Regulation of arterial pressure by the paraventricular nucleus in conscious rats: interactions among glutamate, GABA, and nitric oxide.

1.7 MB·English

by  Martins-Pinge, Marli C.

ORIGINALRESEARCHARTICLE
published:09January2013
doi:10.3389/fphys.2012.00490
Regulation of arterial pressure by the paraventricular
nucleus in conscious rats: interactions among
glutamate, GABA, and nitric oxide
MarliC.Martins-Pinge1†,PatrickJ.Mueller2,3†,C.MichaelFoley2,3†,CherylM.Heesch2,3 and
EileenM.Hasser2,3,4*
1DepartmentofPhysiologicalSciences,CenterofBiologicalSciences,StateUniversityofLondrina,Londrina,Brazil
2DepartmentofBiomedicalSciences,UniversityofMissouri,Columbia,MO,USA
3DaltonCardiovascularResearchCenter,UniversityofMissouri,Columbia,MO,USA
4DepartmentofMedicalPharmacologyandPhysiology,UniversityofMissouri,Columbia,MO,USA
Editedby: Theparaventricularnucleus(PVN)ofthehypothalamusisanimportantsiteforautonomic
JohnW.Osborn,University andneuroendocrineregulation.Experimentsinanesthetizedanimalsandinvitroindicate
ofMinnesota,USA aninteractionamonggamma-aminobutyricacid(GABA),nitricoxide(NO),andglutamatein
Reviewedby: thePVN.ThecardiovascularroleofthePVNandinteractionsoftheseneurotransmittersin
RogerDampney,TheUniversity
conscious animals have not been evaluated fully. In chronically instrumented conscious
ofSydney,Australia
MatthewZahner,PfizerGlobal rats, mean arterial pressure (MAP) and heart rate (HR) responses to microinjections
R&D,USA (100nl) in the region of the PVN were tested. Bilateral blockade of ionotropic excitatory
*Correspondence: aminoacid(EAA)receptors(kynurenicacid,Kyn)inthePVNproducedsmallbutsignificant
EileenM.Hasser,Dalton decreases in MAP and HR. GABA receptor blockade (bicuculline,Bic), and inhibition of
A
CardiovascularResearchCenter,
NO synthase [(NOS), N-(G)-monomethyl-L-arginine, L-NMMA] each increased MAP and
134ResearchParkDrive,
UniversityofMissouri,Columbia, HR. The NOdonorsodiumnitroprusside(SNP) produced depressorresponsesthatwere
MO65211-3300,USA. attenuatedbyBic.NOSinhibitionpotentiatedbothpressorresponsestotheselectiveEAA
e-mail:[email protected] agonist, N-methyl-D-aspartic acid (NMDA), and depressor responses to Kyn. Increases
†Presentaddress: in MAP and HR due to Bic were blunted by prior blockade of EAA receptors. Thus,
MarliC.Martins-Pinge,Department
pressorresponsestoGABAblockaderequireEAAreceptorsandGABAneurotransmission
ofPhysiologicalSciences,Centerof
BiologicalSciences,StateUniversity contributestoNOinhibition.TonicexcitatoryeffectsofglutamateinthePVNaretonically
ofLondrina,Londrina,PR,Brazil. attenuated by NO. These data demonstrate that, in the PVN of conscious rats, GABA,
CampusUniversitario,s/n,Rodovia
glutamate, and NO interact in a complex fashion to regulate arterial pressure and HR
CelsoGarciaCid,Km380,Portalde
undernormalconditions.
VersallesII,86051-990,Londrina,
PR,Brazil.
Keywords:autonomic,sympathetic,NMDA,PVN,hypothalamus
PatrickJ.Mueller,Departmentof
Physiology,WayneStateUniversity
SchoolofMedicine,540E.Canfield,
Detroit,MI48201,USA.
C.MichaelFoley,CardiacSafety
Scientist,SafetyAssessment,
CovanceLaboratories,Inc.,3301
KinsmanBlvd.,Madison,WI53704,
USA.
INTRODUCTION (RVLM), regions critical in control of the sympathetic nervous
The paraventricular nucleus (PVN) of the hypothalamus is a system(Badoer,1996;PynerandCoote,2000;Hardy,2001).
critical site of integration for autonomic- and neuroendocrine- Nitricoxide(NO)hasbeenshowntoactasagaseousneuro-
mediated cardiovascular responses (Swanson and Sawchenko, modulatorto influencesynapticfunctioninthecentral nervous
1983). In addition to its role in the hypothalamoneurohy- system (Bredt et al., 1990; Hirooka et al., 1996; Martins-Pinge
pophysialsystem,thePVNisinvolvedinregulationoftheauto- etal.,1997;Zanzinger,1999).NOsynthase(NOS)isexpressedin
nomicnervoussystem(PynerandCoote,2000;Pateletal.,2001; thePVN(Zhangetal.,1998;KantzidesandBadoer,2005;Mueller
Li and Patel, 2003; Coote, 2004). Morphological and electro- et al., 2006) and NO appears to play a role in endocrine and
physiological studies have shown that the PVN is reciprocally autonomicregulationofcardiovascularresponses.Invitrostud-
connectedtoregionsofthebrainthatareinvolvedincardiovascu- iesindicatethat neuronalactivity withinthe PVNismodulated
larregulation(Saperetal.,1976;SwansonandSawchenko,1983). by NO (Li et al., 2003; Stern, 2004). In addition, administra-
Importantly,thePVNprojectstoboththeintermediolateralcell tion ofanNO donorinto thePVN decreasesrenal sympathetic
column of the spinal cord and the rostral ventrolateral medulla nervedischarge,arterialpressure,andheartrate(HR;Hornetal.,
www.frontiersin.org January2013|Volume3|Article490|1
Martins-Pingeetal. NO,GABA,andGlutamateinPVN
1994;ZhangandPatel,1998).Conversely,microinjectionofNOS food(FormulabDiet,0.28%sodium,Purina,St.Louis,MO)ad
blockersintothePVNproducespressorandsympathoexcitatory libitum.
responses(ZhangandPatel,1998;Wangetal.,2005).Thesedata,
primarilyfromanesthetizedanimals,suggestthatNOhasatonic INSTRUMENTATION
effectwithinthePVNtoinhibitrestingsympatheticactivityand Initially, ratswereanesthetized withIsoflurane[AErane, Baxter,
arterialpressure. Deerfield,IL(5%in100%O2,2Lperminuteforinductionand
TheeffectsofNOwithinthePVNappeartoinvolveacomplex maintenanceat2–2.5%)],placedinastereotaxicapparatus(Kopf,
interaction with the neurotransmitters glutamate and gamma- Tujunga,CA)andusingaseptictechniquebilateralguidecannu-
aminobutyricacid(GABA).Ithasbeenproposedthatactivation lae(22gauge)directedtotheregionofthePVNwereimplanted.
