, USA). Homogenate containing 10 μgprotein was separated alongside Bio-Rad Precision PlusProteins Standards (Bio-Rad Laboratories) by 7.5%SDS-polyacrylamide gel electrophoresis using the MiniProtein II System (Bio-Rad Laboratories) as previouslydescribed (Laemmli, 1970). The separated proteinswere transferred to nitrocellulose membrane (Bio-RadLaboratories) using the Mini Trans-Blot Cell (Bio-RadLaboratories). The blot was blocked in 10% milk in PBSand then incubated with sheep anti-nNOS (1:20 000) in10% milk at 4◦C for 24 h. After a thorough wash, the blotwas incubated with horseradish peroxidase-conjugatedanti-sheep antibody (1:10 000, Jackson ImmunoResearchLaboratories, Inc., West Grove, PA, USA) at 25◦Cfor 4 h. Protein bands were visualized with ECL PlusWestern Blotting Reagents (GE Healthcare/AmershamBiosciences, South San Francisco, CA, USA) and exposedto X-ray films. We used glyceraldehyde-3-phosphatedehydrogenase (GAPDH) as an internal control forWestern analysis of rat NTS. Goat anti-GAPDH anti-body (1:20 000) was purchased from GenScript (cat. no.A00191; Piscataway, NJ, USA).

Immunofluorescent and other staining

Procedures similar to those described in our previouspublications (Lin et al. 2007; Lin et al. 2008; Lin et al.2010) were used for immunofluorescent staining. Insummary, rats were deeply anaesthetized with pento-barbital (50 mg kg−1) and killed by perfusion throughthe heart with PBS followed by 4% paraformaldehyde.After killing, brains were removed, post-fixed andcryoprotected; and frozen 20 μm coronal slices werecut with a cryostat. For immunofluorescence analysis ofnNOS, tissue sections were washed with PBS, blocked with10% donkey normal serum (Jackson ImmunoResearchLaboratories) and then incubated with anti-nNOS anti-body (1:1000, made in sheep, from Dr Piers C. EmsonBarbraham Institute, Cambridge, England) (Lin et al.2000; Lin & Talman, 2001; Lin et al. 2004) in 10%donkey normal serum. After thorough washing withPBS, sections were incubated with rhodamine red X(RRX)-conjugated donkey anti-sheep IgG (1:200, Jackson

ImmunoResearch Laboratories). They were then washed,transferred to slides, air-dried, and mounted withProlong Gold Antifade reagents (Invitrogen/MolecularProbes). For immunofluorescence analyses of eNOS,N-methyl-D-aspartate receptor type 1 (NMDAR1),glutamate receptor type 2 (GluR2), vesicular glutamatetransporter type 1 (VGluT1), vesicular glutamate trans-porter type 2 (VGluT2), protein gene product 9.5(PGP9.5, a neuronal marker), tyrosine hydroxylase (TH),glial fibrillary acidic protein (GFAP), neurofilament 160(NF160), and macrophage the following primary anti-bodies were used in place of nNOS: mouse anti-eNOS anti-body (1:10, cat. no. 610296, BD Biosciences, San Jose, CA)Mouse anti-NMDAR1 (1:25, cat. no. MAB363, Chemicon,USA) (Lin & Talman, 2002), mouse anti-GluR2 (1:25,cat. no. MAB397, Chemicon) (Lin et al. 2008), rabbitanti-PGP9.5 (1:100, cat. no. AB1961ASR, Chemicon)(Lin et al. 2010), rabbit anti-TH (1:100, cat. no. AB152,Chemicon), rabbit anti-VGluT1 and rabbit anti-VGluT2(both from Dr R. H. Edwards, University of CaliforniaSan Francisco) (Fremeau et al. 2001; Lin & Talman, 2006),mouse anti-GFAP (1:100, cat. no. G3893, Sigma-Aldrich,USA), mouse anti-NF160 (1:25, cat. no. MAB5254,Chemicon), mouse anti-macrophage (1: 100, cat. no.CBL260, Millipore, Billerica, MA, USA). Appropriatefluorescent secondary antibodies made in donkey againstrespective primary antibodies were also used in placeof RRX-conjugated anti-sheep antibody. Multiple-labelimmunofluorescent staining was performed for thoseprimary antibodies that were raised in different speciesin some sections to reduce the number of animals needed.In this case, primary antibodies were mixed in incubationmedium as described in our earlier publications (Lin &Talman, 2005; Lin et al. 2007; Lin et al. 2008). In addition,we performed nuclear staining of the NTS by incubatingsections with 0.5 μM TO-PRO-3 (Invitrogen) in PBS for15 min. We analysed stained sections with a Zeiss LSM510 or LSM 701 confocal laser-scanning microscope asdescribed in our earlier publication (Lin et al. 2000; Lin &Talman, 2002; Lin et al. 2004). Digital confocal images wereobtained and processed with software provided with theZeiss LSM 510 or LSM 710. We also performed Nissl stainand examined the stained sections with a Nikon Optiphotmicroscope.