oftheNMDAsubtypeofionotropicglutamatereceptorsincreases Coordinates for cannulae placement were similar to previous
release of NO in the PVN, which then negatively modulates work(Kenneyetal.,2001;Lietal.,2001;Zhangetal.,2002;Chen
NMDA-mediated increases in sympathetic nerve discharge (Li andToney,2003):1.8mmposteriortobregma,0.5mmlateralto
etal.,2001).DepressorandsympathoinhibitoryresponsestoNO midline,and7.6mmbelowthesurface,withtheincisorbarsetto
donorsareblockedbytheGABA receptorantagonistbicuculline 5mmabovetheinterauralplaneandthecannulavertical.Studies
A
(Bic;ZhangandPatel,1998).Thisisconsistentwiththeconcept fromourlaboratorieshaveshownsimilarcoordinatestoproduce
thatNOmaybluntresponsestoexogenousNMDAbyincreasing injectionssiteslocalizedtothePVN(Kvochinaetal.,2009;King
GABAtransmission.However,itisnotknownwhetherendoge- etal.,2012).Ratswereallowedtorecoverfor12days,anesthetized
nousNOtonicallymodulatesbasalexcitatoryeffectsofglutamate againwith Isofluraneand asepticallyinstrumented witharterial
withinthePVN. and venous catheters for measurement of arterial pressure and
TheroleofNOisknowntodifferdramaticallyduringdiffer- HRandinjectionofdrugs,respectively.Thecatheterswererouted
entlevelsofneuronalactivityandunderavarietyofphysiological subcutaneouslytothenapeoftheneck,exteriorized,andsecured
and pathophysiological conditions (Villar et al., 1994; DiCarlo to the skin with suture; catheters were filled with heparinized
etal.,2002;Felderetal.,2003;LiandPatel,2003;Muelleretal., saline (100U/ml) and sealed with a stylet. Ratswere allowed to
2003,2006;Heeschetal.,2009).However,theinteractionsamong recoverfor2daysbeforethebeginningofexperiments.
NO,GABA,andglutamatehavebeenstudiedprimarilyinanes-
thetized animals (Zhang and Patel, 1998; Li et al., 2001; Patel EXPERIMENTALPROTOCOLS
et al., 2001; Akine et al., 2003). Anesthesia is well-known to On the day of experimentation, arterial pressure, mean arte-
alter neurotransmission, including both GABAergic and gluta- rial pressure (MAP), and HR were monitored (PowerLab;
matergic neurotransmission (Franks and Lieb, 1982; Jin et al., ADInstruments, Colorado Springs, CO) for at least 30min to
2009; Olsen and Li, 2011), and autonomic and cardiovascu- ensure stable baseline parameters prior to any experimental
larregulation (Schadt and Ludbrook, 1991; Moffitt et al., 1998, manipulation. Microinjections were made using stainless steel
1999; Araújo et al., 1999; Sakima et al., 2000; Machado, 2001). injectors (33 gauge) connected by PE tubing to a 0.5μl syringe
Furthermore,anesthesiaalterscardiovascularresponsesmediated (Hamilton,Reno,NV).Injectorswerefilledwiththeappropriate
bythePVN(Kannanetal.,1987,1989).Giventhisevidence,itis solutionandinserted intotheguidecannulaepriortoinjection.
apparentthatGABA,glutamate,andNOfunctionmaydifferin Protocolsdesignedtoevaluatetoniceffectsofendogenoustrans-
the conscious state. Therefore, it is critical to evaluate the rela- mitters utilized bilateral microinjections of appropriate agents
tiverolesofGABA,glutamate,andNO,andtheirinteractions,in to block the effects of the specific transmitter. Because the time
consciousanimalsinordertodeterminetheimportanceofthese courseoftheagonistsusedisrelativelyshort,protocolsexamin-
transmittersinthePVNincontrolofthecardiovascularsystemin ing effects of agonists utilized unilateral microinjections. As in
theconsciousstate. other studies (Chen and Toney, 2003; Chen et al., 2003; Li and
This study in conscious rats tested the hypothesis that, in Pan,2007a),thePVNinitiallywasidentifiedinallratsbypressor
regardtocontrolofarterialpressure,bothNOandGABAinthe responsestoBic(1mM)withmildornobehavioralresponse.
PVN are tonically inhibitory, while glutamate is tonically exci-
TonicexcitationofthePVN
tatory. We also hypothesized that the tonic inhibitory effects of
Tonic effects on MAP and HR of ionotropic glutamate recep-
NOrequireGABAergicmechanismswithinthePVN.Finally,we
tor activation in the PVN in conscious rats were evaluated by
tested the hypothesis that endogenous NO modulates the tonic
examiningresponsestobilateralmicroinjectionofkynurenicacid
excitatoryeffectsofglutamate.
[Kyn, 40mM;100nl,(LiandPan,2007a)],ageneralionotropic
glutamate receptor antagonist. In control experiments, similar
EXPERIMENTALPROCEDURES
volumesofsaline(0.9%)wereinjectedbilaterally.
Allprocedureswereperformedaccordingtotheguidelinesstated
in the National Institutes of Health “Guide for the Care and TonicinhibitionofthePVN
Use of Laboratory Animals.” All protocols were approved by These protocols examined the tonic effects of NO and GABA
A
the University of Missouri-Columbia Animal Care and Use receptor activation within the PVN on cardiovascular func-
Committee. Twenty-nine maleSprague-Dawleyrats(280–350g, tion. Bilateral microinjections (100nl) of L-NMMA [N-(G)-
Harlan Sprague Dawley, Indianapolis, IN) were used in these monomethyl-L-arginine,2mM(Lietal.,2001),aNOSinhibitor,
studies.Theratswereallowedtoadaptinhouseforatleast1week orBic1mM,(Lietal.,2006)],aGABA receptorantagonist,were
A
beforeanyinstrumentation.Ratswereallowedaccesstowaterand madeinthePVNandhemodynamicresponsesmonitored.