Image analysis for nNOS immunostaining

We quantified nNOS-IR in the NTS, NA, CVLMand RVLM with the NIH ImageJ software (apublic domain program available from the NIH,http://rsb.info.nih.gov/ij/). These analyses included allnNOS-IR observed in cells and processes in each of theareas and normalized by the area selected for analysis.We also counted cells that were positive for nNOS-IRin the NTS, nucleus ambiguus (NA), nodose ganglion



(NG), caudal ventrolateral medulla (CVLM) and rostralventrolateral medulla (RVLM). We used two to threesections from each animal for each of the areas analysed.NTS sections within 200 μm of the centre of the injectionsite were used (Bregma −13.40 to −13.80 mm) foranalysis. Sections selected for the NA were from Bregma−12.50 to −12.90 mm, RVLM from Bregma −12.70 to−13.10 mm, CVLM from Bregma −14.00 to −14.30 mm.Student’s two tailed t test was used to determine ifnNOS-IR was statistically significantly different betweenAAV2nNOSshNOS and PBS control groups in differentareas. Significance was accepted at P values ≤ 0.05.

Delivery of vectors into NTS for baroreflex analysis

We bilaterally microinjected (200 nl) AAV2nNOSshRNAinto NTS or, for controls, bilaterally injectedeither phosphate buffered saline, AAV2nNOScDNA, orAAV2eGFP. Animals were all allowed to recover fromsurgery and to remain in their home cage for 2 weeksbefore returning to the lab for instrumentation andstudy of baroreflex responses. After injection and removalof the pipette, buprenorphine (0.01–0.05 mg kg−1) wasadministered subcutaneously, wounds were closed andanaesthesia was stopped. After full recovery fromanaesthesia each animal was returned to its home cagewhere it remained until it was brought to the laboratory forinstrumentation and subsequent evaluation of baroreflexfunction (see below).

Instrumentation and baroreflex testing

Animals were instrumented 2 weeks after injections intoNTS. As we have previously described (Riley et al.2002), adult (approximately 300 g) male Sprague–Dawleyrats were anaesthetized with isoflurane as above. Whileanaesthetized the animals were instrumented with afemoral arterial cannula for recording of arterial pressure(AP), mean AP (MAP), and heart rate (HR) and witha femoral venous cannula for delivering propranolol,atropine, or drugs used to test the baroreflex.

The arterial baroreflex was assessed as previouslydescribed (Riley et al. 2002) in animals that wereanaesthetized with protocols that we have shown donot interfere with baroreflex responses (Talman et al.1980b). After instrumentation for recording physio-logical variables, chloralose anaesthesia (60 mg kg−1

loading dose, 20 mg kg−1 h−1; I.V.) was induced, isofluraneanaesthesia was discontinued, and 15 min later baroreflextesting began. At 15 min intervals throughout the periodwhile animals were anaesthetized with chloralose, weassessed the level of anaesthesia by performing tail pinchtesting and assessing changes in blood pressure or heartrate as well as any sign of motor response to the noxious

stimulus as we have previously reported (Talman et al.1991). Supplemental anaesthetic doses (20 mg kg−1) wereadministered before proceeding at any time when changesin blood pressure or heart rate or limb movementwere detected with the tail pinch. Reflex tachycardicresponses to depressor effects of randomly chosen doses(0.25–4 μg) of sodium nitroprusside (injected I.V.) wereassessed as were reflex bradycardic responses to pressoreffects of randomly administered doses (0.0625–1 μg)of phenylephrine (injected I.V.). The full range of dosesfor each animal was defined by AP responses so thatin each animal we sought to achieve changes of MAPranging from ±10 mmHg to ±50 mmHg. Each dose andeach agent was administered after return of AP and HRto basal levels. Because results suggested that decreasedexpression of nNOS in NTS interfered with the reflextachycardia and not reflex bradycardia, in some animalswe tested baroreflex responses 15 min after administrationof propranolol (1 mg kg−1 I.V.) to block sympatheticallymediated reflex responses in order to determine whetherreflex tachycardic responses in animals that had notreceived AAV2nNOSshRNA, but had received propranolol(n = 5), would differ from reflex tachycardic responsesin animals that had received AAV2nNOSshRNA alone(n = 7). A persistent difference between those groupsmight unmask potentially obscured parasympatheticresponses in animals treated with AAV2nNOSshRNA.In another group of animals baroreflex responses wereassessed after treatment with atropine (1 mg kg−1 I.V.) inorder to determine if reflex tachycardia or bradycardiawas similarly affected by muscarinic blockade in controlrats (n = 6) and in those treated with AAV2nNOSshRNA(n = 6). Rats were killed after baroreflex testing with anoverdose of pentobarbital (150 mg kg−1 I.V.).

Statistical analyses for baroreflex responses

Data are expressed as means ± standard error of themean (SEM) and were analysed by analysis of variance(ANOVA) with Tukey’s post hoc comparison or Bonferroniadjustment. To analyse slopes of baroreflex responses weused random coefficient regression to estimate the meanslope of regression lines for the relationship of changein HR to change in MAP within animals in a treatmentgroup, and compared mean slopes among the treatments.Significance was accepted at P values ≤ 0.05.

Results

Downregulation of nNOS in HEK cells withAAVp-nNOSshRNA

We first sought to establish that an shRNA that wedeveloped as described above efficiently downregulated



expression of nNOS in vitro. In human embryonic kidneycells (HEK293 cell line), which do not express nNOS,we induced expression of the protein by exposing thecells for 48 h to AAVp-nNOScDNA. Utilizing real timeRT-PCR we found that treated HEK cells demonstratednNOS mRNA expression after this treatment (Fig. 1A).We saw 94% reduction of induced nNOS mRNA whenthe cells were also incubated with AAVp-nNOSshRNA.Consistent with RT-PCR results, Western blot analysis alsoshowed induced expression of nNOS protein after HEKcells were incubated with AAVp-NOScDNA and reductionof nNOS after exposure of the cells to AAVp-nNOSshRNA(Fig. 1B).

Downregulation of nNOS in NTS withAAV2nNOSshRNA

To test efficacy of transduction in vivo, we firstintroduced the AAV2nNOSshRNA unilaterally (n = 6)into the NTS and documented marked loss of nNOSimmunofluorescence at the site of injection 2 weeks later(Fig. 2), when compared with that in animals (n = 5)in which PBS had been injected into the NTS (Table 1).We also observed a significant decrease (P < 0.00001) innNOS-immunoreactivity (IR) in the contralateral side ofthe NTS with grey value of 12.5 ± 1.6. The decrease innNOS-IR in the contralateral NTS most likely was dueto transport of AAV2nNOSshRNA from the injectionside to the contralateral side as AAV2 vectors undergoanterograde and retrograde transport (Lin et al. 2011).Further, we observed a significant decrease (P < 0.00001)in the number of cells that were positive for nNOS-IR inthe ipsilateral NTS (Table 2). The number of cells thatwere positive for nNOS-IR in the contralateral NTS alsowas significantly decreased (P < 0.00001), to 3.6 ± 1.1 persection of NTS. Similar to the results in our previous

Figure 1. Expression of nNOS was reduced in HEK293 cells byAAVp-nNOSshRNAA, real time RT-PCR results show that AAVp-nNOSshRNA led to 94%reduction (mean of 3 set of experiments) in induced nNOS mRNA(column AAV2shRNA). A control short hairpin RNA did not affectnNOS expression (data not shown). B, Western blot shows that adecrease in nNOS protein was also observed by AAVp-nNOSshRNA(lane AAV2shRNA). The nNOS protein band (160 kDa) is indicated bythe arrow on the left side of the blot.

study (Lin et al. 2011), injection of PBS did not changenNOS-IR in the NTS, while injection of AAV2nNOScDNAincreased nNOS-IR in the NTS, when compared with thatof un-injected rats.

As mentioned above, we have shown that AAV2 vectorsundergo anterograde and retrograde transport (Lin et al.2011). Therefore, we also examined the NG, RVLM, NA,and CVLM to determine if AAV2nNOSshRNA changesnNOS expression in these areas. Compared to that ofPBS injected controls, we saw a significant decrease(P < 0.00001) in the number of nNOS-IR positive cellsin the ipsilateral NG (Fig. 2 and Table 2), when comparedto that of PBS injected group. The number of nNOS-IRpositive cells in the contralateral NG also showed asignificant decrease (P < 0.00001) to 21 ± 5.5 per NGsection. However, we did not see significant changes innNOS-IR (Table 1) or nNOS-IR positive cell numbers(Table 2) in the RVLM, CLVM and NA animals thathad received PBS injection vs. those that had receivedAAV2nNOSshRNA injection.

In contrast to effects of AAV2nNOSshRNA on nNOSin the NTS we found no change in immunofluorescentIR for eNOS in the same region over the same periodafter transfection (Fig. 3). IR for eNOS was prominentlylocated in endothelial cells of blood vessels in the NTSwhile that for nNOS was present largely in neurons as wehave previously reported (Lin et al. 2007). Similarly, wefound no changes in immunofluorescent IR for PGP9.5(Fig. 3), TH (Fig. 3), NMDAR1, GluR2, VGluT1, VGluT2and NF160. There was a minimal decrease in GFAP IR. Inaddition, we found that nuclear staining of the injectedNTS was similar to that of a normal rat. Thus, therewas no evidence for cell loss after AAV2nNOSshRNAinjection. The injected NTS was also negative for themacrophage staining, an inflammation marker. Nisslstaining of the injected NTS also did not show any sign ofnecrosis.