FrontiersinPhysiology|IntegrativePhysiology January2013|Volume3|Article490|2
Martins-Pingeetal. NO,GABA,andGlutamateinPVN
Transmitterinteractions
To examine the interactions among NO, GABA, and glutamate
in the PVN of conscious rats, the response to manipulation of
thedifferentreceptorsystemswasevaluatedinthepresenceand
absence of other systems. Hemodynamic responses to unilat-
eralmicroinjectionoftheNOdonorsodiumnitroprusside[SNP,
1M; 100nl, (Zhang and Patel, 1998) were evaluated alone and
in the presence ofBic(1mM; 100nl), orsaline(0.9%; 100nl)].
Similarly, the effects of unilateral microinjections of the selec-
tiveionotropicglutamatereceptoragonistN-methyl-D-aspartate
[NMDA,1mM;100nl,(Lietal.,2001)]wereevaluatedaloneand
inthepresenceoftheNOSinhibitorL-NMMA(2mM;100nl).
To examine the interaction of tonic endogenous ionotropic
glutamatereceptoractivationwithendogenousNO,theresponse
tobilateralPVNinjectionofKyn(40mM,100nl)wasevaluated
inthepresenceandabsenceofinhibitionofNOSwithL-NMMA
(2mM; 100nl). Finally, the interaction of endogenous gluta-
mateandGABAwasevaluated.Hemodynamiceffectsofbilateral FIGURE1|PVNinjectionsite.Brightfieldphotomicrograph(Left)indicates
thelocationofaninjectionsitewithinthePVNofanindividualrat.100nlof
blockade of GABA receptors by microinjection of Bic (1mM;
A thedyePontamineskybluewasinjectedattheconclusionofall
100nl)wereexamined aloneandinthepresenceofblockadeof experiments.AschematicofthePVN(PaxinosandWatson,2007)is
ionotropicglutamatereceptorswithKyn(40mM;100nl). superimposedforreference.
Thetimeallowedforrecoveryfollowingtheinitialadministra-
tion of a specific agent was based on previous studies showing
recovery, as well as repeatable responses (Li et al., 2001; Chen drugs used for microinjection were relatively short-acting with
andToney,2003;Chenetal.,2003). Thesecond administration recoverytypicallywithin30–45minorless.
of each drug was made 5min after antagonist administration.
A maximum of 3 drug administrations was performed in each ANALYSISOFDATA
PVNanddrugswereappliedusingamicrosyringeconnectedby Allresultsareexpressedasmean±SEM.Responsestodrugsare
PEtubingtoaninjector.Attheendoftheexperiment,Pontamine expressedasthedifferencebetweenthebasalvalueandthepeak
skybluedye(2%;100nl)wasmicroinjectedintotheexperimen- increaseordecreasefollowingeachdoseofdrug.Student’stwo-
tal site of the brain for subsequent histological estimation of tailed paired t-test was used for comparison of hemodynamic
injectionsites. parametersbetween baselineconditionsandafterdrug.Wealso
At the end of the experiment, rats were euthanized by over- used Student’s two-tailed paired t-test to compare the response
doseofsodiumpentobarbital;guidecannulaeweredetachedfrom (change)totwodifferentdrugsortoagivendruginthepresence
theircementedpositionontheskullandbrainswerefixedin4% and absence of another perturbation in the same rat. P<0.05
formaldehyde for at least 48h. Serial coronal sections (40μm) wasconsideredtoindicatestatisticalsignificance.
were cut using a cryostat, thaw mounted on microscope slides
and stained with 1% aqueous neutral red. Figure1 shows the RESULTS
injectionsitewithinthePVNinonerat.Unfortunately,inmost BASELINEHEMODYNAMICPARAMETERS
ratsremoving theguidecannulaepriorto fixationofthebrains There were no significant differences in baseline hemodynamic
produced non-specific damage. However, injection tracts, the parametersbeforeanyprotocolsinvolvingmicroinjectionofsub-
rostral-caudalspread ofthe dye and dye spotswere confined to stancesintothePVNinconsciousrats(Table1).Averagebaseline
the brainparenchyma in regions consistent with PVN injection MAPwas117 ± 2mmHg, andHR was347± 4bpm(n=29).
sitesreportedinpreviousstudiesfromourlaboratoriesandothers Asdescribedbyothers(Martinetal.,1991),aftermicroinjection
(MartinandHaywood,1998;Kenneyetal.,2001;Lietal.,2001; ofBicandNMDAintothePVN,weoftenobservedmildbehav-
Chenetal.,2003;Kvochinaetal.,2009;Kingetal.,2012). ioral responses, including alerting and facial grooming. These
behaviorswerenotaggressiveandweredistinctly differentfrom
DRUGS the defense responses described after activation ofthe posterior
Bic methiodide, SNP, L-NMMA, and Kyn were obtained from hypothalamus(ShekharandDiMicco,1987;Wibleetal.,1988).
Sigma Chemical (St. Louis, MO). NMDA was obtained from
Tocris (St. Louis, MO). With the exception of Kyn, all drugs TONICIONOTROPICGLUTAMATERGICEXCITATIONWITHINTHEPVN
were dissolved in sterile saline. Kyn was first dissolved in a few Figure2AisanoriginalrecordshowingMAPandHRresponses
dropsof1NNaOHbeforebeingdilutedinsaline.Alldrugswere to bilateral microinjection of the broad spectrum excitatory
pHadjustedto7.3–7.5andfilteredbeforemicroinjection.Doses amino acid (EAA) receptor antagonist, Kyn, in the PVN of a
of drugs for microinjection were taken from previous studies conscious rat. Bilateral blockadeof ionotropic glutamate recep-
(Martin et al., 1991; Zhang and Patel, 1998; Chen and Toney, torswithKyn(n=4)producedasmallbutsignificantdecrease
2001;Chenetal.,2003;Lietal.,2006;LiandPan,2007a,b).Most in MAP (−6 ± 2mmHg) and HR (−15 ± 3bpm); Table1.
www.frontiersin.org January2013|Volume3|Article490|3
Martins-Pingeetal. NO,GABA,andGlutamateinPVN
Table1|Meanarterialpressure(MAP)andheartrate(HR)beforeandafterdrugsmicroinjectedinthePVN.