RT-PCR analysis of NTS tissue punches demonstratedthat nNOS mRNA was significantly reduced (P < 0.001,Fig. 4, n = 5) in animals that had been treated withAAV2nNOSshRNA when compared with those in whichPBS alone (n = 5) had been injected into NTS. However,mRNA for eNOS was not affected by AAV2nNOSshRNA(Fig. 4). Consistent with results of immunostaining andRT-PCR, Western blot analysis of NTS tissue (Fig. 5) alsoshowed a decrease of nNOS protein to 64 ± 7% (n = 6)in rat NTS after AAV2nNOSshRNA. The reason thatWestern blot analysis showed only a moderate decrease (to64% of control) in nNOS protein when immunostainingshowed more decrease (to 20% of control) is likely to bebecause tissue we obtained for Western blot by micro-punches included some tissue that lay outside the zoneof greatest shRNA effect in NTS. Effects on nNOS in thistissue would have diluted the effect seen by nNOS-IR inNTS. In addition, Western blot analysis is known to be



more qualitative than quantitative (Alegria-Schaffer et al.2009).

Baroreflex testing after bilateral nNOS shRNA

Animals in all groups (PBS, AAV2nNOSshRNA,AAV2nNOScDNA, and AAV2eGFP) showed no signsof distress or altered activity during the 2 weeks ofobservation. Furthermore, prior to baroreflex testing inanimals anaesthetized with chloralose, basal MAP andHR did not differ between groups (Table 3). Whencompared to baroreflex responses in PBS control animals(n = 6) there was no change in response seen in eitherof the other two control groups (n = 7 for both groups,Figs 6 and 7). In contrast, in those animals treatedwith AAV2nNOSshRNA (n = 7, Figs 6 and 7) reflextachycardia in response to graded reductions in MAP,a predominantly sympathetic effect, was reduced whencompared with PBS (P = 0.007), AAV2eGFP (P = 0.02),and AAV2nNOScDNA (P = 0.009). However, betweengroups there were no significant differences in theparasympathetic limb of the baroreflex manifested byreflex bradycardic responses to increases in MAP.

Additional animals in which either PBS orAAV2nNOSshRNA had been injected into the NTS under-went baroreflex testing after intravenous administrationof propranolol (PBS n = 5; AAV2nNOSshRNA n = 5)and results were compared with the same groups ofanimals (above) that had not received propranolol (seeTable 4). We hypothesized that reflex tachycardic responses

Table 1. nNOS-IR as grey value (mean ±standard deviation) in the NTS, CVLM, NA andRVLM after injection of PBS or AAV2nNOSshRNA

PBS AAV2nNOSshRNA

NTS 48.5 ± 5.7 9.5 ± 1.6 (P < 0.00001)CVLM 53.9 ± 5.7 54.6 ± 6.2RVLM 55.9 ± 9.9 58.4 ± 6.3NA 70.8 ± 7.8 68.9 ± 6.9

Table 2. Number of cells stained for nNOS-IR(mean ± standard deviation) per section in theNTS, NG, CVLM, NA and RVLM after injection ofPBS or AAV2nNOSshRNA

PBS AAV2nNOSshRNA

NTS 40.1 ± 4.3 3.4 ± 1.1 (P < 0.00001)NG 76.2 ± 6.8 14.7 ± 4.1 (P < 0.00001)CVLM 4.8 ± 0.7 5.0 ± 1.1RVLM 4.4 ± 1.3 4.6 ± 1.7NA 2.9 ± 0.6 3.5 ± 2.1

in animals that had been treated with propranololwould be identical to reflex tachycardic responses inanimals treated with AAV2nNOSshRNA if the latterwere eliminating sympathetically mediated chronotropiceffects. We found that reflex bradycardic responses topressor effects of phenylephrine did not differ betweenany of the four groups but that reflex tachycardic responses

Figure 2. Confocal immunofluorescence imagesshowing a marked decrease in nNOS-immunoreactivity (IR) in the NTS and nodoseganglion (NG) after AAV2nNOSshRNA injectionAAV2nNOSshRNA (B) injected into the NTS decreasedthe number of cells and fibres that were positive fornNOS-IR in the NTS and dorsal motor nucleus of vagus(DMV) 2 weeks after the injection when compared witha PBS-injected control rat (A). Similarly, we also saw adecrease in nNOS-IR in the NG (D) as compared to thatof a PBS-injected rat (C). Scale bar = 100 μm.



to depressor effects of nitroprusside differed significantlybetween groups. The significant difference was achievedbecause propranolol attenuated reflex tachycardia inanimals that had received PBS into the NTS. Thus, reflex

tachycardia in animals treated with PBS alone differedsignificantly from that in animals treated with PBS pluspropanolol, those treated with AAV2nNOSshRNA, orthose treated with AAV2nNOSshRNA plus propranolol.