Groups Before After
MAP(mmHg) HR(bpm) MAP(mmHg) HR(bpm)
Bic(n=7) 119±6 346±9 141±5* 467±17*
L-NMMA(n=6) 113±5 332±10 133±2* 410±23*
Kyn(n=4) 128±6 371±7 121±4* 354±8*
SNP(n=6) 120±2 344±16 107±3* 385±11*
SNP(afterBic;n=6) 121±4 355±17 114±5* 395±21*
Bic(afterKyn;n=6) 125±4 357±14 134±5* 408±15*
Kyn(afterL-NMMA;n=4) 134±8 383±8 121±7* 354±14
NMDA(n=4) 124±2 361±12 135±2* 446±17*
NMDA(afterL-NMMA;n=4) 124±3 364±5.1 142±2* 466±11*
∗p<0.05comparedtovaluesbeforemicroinjection.
Bilateral microinjection of physiological saline (n=5) pro- NITRICOXIDEATTENUATESTONICGLUTAMATERGICEXCITATIONOF
duced no significant effect on MAP (−1 ± 1mmHg) or HR THEPVN
(−4±17bpm). As in the previous experiments, bilateral microinjection of Kyn
into the PVN produced small but significant decreases in MAP
TONICINHIBITIONWITHINTHEPVN and HR (Figure5). After a 45min recovery period, bilateral
Originaltracingsofhemodynamicresponsestobilateralmicroin- microinjection of Kyn was repeated in the presence of bilateral
jection of the GABA receptor antagonist, Bic, or the NOS NOS blockade (L-NMMA). The baseline MAP and HR 5min
A
inhibitor, L-NMMA, into the PVN of individual conscious rats after L-NMMA, immediately before the second Kyn microin-
are shown in Figures2B,C, respectively. Group data (Figure3, jection, were not different from the baseline before Kyn alone
Table1) indicate that bilateral disinhibition of the PVN by (Table1).L-NMMAenhancedthedepressoreffectofionotropic
microinjection of Bic (n=7) produced a significant increase glutamatereceptorblockadeonMAPwithoutsignificantlyalter-
in both MAP and HR. Bilateral blockade of NOS in the PVN ingtheHRresponses(Figure5).
withL-NMMA(n=6)alsoinducedapressorresponseaccom- Unilateralmicroinjectionoftheionotropicglutamateagonist,
panied by an increase in HR. The pressor responses to Bic and NMDA, into the PVN of conscious rats produced an increase
L-NMMAwerenotsignificantlydifferent,buttheHReffectswere in MAP and HR (Figure6, Table1). Unilateral microinjection
significantlygreaterforBic. of L-NMMA did not alter baseline cardiovascular parameters.
However,duringNOSinhibitionthepressorresponsetoNMDA
was significantly augmented (Figure6), consistent with a role
ACTIVATIONOFGABA RECEPTORSCONTRIBUTESTOINHIBITORY
A of NO to bluntthe excitatory effects ofNMDAreceptor activa-
EFFECTSOFNITRICOXIDEINTHEPVNOFCONSCIOUSRATS
tion.ThetachycardiainresponsetoNMDAwasnotsignificantly
Initially,responsestotwoconcentrationsoftheNOdonor,SNP,
alteredbyL-NMMA.
were determined and repeatability of the response to multiple
injections of SNP was tested. Unilateral microinjection of SNP
TONICGLUTAMATERGICINPUTCONTRIBUTESTOEXCITATIONOFTHE
(0.5Mand1M;n=5)intothePVNproducedrapiddose-related
PVNDUETOGABA RECEPTORBLOCKADE
decreasesinMAP(−11±2mmHgand−17±3mmHg,respec- A
Figure7 contains mean data demonstrating responses to bilat-
tively). The depressor response was associated with tachycardia
eral Bic before and after ionotropic glutamate receptor block-
(51±15and54±3bpm,respectively). Responsestounilateral
ade. Bilateral PVN microinjection of Bic produced significant
microinjection of SNP (0.5M) were repeatable and not altered
increases in MAP and HR. After recovery, Kyn was injected
after microinjection of saline into the PVN (MAP: −8 ± 2 vs.
bilaterally, and 5min later Bic was injected a second time.
−10±2mmHg;HR:45±5vs.32±8).
Following ionotropic glutamate receptor blockade, the pressor
In separate rats (n=6) responses to SNP (1M) were deter-
andtachycardicresponsestoBicweresignificantlyattenuated.
mined before and after injection of Bic (Figure4, Table1).
UnilateralblockadeofGABA receptorswithBicinitiallytended DISCUSSION
A
toproducesmallincreasesinMAPandHR;however,5minlater PURPOSE
hemodynamicparametershadreturnedtocontrol.Importantly, Thisstudywasdesignedtoexamine,inconsciousrats,theinflu-
baseline MAP and HR before injection of SNP alone or injec- ence of the PVN on arterial pressure and HR and to evaluate
tionafterunilateralBicwerenotstatistically different(Table1). potential interactions of NO, glutamate, and GABA in mediat-
InthepresenceofBic,theMAPresponsetoSNPwasattenuated ingtheseeffects.Wealsotestedthehypothesisthatendogenously
by∼50%(Figure4).ThetachycardicresponseproducedbySNP releasedNObluntsthetonicexcitatoryeffectsofglutamatewithin
wasnotalteredbypreviousBicmicroinjection. the PVN. The use of conscious rats is an important aspect of
FrontiersinPhysiology|IntegrativePhysiology January2013|Volume3|Article490|4
Martins-Pingeetal. NO,GABA,andGlutamateinPVN
FIGURE3|EffectsofbilateralblockadeintheregionofthePVNof
GABAAreceptors(bicuculline;Bic,1mM;openbars;n=7),orNO
synthase(NOS)(L-NMMA;2mM,filledbars;n=6)onMAPandHRin
separategroupsofconsciousrats.BilateralmicroinjectionofbothBicand
L-NMMAsignificantlyincreasedMAPandHR.ThepressorresponsetoBic
andL-NMMAwassimilar,whilethetachycardicresponsetoL-NMMAwas
significantlylessthantheresponsetoBic(∗p≤0.05).
ofGABAandNOappeartobesynergistic. Finally,thesestudies
FIGURE2|Originalrecordsfromindividualconsciousratsshowingthe
arterialpressure(AP),MeanAP(MAP),andheartrate(HR)responses demonstrateforthefirsttimethatunderbasalconditionsinthe
tobilateralmicroinjections(100nl)intothePVNof:(A)Kynurenicacid consciousrat,endogenousNOattenuatesthetonicpressoreffects
(Kyn,40mM);(B)Bicuculline(Bic,1mM);(C)L-NMMA(2mM). ofglutamatewithintheregionofthePVN.