Figure 3. Confocal images of eNOS-IR (A and B),TH-IR (C and D) and PGP9.5-IR (E and F) in the ratNTS show no difference between PBS-injectedcontrol (A, C and E) and AAV2nNOSshRNA injected(B, D and F) ratsArrows in A and B indicate eNOS-IR in the inner lining(endothelial layer) of blood vessels. Scale bar = 50 μmin A and B, 100 μm in C–F.

Figure 4. Real time RT-PCR results showing significant reduction (∗∗P < 0.001) of nNOS mRNA in therat NTS 2 weeks after AAV2nNOSshRNA (column AAV2shRNA) injection when compared to that of PBSinjected controlsThere was no difference in eNOS mRNA expression.



There was no difference in reflex tachycardic responsesbetween any of the latter three groups.

By comparison, treating animals with the muscarinicantagonist atropine significantly reduced reflexbradycardic responses to the pressor effects ofphenylephrine in animals treated with PBS and inthose treated with AAV2nNOSshRNA. The slope of reflexbradycardia, which had not differed between the PBS andAAV2nNOSshRNA group without atropine (see Table 4),while significantly decreased in both groups, was againnot significantly different between the two groups afteratropine (Table 4). Thus, reflex bradycardic responseswere similarly reduced (no significant difference) byatropine in the PBS and AAV2nNOSshRNA groups.

Discussion

The current study, utilizing interference RNA technology,makes the following novel contributions. First, theAAV2nNOSshRNA, used for the first time in this study,downregulated expression of nNOS locally while notinfluencing expression of eNOS, which, being foundin immediate proximity of neurons expressing nNOS(Lin et al. 2007), could also contribute NO· to cellulartransmission at the site. The shRNA affected nNOS inNTS with no discernible pathological changes in thetissue as a result of injections. As further evidence ofselectivity of the shRNA, we found that, in additionto eNOS, other neuronal markers in NTS were notaffected. Thus there was no change in glutamate receptors,in vesicular glutamate transporters, in a marker ofcatecholamine neurons, in neurofilament, or in immuno-labelling for NTS neurons with PGP9.5. Furthermore,there was no sign of inflammation in NTS aftertreatment. Decreased expression of nNOS in NTS ledto attenuated baroreflex activity suggesting that NO·from nNOS serves an excitatory function in baroreflextransmission at the NTS level. However, at the sitein NTS where we altered expression of nNOS onlyreflex tachycardia, seen when blood pressure was acutelylowered, was attenuated while reflex bradycardia inresponse to acutely raised arterial pressure was not.Attenuation of reflex tachycardia was to the samedegree as that achieved by systemic administration ofpropranolol, which blocks sympathetic chronotropiccardiac effects. In addition, propranolol did not furtherreduce reflex tachycardic effects in animals treated withnNOSshRNA. This finding suggests that nNOSshRNAhad reduced sympathetic chronotropic effects on theheart to such an extent that pharmacological blockade ofsympathetic effects mediated through cardiac β-receptorsproduced no further reduction in those sympatheticchronotropic events. Thus, although some effect mediatedthrough altered withdrawal of parasympathetic tone

when baroreceptors were unloaded cannot be completelyexcluded, the data are most consistent with loss of nNOSin the NTS having maximally, if not exclusively, affectedthe sympathetic limb of the baroreflex. In analysingbaroreflex function we utilized both linear regressionanalysis (reported in this paper) and logistic analysis withsigmoid curve fitting (not shown in this paper). For eachmethod we analysed changes in HR with respect to changesin MAP given that plotting changes as opposed to plottingraw data for HR and MAP provides better regression andcurve fitting (Head & McCarty, 1987). With the formeranalysis we found that animals treated with nNOSshRNAmanifested significant changes in baroreflex responsesover the full range of decreases in AP with resulting reflexincreases in HR and of increases in AP with resulting reflexdecreases in HR. With sigmoid curve fitting we found asimilar trend that did not reach significance. However,sigmoid curve fitting after logistic analysis of data did notallow separation of the reflex tachycardic from the reflexbradycardic limbs of the reflex as did linear regression andthus failed to identify changes that only occurred in reflextachycardia. Although Head & McCarty (1987) urged useof sigmoid curve fitting applied when they had achieved afull range of changes in blood pressure (60 to 160 mmHg)and heart rate, we avoided such full range intentionally inour study given that other publications (Minisi et al. 1989)suggest that the larger the change in arterial pressure themore likely the activation of non-baroreflex responses. Asa result, the full range of our data from baroreflex testingdid not reach a high or low asymptote in each animal,thus precluding sigmoid curve fitting analysis. Therefore,we applied linear regression to each limb of the reflexseparately and uncovered significant changes only in reflextachycardic responses in the nNOSshRNA treated animals

Figure 5. Western blot analysis showing decreased nNOSexpression in rat NTS 2 weeks after AAV2nNOSshRNAinjectionThe nNOS protein band (160 kDa, top blot) and the loading controlband (GAPDH, 36 kDa, bottom blot) from the same blot analysis areindicated by the arrows on the right side of the blot.