InthePVNanumberofexcitatoryandinhibitoryneurotrans-
mitters converge to influence neuronal activity (Swanson and
these studies in that anesthesia is well-known to alter neuro- Sawchenko, 1983). Studies in vitro and in anesthetized animals
transmission, autonomicand cardiovascularfunction. Blockade suggestaninteractionamongglutamate,GABA,andNOinreg-
ofionotropic glutamatereceptors with Kyn inthe regionof the ulation of the PVN (Horn et al., 1994; Zhang and Patel, 1998;
PVNproducedasmallbutsignificantdecreaseinMAPandHR, Stern, 2004). The current experiments indicate that an interac-
suggestingthat,inconsciousrats,ionotropicglutamatergictrans- tion between glutamate and GABA, as well as an interaction of
mission in the PVN participates in tonic excitatory drive to the NOwithbothglutamateandGABA,contributestoPVNcontrol
vasculature and heart. Furthermore, the increase in MAP and ofarterialpressureinconsciousrats.Takentogether,thedatasug-
HRduetobilateralblockadeofeitherGABA receptorsorinhi- gest that in conscious rats the influence of the PVN on arterial
A
bition of NOS indicates that in the conscious state, similar to pressure and HR reflects a complex balance among a variety of
anesthetized animals,thePVNisundertonicinhibitionbyboth excitatory and inhibitory inputs. A schematic of these potential
GABA via GABA receptors and by NO. The inhibitory effects interactionsisshowninFigure8andwillbedescribedbelow.
A
www.frontiersin.org January2013|Volume3|Article490|5
Martins-Pingeetal. NO,GABA,andGlutamateinPVN
FIGURE5|EffectsofbilateralPVNblockadeofionotropicglutamate
receptorsusingkynurenicacid(Kyn,40mM)alone(openbars)orafter
bilateralinhibitionofNOSwithL-NMMA(2mM,filledbars)in
consciousrats(n=4).BilateralmicroinjectionofKynproducedsmallbut
significantdecreasesinMAPandHR.PriorinjectionofL-NMMA
FIGURE4|MAPandHReffectsofunilateralmicroinjectionoftheNO significantlyenhanceddepressorresponsestoKyn(∗p≤0.05)withno
donorsodiumnitroprusside(SNP,1.0M)intothePVNofconscious
significanteffectonHRresponses.
rats(n=6).SNPwasinjectedbefore(openbars)andafter(filledbars)
blockadeofGABAAreceptorswithbicuculline(Bic,1mM).SNPalone
producedsignificantdecreasesinMAP,accompaniedbytachycardia.Bic
significantlyattenuatedthedepressoreffectofSNP(∗p≤0.05)without support for tonic GABAergic inhibition in conscious rats and
alteringthetachycardia. also indicates that the region of the PVN is under tonic inhi-
bition dueto NO in the conscious state (Figure8B). Similarto
experiments in anesthetized animals (Zhang and Patel, 1998),
TONICEFFECTSOFTHEPVN bilateralmicroinjectionoftheNOSblockerL-NMMAincreased
ThePVNisacriticalsiteofneuroendocrineintegrationandplays MAPandHR.Inthepresentstudy,wealsoinvestigatedthecon-
an important role in modulating sympathetic tone and arterial tribution of EAAs in the region of the PVN to maintenance of
pressure under a variety of physiological and pathophysiologi- basalarterialpressureandintheresponsetoinhibitionofGABA
A
calconditionsincludingheartfailure,diabetes,andhypertension receptorsinconsciousrats.Inourexperiments,bilateralblockade
(PatelandZhang,1996;MartinandHaywood,1998;Patel,2000; of ionotropic glutamate receptors produced a small but signifi-
DiCarloetal.,2002;Felderetal.,2003;LiandPatel,2003;Mueller cant reduction in arterial pressure and HR, suggesting that the
etal.,2006;Heeschetal.,2009).However,itsroleinbasalcardio- balanceofinputsissuchthatEAAsprovideasmalltonicexcita-
vascularcontrolisnotentirelyclear,althoughitlikelyreflectsthe toryeffectwithinthePVN,whichcontributestomaintenanceof
integrationofbothexcitatoryandinhibitoryinfluences.Bilateral basalarterialpressureandHR(Figure8C).Similarexperiments
inhibition of GABA receptors with Bic in the PVN region of in anesthetized rats (Chen et al., 2003) and conscious rabbits
A
conscious(Martinetal.,1991)oranesthetized(ZhangandPatel, (Badoer et al., 2002) showed that blockade of ionotropic EAA
1998; Chen and Toney, 2003; Chen et al., 2003) rats increases receptors in the PVN had no significant effect onbaseline arte-
MAPandHR,suggestingthatthePVNisundertonicGABAergic rial pressure. The difference in these studies may be due to the
inhibition (Figure8A). The current study provides additional presence ofanesthesia, unilateralversusbilateralinjections, and
FrontiersinPhysiology|IntegrativePhysiology January2013|Volume3|Article490|6
Martins-Pingeetal. NO,GABA,andGlutamateinPVN
FIGURE6|MAPandHReffectsofunilateralmicroinjectionofthe FIGURE7|EffectsofbilateralblockadewithinthePVNofGABAA
selectiveionotropicglutamatereceptoragonistN-methyl-D-aspartic receptorswithbicuculline(Bic,1mM)onMAPandHRinconscious
acid(NMDA,1mM)intothePVNofconsciousrats(n=4).NMDAwas rats(n=6)alone(openbars)orafterblockadeofionotropicglutamate
injectedbefore(openbars)andafter(filledbars)blockadeofNOSwith receptorswithkynurenicacid(Kyn,40mM;filledbars).Bilateral
L-NMMA(2mM).NMDAaloneproducedsignificantincreasesinMAPand microinjectionofBicaloneproducedsignificantincreasesinMAPandHR.
HR.PressorresponsestoNMDAweresignificantlyenhancedbyprior PressorandtachycardicresponsestoBicweresignificantlyattenuatedby
injectionofL-NMMA(∗p≤0.05). priormicroinjectionofKyn(∗p≤0.05).
possiblespeciesdifferences.Alternatively,aspreviouslysuggested
(Badoer et al., 2002), the PVN may exert both excitatory and sympathoinhibitoryeffectsofNOintheconsciousstatearecon-
inhibitory effects onsympathetic nerveactivity, andtherelative sistent with previouswork examining responses to SNP(Zhang
influenceoftheseeffectsmayvarydependingonspeciesandstate andPatel,1998)orNOSblockade(Mastelarietal.,2011)inthe
oftheanimal.Nevertheless, thecurrentdataareconsistentwith PVN. Other studies in conscious Wistar rats reported no effect
the hypothesis that the relatively small tonic excitatory effects (Hashiguchi et al., 1997) or pressor responses (Busnardo et al.,
exerted by the PVN involve, at least in part, ionotropic gluta- 2010)toPVNmicroinjectionofNOdonors.Whilethereasonsfor
matergic transmission. Other potential sources of excitation in thisdiscrepancyarenotclear,differencesincludingdrugconcen-
the PVN (Figure8D) were not evaluated in the current experi- trationandstraincouldcontribute.Takentogether,theprepon-
ments.However,previousstudiesinanesthetizedratssuggestthat deranceofevidencesuggeststhatNOisaninhibitorymodulator
angiotensin II also contributes to excitation of the PVN under of cardiovascular control in the PVN in both conscious and
bothphysiologicalandpathophysiologicalconditions(Chenand anesthetized animals. Microinjection of SNP into the region of
Toney,2003;FreemanandBrooks,2007). the PVN was associated with tachycardia in the present exper-
iments rather than bradycardia as previously reported (Zhang
NITRICOXIDEANDGABA and Patel, 1998; Wang et al., 2005). The mechanism for this
UnilateralmicroinjectionoftheNOdonorSNPintotheregionof tachycardia is not completely clear. It is possible that it was a
thePVNofconsciousratsdecreasedMAP,andblockadeofNOS baroreflex-mediatedresponsetothedecreaseinarterialpressure.
withL-NMMA-inducedpressorandtachycardicresponses.These Alternatively, itmaybeadirecteffectofSNPinthePVNrather
www.frontiersin.org January2013|Volume3|Article490|7
Martins-Pingeetal. NO,GABA,andGlutamateinPVN
our GABA receptor blockadewas not complete in the current
A
study. Nonetheless, it appears that in conscious rats, NO exerts
primarily an inhibitory role in the PVN and this inhibition is
dependentatleastinpartonGABA.However,itispossiblethat
thecontributionofGABAtoNO-mediatedeffectsmaybelessin
consciouscomparedtoanesthetizedanimals.
NITRICOXIDEANDGLUTAMATE
ExcitatoryresponsestoactivationoftheNMDAsubtypeofglu-
tamate receptors in the region of the PVN were significantly
enhanced by prior blockade of NOS. These data in conscious
ratsextendpreviousfindingsinanesthetizedrats(Lietal.,2001)
demonstratingthattheexcitatory effectsofexogenouslyadmin-
isteredNMDAinthePVNarebluntedbyNO(Figure8,dashed
line).Althoughnotspecificallyexaminedinourstudy,thiseffect
ofNOmayalsoinvolveaneffectmediatedbyGABA(Figure8E)
ratherthanmodulationofglutamatergicsignaling,asNOappears
to increase directly glutamate release (Horn et al., 1994) and
AMPA-mediated currents (Roychowdhury et al., 2006) in the
PVN. The effects ofNO to modulate GABA function appearto
predominate, possiblydueto greater tonic GABAergicinputon
FIGURE8|Schematicrepresentationofpotentialinteractionsof PVNneurons.
inhibitoryandexcitatoryinputstothePVN.Thecellintheschematic WealsoexaminedtheinfluenceofNOontheeffectsoftonic
representsaPVNneuroninvolvedincontrolofsympatheticnervous
endogenous ionotropic glutamate receptor mechanisms within
systemactivity.Asdescribedinthetext,thisneuronreceivesinhibitory
influencesduetoGABA,viaGABAAreceptors(A),andNO(B),which thePVN.TheNOSinhibitorL-NMMAenhancedthedepressor
appearstoexertitseffectsatleastinpartthroughGABA(E).Excitatory response to bilateral inhibition of ionotropic glutamate recep-
inputsincludebothexcitatoryaminoacid(EAA,C)andnon-ionotropicEAA tors with Kyn. Importantly, the depressor response to Kyn was
receptormediated(non-EAA)(D)inputs.NO,likelyactingthroughGABA,
enhancedeventhoughL-NMMAwouldbeexpected toincrease
bothbluntstheresponsetoactivationofNMDAreceptors,andtonically
arterial pressure over the same time period. These data sug-
inhibitstheeffectsofongoingEAAexcitation.
gestthatinconsciousratsnotonlydoesNOblunttheresponse
toexogenouslyadministered NMDA,buttheendogenous,tonic
excitatory effects of glutamate in the PVN also are under tonic
thanbaroreflex-mediated, becausealthoughthedepressoreffect
negativemodulationbyendogenousNO.Toourknowledge,this
ofSNPwasdiminishedinthepresenceofBic,thetachycardiawas
is the first demonstration of an effect of endogenous NO on
unchanged.Thus,thetachycardialikelywasnotdependentonthe
tonic EAA-mediated excitation within the PVN. Since we also
magnitude of the depressor response. Furthermore, it does not
found that the pressor response to exogenous NMDAis poten-
appeartorequireGABA receptors,becauseitwasnotalteredby
A
tiated following blockade of NOS, tonic effects of endogenous
pretreatmentwithBic.
NO also may involve the NMDA subtype of glutamate recep-
Full expression of the depressor effect of NO in the PVN
tors.Thus,theoveralleffectofNOwithinthePVNisinhibitory,
regionofawakeratsappearstorequireGABAergictransmission
(Figure8E), since responses to SNP were attenuated ∼50% by sinceNOattenuatestonicexcitationduetobothexogenousand
tonic endogenous glutamate, and facilitates inhibitory GABA
Bic.Inpreviousworkinanesthetized animals(ZhangandPatel,
neurotransmission.