Table 3. Basal cardiovascular variables

PBS (n = 6) AAV2eGFP (n = 7) AAV2cDNA (n = 7) AAV2shRNA (n = 7)

MAP (mmHg) 84.2 ± 3.4 88.1 ± 1.7 84.7 ± 3.1 92.6 ± 2.4HR (beats min−1) 315.5 ± 17.1 318.4 ± 6.0 302.6 ± 8.2 302.4 ± 7.8

There were no significant differences in MAP or HR between groups.

when compared with any other group. Our observationsuggests that it is important to assure adequate evaluationof each limb of baroreflex responsiveness to assure that fullrange regression (either linear or sigmoid curve fitting)is not being influenced by changes in only one limb.We recognize that others (Head & McCarty, 1987) haveused sigmoid curve fitting after logistic analysis to showchanges in baroreflex slope when only the sympatheticor parasympathetic influences had been blocked whilethe same approach, when used by us, did not despiteclear differences seen with linear regression analysis. Itis not tenable that the changes we report with linearregression are due to shifts of the stimulus responses inthat basal blood pressures did not differ and the only slope

that did differ was that representing largely sympatheticeffects. Our findings raise the possibility that at the siteof injection into NTS there are distinct cellular elementsinvolved in processing the several autonomic componentsof baroreflex activity. Thus, the product of the endothelialand neuronal isoforms of NOS may have opposing effects,eNOS being inhibitory and nNOS being excitatory, onbaroreflex function.

Utilizing viral vectors and dominant negatives tosuppress expression of eNOS, others have ascribed aninhibitory effect of NO· generated from eNOS. The eNOSeffect was linked to angiotensin in NTS and to suppressionof the baroreflex (Paton et al. 2001) and was presumed tobe tonic in that dominant negative suppression of eNOS in

Figure 6. Reflex increases (upper left hand part of regression curves) in heart rate (HR, ordinate) inresponse to decreases in mean arterial pressure (MAP, abscissa) produced by infusion of nitroprussideare significantly (P < 0.05) attenuated in animals treated with nNOSshRNA (shRNA; r = −0.52) whencompared with those seen in animals treated with PBS (r = −1.69), AAV2eGFP (eGFP; r = −1.50), orAAV2nNOScDNA (cDNA; r = −1.63) while reflex decreases (lower right hand part of regression curves)in HR in response to increases in MAP produced by infusion of phenylephrine do not differ betweengroupsSlopes (�HR/�MAP) for the tachycardic and bradycardic reflex responses are shown graphically in the bar graphinsert at the lower left hand corner with data expressed as means ± SEM. A comparison of slopes of the fullbaroreflex analysis (including reductions and increases in MAP and reflex changes in HR; not shown) did showthat responses in the PBS (r = −1.27), eGFP (r = −1.21), and cDNA nNOS (r = −1.20) groups did not differ fromeach other but responses in the shRNA group (r = −0.73) differed significantly (P = 0.002; 0.004; and 0.007respectively) from responses in each of the other groups. Basal MAP and HR did not significantly differ betweengroups (see Table 3).



NTS led to reduced blood pressure in hypertensive animals(Waki et al. 2006). However, the inhibitory action of NO·in NTS as shown in the aforementioned studies had itselfbeen questioned given findings that local overexpressionof eNOS in NTS led to hypotension and bradycardia,responses that suggest a positive baroreflex effect of NO·(Sakai et al. 2000; Hirooka et al. 2001). Both of the latterstudies used adenovirus as a vector and therefore likelytargeted glia as well as neurons (Allen et al. 2006). Others,however, have used pharmacological methods to study thequestion and have found that NO·positively modulates thebaroreflex at the NTS level (Talman & Nitschke Dragon,2004; Dias et al. 2005). In our own previously publishedwork in which we used AR-R 17477 to block, somewhatselectively, nNOS, we found attenuation of baroreflexresponses with acute inhibition of nNOS. However, AR-R17477, while 100-fold more effective an antagonist ofnNOS than of eNOS, is not completely devoid of activityon eNOS (Johansson et al. 1999). Thus, interpretation ofpublished studies is somewhat limited with respect to therole of NO· as a neurotransmitter or neuromodulator incardiovascular signal transduction in NTS, and methodsthat would avoid pharmacological effects on the enzymewere needed. It is relevant that acute blockade of nNOSwith pharmacological agents led to reduced baroreflexfunction as we report here with a method that led toaltered nNOS over the course of several weeks duringwhich transduction occurred. The two approaches, then,were complementary.