1998), Bic abolished responses to SNP. Thus, it appears that in
theabsenceofanesthesia,aportionoftheresponsetoNOdonors
is independent of GABA. Another possibility may be related to GABAANDGLUTAMATE
the dose of Bic used. In the present study, we used a relatively EndogenousGABAandglutamatealsoappeartointeractinthe
low dose of Bic (1mM). As reported by others (Martin et al., PVN of conscious rats. Bilateral removal of GABAA receptor-
1991),weobservedthatBicinjectionintothePVNofconscious mediated inhibition in the region of the PVN produced pres-
rats resulted in mild behavioral responses (such as licking and sor and tachycardic responses, effects observed in other studies
grooming).Wefoundthathigherdosestendedtoelicitlocomotor usingconsciousrats(Martinetal.,1991;deAbreuetal.,2009).
activitythatinterferedwithmeasurementsofarterialpressureand Experimentsinanesthetizedanimalssuggestthatthisresponseis
HR.Therefore, wechoseadosewithinthelowerrangeofthose due to an unmasking of excitatory inputs, including glutamate
previouslyusedinanefforttocircumventthisconcern.Itispos- and angiotensin II (Chen and Toney, 2003; Chen et al., 2003;
siblethatthisrelativelylowdoseofBicdidnotcompletelyblock LiandPan,2007a).Inthecurrentstudy,excitatoryresponsesto
GABAAreceptors.Althoughpreviousstudiesinanesthetizedrats GABAA blockade were blunted during inhibition of ionotropic
indicate that cardiovascular responses to 40mM and 1mM Bic glutamatereceptors.Thesedatasuggestthatintheconsciousrat,
microinjectedintothePVNweresimilar(ZhangandPatel,1998; glutamate provides a substantial portion of the excitation that
Lietal.,2006),wecannotcompletelyeliminatethepossibilitythat is evident when GABAA inhibition is removed (Figure8C), or
FrontiersinPhysiology|IntegrativePhysiology January2013|Volume3|Article490|8
Martins-Pingeetal. NO,GABA,andGlutamateinPVN
glutamatergicinputsmaybetonicallyinhibitedbyGABA.Inthis responsesinthePVN,disinhibition(Bic)orexcitation(NMDA)
regard, following Bic in the PVN, release of glutamate and the producedpressorresponseswithlimitedbehavioralactivation.In
frequencyofglutamatergicEPSCsareincreased(Lietal.,2006). addition, our coordinates are consistent with those used previ-
Alternatively,GABAergicneuronsintheregionofthePVNmay ously by us, in which injection sites were confirmed (Kvochina
be excited tonically by glutamate. Althoughsimultaneous disfa- et al., 2009; King et al., 2012). Collectively, this evidence sug-
cilitation due to Kyn could contribute to the blunted response gests that responses were due primarily to changes in neuronal
toBic,thismostlikelyinvolvesaninteractionbetweenglutamate activationintheregionofthePVN.
and GABA as previously suggested (Chen et al., 2003) because
the minor effects of Kyn alone are not sufficient to account for SUMMARY
the magnitude of the diminished response. Future experiments The combined data from these studies in conscious rats is
are required to evaluate the cellular mechanisms of these inter- consistent with previous work in anesthetized animals and
actions, aswellasapotential roleforothertransmitters suchas suggests that the PVN is under tonic excitation due to endoge-
angiotensinII. nous activation of ionotropic glutamate receptors. In addi-
tion, both NO and GABA have tonic inhibitory effects in
LIMITATIONS the region of the PVN in conscious rats and this inhibi-
As with any study our conclusions have certain caveats that tion masks the majority of tonic glutamatergic excitation.
must be taken into consideration. For example, in the cur- Importantly, we found that in conscious rats, endogenous NO
rent study we measured arterial pressure and HR in conscious appears to tonically blunt the ongoing activation of the PVN
rats. It is most likely that responses to microinjections are due by EAAs. The nature of these tonic effects involves a com-
primarily to changes in sympathetic nervous system activity. plex interaction among glutamatergic, GABAergic, and NO
Although this assumptionis consistent with previousstudiesin mechanisms.
anesthetized animals (Zhang and Patel, 1998; Li et al., 2001),
numerous neural and humoral factors influence arterial pres- ACKNOWLEDGMENTS
sure, and the influence of the PVN on sympathetic activity The authors would like to acknowledge the outstanding tech-
may vary depending on the specific sympathetic nerve evalu- nical expertise of Sarah A. Friskey. We would also like to
ated. Therefore, future experiments are necessary to confirm thank the Neurohumoral Control of the Circulation group
the involvement of the sympathetic nervous system, includ- at the Dalton Cardiovascular Research Center for valuable
ing effects on sympathetic activity to specific regions, vs. other input on this project. This study was supported by the
neurohumoralfactors. National Institutes of Health HL 55306 and HL 64669
Also,becauseofnon-specificdamageduringremovalofguide (Eileen M. Hasser). This investigation was conducted in
cannulae, we cannot say unequivocally that all injections were a facility constructed with support from Research Facilities
centeredinthePVN.However,injectionsiteswereconfinedtothe Improvement Program Grant Number CO6 RR-16498 from
brainparenchyma, andthe primarycardiovascularregionnear- the National Center for Research Resources, National Institutes
esttheinjectionswasinfactthePVN.Inallrats,consistentwith ofHealth.
REFERENCES oxide synthase indicating a neural Chen,Q.H.,andToney,G.M.(2003). hypertensive rats. Brain Res. 955,
Akine,A.,Montanaro,M.,andAllen,A. role for nitric oxide. Nature 347, Responses to GABA-A receptor 153–160.
M.(2003).Hypothalamicparaven- 768–770. blockade in the hypothalamic Felder, R. B., Francis, J., Zhang, Z.
tricularnucleusinhibitiondecreases Busnardo,C.,Crestani,C.C.,Tavares, PVN are attenuated by local H., Wei, S. G., Weiss, R. M., and
renal sympatheticnerveactivityin R.F.,Resstel,L.B.,andCorrea,F.M. AT1receptorantagonism.Am.J.Physiol. Johnson, A. K. (2003). Heart fail-
hypertensiveandnormotensiverats. (2010). Cardiovascular responses Regul. Integr. Comp. Physiol. 285, ureandthebrain:newperspectives.
Auton.Neurosci.108,17–21. to L-glutamate microinjection R1231–R1239. Am.J.Physiol.Regul.Integr.Comp.
Araújo, G. C., Lopes, O. U., and into the hypothalamic paraven- Coote,J.H.(2004).Aroleforthepar- Physiol.284,R259–R276.
Campos, R. R. Jr. (1999). tricular nucleus are mediated aventricularnucleusofthehypotha- Franks,N.P.,andLieb,W.R.(1982).