However, to avoid pharmacological manipulation of theNTS, we developed an shRNA that decreased expressionof nNOS in NTS but did not affect expression of eNOS.Similar methodology, though with a different shRNA, hasbeen used by others in the basal forebrain (Mahairakiet al. 2009); but, to our knowledge, the use of nNOSshRNA in the current paper is the first application of that

technology in assessing physiological reflex function ata central level in vivo in a mammalian species. In con-trasting experiments we used an nNOS cDNA (Silvagnoet al. 1996) (provided by Dr David Bredt), which wehave shown (Lin et al. 2011) produces an 11-fold increasein nNOS seen when punches of tissue from NTS wereanalysed by RT-PCR. Both the nNOS shRNA and nNOScDNA were introduced into the NTS via AAV2, which hasbeen shown to selectively transfect neurons (Nomoto et al.2003; Lin et al. 2011).

In this paper we report for the first time the use ofa shRNA that we developed to downregulate expressionof nNOS in central neurons of the NTS. We showthat loss of nNOS expression in NTS is associatedwith selective loss of largely sympathetically mediatedreflex tachycardia induced by acute depressor effectsof sodium nitroprusside without loss or attenuation oflargely parasympathetically mediated reflex bradycardicresponses to pressor effects of phenylephrine. Therelative contributions of sympathetic and parasympatheticinfluences on reflex tachycardia and bradycardia wereconfirmed in this study in that reflex bradycardia wasreduced to a greater extent by β-adrenergic blockadethan by muscarinic blockade, and reflex bradycardia wasblocked by the latter but not significantly affected by theformer. Further supporting persistence of cardiac vagalinfluences in animals treated with AAV2nNOSshRNAare our findings that the slope of reflex bradycardicresponses in the PBS (−0.98 beats min−1 mmHg−1) andin the AAV2nNOSshRNA (−0.91 beats min−1 mmHg−1)groups did not differ from each other and did not differfrom the slope (−0.92 beats min−1 mmHg−1) found byGuyenet et al. (1987) in animals that had been treatedwith a β-blocker. Thus, we show that loss, or significantreduction, of nNOS in NTS decreases baroreflex responses,an effect that supports the hypothesis that NO·

Figure 7. Representative physiographic traces ofchanges in arterial pressure (AP), mean arterialpressure (MAP), and heart rate (HR) in animals thathad received bilateral injections of PBS (leftpanels), AAV2nNOScDNA (middle panels) orAAV2shRNA (right panels) show similar reflextachycardic responses to depressor effects ofintravenous nitroprusside infusion in the twocontrol groups, but diminished reflex tachycardiadespite similar depressor responses in an animaltreated with shRNA



Table 4. Effects of sympathetic or vagal inhibition on baroreflex responses

PBS AAV2shRNA

Basal Basal Parasympathetic Sympathetic Basal Basal Parasympathetic SympatheticMAP HR slope slope MAP HR slope slope

(mmHg) (beats min−1) (�HR/�MAP) (�HR/�MAP) (mmHg) (beats min−1) (�HR/�MAP) (�HR/�MAP)

Withoutinhibition

84.2 ± 3.4 315.5 ± 17.1 −1.0 ± 0.1 −1.7 ± 0.3 92.6 ± 2.4 302.4 ± 7.8 −0.9 ± 0.1 −0.5 ± 0.0∗

Withpropranolol

86.2 ± 2.7 281.6 ± 13.4 −0.5 ± 0.4 −0.7 ± 0.1∗ 91.2 ± 4.9 297.0 ± 7.1 −0.8 ± 0.5 −0.6 ± 0.1∗

With atropine 91.8 ± 2.3 311.8 ± 4.0 −0.4 ± 0.1∗† −0.7 ± 0.1∗ 95.2 ± 5.1 328.7 ± 10.0 −0.5 ± 0.1∗† −0.3 ± 0.1∗

∗Significantly (P < 0.05) different from PBS without propranolol or atropine. †Significantly (P < 0.05) different from AAV2shRNAwithout propranolol or atropine. PBS without inhibition; n = 6, AAV2shRNA without inhibition; n = 7. PBS with propranolol; n = 5,AAV2shRNA with propranolol; n = 5. PBS with atropine; n = 6, AAV2shRNA with atropine; n = 6.

synthesized by nNOS plays an excitatory role in baroreflextransmission in NTS. Although baroreflex testing wasperformed under anaesthesia with α-chloralose, thereis no reason to think that the anaesthetic protocolinterfered with baroreflex responses in that we havepreviously shown that baroreflex responses with theanaesthetic as we use it are identical to those in awakeunrestrained animals (Talman et al. 1980b). The baroreflexeffect of the nNOSshRNA could not be attributed tocollateral effects on eNOS in that we found no changein expression of the latter enzyme in NTS after applicationof the shRNA. This study shows another exampleof selective alteration of predominantly sympatheticvs. predominantly parasympathetic baroreflex responsesthrough selective alteration of the expression of a specificputative transmitter or receptors in NTS (Colombariet al. 1997; Machado et al. 1997). Earlier studiesshowing selective reduction of cardiovagal elementsof baroreflex transmission did so after inhibition ofthe NMDA-type glutamate receptor while sympatheticelements of baroreflex transmission were spared, thussuggesting that the latter was mediated through actions atnon-NMDA receptors in NTS. However, as noted we havefound that cardiovascular responses to local application ofNMDA itself in the NTS are blocked by pharmacologicalinhibition of nNOS in NTS. Thus, our studies cannoteliminate the possibility that alteration of sympatheticeffects by nNOS shRNA occurs through effects on neuronsexpressing NMDA receptors. In fact, it is likely that is thecase in that we have found a high degree of colocalizationof nNOS and NMDA receptors in NTS neurons (Lin &Talman, 2002). The physiological effects of nNOSshRNAin NTS are likely due to a local effect rather than an effectof the shRNA at a distant site. We know from our earlierstudies (Lin et al. 2011) that AAV2 is retrogradely trans-ported to the NG where it may transduce signals uniformlyin neurons within that ganglion. Indeed in this studynNOS was downregulated in ganglionic neurons. Thusthe decrease in nNOS expression in the NTS after shRNAapplication could have happened at both presynaptic

and postsynaptic sites. Although we cannot completelyexclude a contribution to the physiological effects bychanges in nNOS in baroreceptor afferents, it would beunlikely that altering function of those NG neurons woulddifferentially affect one element of baroreflex transmissionat the primary neuron. Such differentiation would be morelikely at the second order neuronal level in the NTS. Theabsence of changes in nNOS expression at other brainstemsites that share reciprocal connections with NTS likewisesupports the local action in NTS.

Our studies further show that upregulation of nNOS inNTS does not enhance baroreflex responses to changes inarterial pressure. We interpret that finding as indicatingthat, in the basal state, NO· production through nNOS isalready optimal and further enhancement of the capacityfor NO· synthesis does not then alter physiologicalresponses that are under NO· control.

Our findings do not conflict with those from other labsthat suggested opposite (inhibitory) baroreflex effects ofNO· when the bioactive molecule is synthesized by eNOS.However, such differences in responses when the samefreely diffusible (Garthwaite, 1995; Lancaster, 1996) agentis released from two separate sources in close proximityto each other do raise a question about the mechanismthat could mediate the two effects. Given that nNOS andeNOS containing structures lie immediately adjacent toeach other in the NTS it is unlikely that those differencescan be explained simply by a different site of actionof NO· released from one vs. the other enzyme. As weand others have pointed out, physiological actions ofNO· may depend upon packaging of the molecule intoa larger bioactive substance such as a nitrosothiol (Ohtaet al. 1997; Lipton et al. 2001). If that were the case, onecould conjecture that different S-nitrosothiols may be themediators of differing effects of NO· in NTS control ofbaroreflex functions.

In summary, our findings provide anatomical, neuro-chemical and physiological validation of a newlydeveloped shRNA for nNOS and with that new toolthey provide support for an excitatory role of NO·



synthesized by nNOS in modulating tachycardic responsesto unloading arterial baroreceptors. The ability to inter-fere selectively with one biosynthetic enzyme with noapparent cellular damage and with no other apparentneurochemical alteration allows one to dissect individualelements of baroreflex control in the NTS in contrast toless discriminating damage to NTS neurons or less selectivepharmacological modification of NTS function. Findingthat reflex responses largely mediated by sympatheticactivation can be altered while leaving unchanged thosereflex responses largely mediated by the parasympatheticlimb of the baroreflex at the NTS level demonstratesthat select neurochemical perturbations can differentiallyaffect the two limbs of the baroreflex at the NTS level.It remains to be determined if that differential effect ismediated through different second order neurons anddifferent projection pathways from NTS.

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Author contributions

All authors approved the final version of the submittedmanuscript. All studies were performed in the authors’laboratories at the University of Iowa. L.-H.L. participated indeveloping the hypothesis, designing experiments, performingimmunofluorescence studies, performing Western analysis,analysing data and writing the manuscript. D.N.D. participatedin designing experiments, performing physiological studies,analysing data, and writing the manuscript. J.J. performedWestern blot analysis and participated in writing the manuscript.X.T. developed the shRNA, performed Western blot analysis,performed RT-PCR, and participated in writing the manuscript.Y.C. participated in designing and performing RT-PCRstudies and in writing the manuscript. C.S. participated in



developing the hypothesis, designing experiments, analysingdata, and writing the manuscript. W.T.T. developed theoriginal hypothesis, designed experiments to test the hypothesis,participated in developing the shRNA, analysed data, and wasthe lead in writing the manuscript.

Acknowledgements

This work was funded in part by NIH Grant RO1 HL 59593 (toW.T.T.), NIH Grant RO1 HL 088090 (to L.-H.L. and W.T.T.), aVA Merit Review (to W.T.T.), and NIH Grant P01 HL084207 (toC.D.S.).


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