Importance of glycinergic and by a local nitric oxide-guanylate lamusintheautonomiccontrolof Molecular mechanisms of general
glutamatergic synapses within cyclasemechanism.BrainRes.1344, heart andkidney.Exp.Physiol.90, anaesthesia.Nature300,487–493.
the rostral ventrolateral medulla 87–95. 169–173. Freeman, K. L., and Brooks, V. L.
for blood pressure regulation in Chen, Q. H., Haywood, J. R., deAbreu,S.B.,Lenhard,A.,Mehanna, (2007). AT1 and glutamatergic
conscious rats. Hypertension 34, and Toney, G. M. (2003). A., de Souza, H. C. D.,de Aguiar receptorsinparaventricularnucleus
752–755. Sympathoexcitation by PVN- Correa,F.M.,Hasser,E.M.,etal. support blood pressure during
Badoer,E.(1996).Cardiovascularrole injected bicuculline requires (2009). Role of paraventricular water deprivation. Am. J. Physiol.
ofparvocellularneuronsinthepar- activation of excitatory amino nucleus in exercise training- Regul. Integr. Comp. Physiol. 292,
aventricularnucleusofthehypotha- acid receptors. Hypertension 42, induced autonomic modulation R1675–R1682.
lamus.Physiology11,43–47. 725–731. in conscious rats. Auton.Neurosci. Hardy,S. G.P. (2001).Hypothalamic
Badoer, E., Ng, C. W., and De, M. Chen, Q. H., and Toney, G. M. 148,28–35. projections to cardiovascular cen-
R. (2002). Tonic sympathoinhibi- (2001). AT1-receptor blockade in DiCarlo, S. E., Zheng, H., Collins, tersofthemedulla.BrainRes.894,
tionarisingfromthehypothalamic thehypothalamicPVNreducescen- H. L., Rodenbaugh, D. W., and 233–240.
PVNintheconsciousrabbit.Brain tral hyperosmolality-induced renal Patel, K. P. (2002). Daily exer- Hashiguchi, H., Ye, S. H., Ross-
Res.947,17–24. sympathoexcitation.Am.J.Physiol. cise normalizes the number of Cisneros, F., and Alexander, N.
Bredt,D.S.,Hwang,P.M.,andSnyder, Regul. Integr. Comp. Physiol. 281, diaphorase (NOS) positive neu- (1997).Centralnitricoxidedonors
S.H.(1990).Localizationofnitric R1844–R1853. rons in the hypothalamus of attenuatecardiovascularandcentral
www.frontiersin.org January2013|Volume3|Article490|9
Martins-Pingeetal. NO,GABA,andGlutamateinPVN
norepinephrineresponsestostress. nucleus. Physiol. Behav. 97, modulation after exercise training Sakima, A., Yamazato, M., Sesoko,
Am.J.Physiol.Regul.Integr.Comp. 171–179. in unanesthetized rats. Brain Res. S., Muratani, H., and Fukiyama,
Physiol.272,R1447–R1453. Li, D. P., and Pan, H. L. (2007a). 1375,68–76. K. (2000). Cardiovascular and
Heesch, C. M., Zheng, H., Foley, Glutamatergic inputs in the Moffitt,J.A.,Foley,C.M.,Schadt,J.C., sympatheticeffectsofL-Glutamate
C. M., Mueller, P. J., Hasser, hypothalamic paraventricular Laughlin,M.H.,andHasser,E.M. and glycine injected into the
E. M., and Patel, K. P. (2009). nucleus maintain sympathetic (1998). Attenuated baroreflex con- rostral ventrolateral medulla of
Nitric oxide synthase activity vasomotor tone in hypertension. trol of sympathetic nerve activity conscious rats. Hypertens. Res. 23,
and expression are decreased in Hypertension49,916–925. aftercardiovasculardeconditioning 633–641.
the paraventricular nucleus of Li,D.P.,andPan,H.L.(2007b).Roleof inrats.Am.J.Physiol.Regul.Integr. Saper, C. B., Loewy, A. D., Swanson,
pregnant rats. Brain Res. 1251, γ-aminobutyricacid(GABA)Aand Comp.Physiol.274,R1397–R1405. L. W., and Cowan, W. M. (1976).
140–150. GABAB receptors in paraventricu- Moffitt, J. A., Schadt, J. C., and Direct hypothalamo-autonomic
Hirooka, Y., Polson, J. W., and lar nucleus in control of sympa- Hasser, E. M. (1999). Altered connections. Brain Res. 117,
Dampney,R.A.L.(1996).Pressor theticvasomotortoneinhyperten- central nervous system processing 305–312.
and sympathoexcitatory effects of sion.J.Pharmacol.Exp.Ther.320, of baroreceptor input following Schadt,J.C.,andLudbrook,J.(1991).
nitric oxide in the rostral ventro- 615–626. hindlimb unloading in rats. Am. Hemodynamic and neurohumoral
lateral medulla. J. Hypertens. 14, Li, Y.-F., Jackson, K. L., Stern, J. E., J. Physiol.HeartCirc. Physiol.277, responses to acute hypovolemia in
1317–1324. Rabeler,B.,andPatel,K.P.(2006). H2272–H2279. consciousmammals.Am.J.Physiol.
Horn,T.,Smith,P.M.,McLaughlin,B. Interactionbetweenglutamateand Mueller,P.J.,Cunningham,J.T.,Patel, 260,H305–H318.
E.,Bauce,L.,Marks,G.S.,Pittman, GABA systems in the integration K. P., and Hasser, E. M. (2003). Shekhar, A., and DiMicco, J. A.
Q. J., et al. (1994). Nitric oxide ofsympatheticoutflowbythepar- Proposed roleoftheparaventricu- (1987). Defense reaction elicited
actions in paraventricular nucleus: aventricularnucleusofthehypotha- larnucleusincardiovasculardecon- by injection of GABA antagonists
cardiovascular and neurochem- lamus. Am. J. Physiol. Heart Circ. ditioning.ActaPhysiol.Scand.177, and synthesis inhibitors into the
ical implications. Am. J. Physiol. Physiol.291,2847–2856. 27–35. posterior hypothalamus in rats.
Regul. Integr. Comp. Physiol. 266, Li,Y.-F.,Mayhan,W.G.,andPatel,K. Mueller,P.J.,Foley,C.M.,Heesch,C. Neuropharmacology26,407–417.
R306–R313. P.(2001).NMDA-mediatedincrease M.,Cunningham,J.T.,Zheng,H., Stern, J. E. (2004). Nitric oxide and
Jin, Y. H., Zhang, Z., Mendelowitz, in renal sympathetic nerve dis- Patel,K.P.,etal.(2006).Increased homeostatic control: an intercellu-
D., and Andresen, M. C. (2009). chargewithinthePVN:roleofnitric nitric oxide synthase activity and larsignallingmoleculecontributing
Presynaptic actions of propofol oxide. Am. J. Physiol. Heart Circ. expressioninthehypothalamus of to autonomic and neuroendocrine
enhance inhibitory synaptic trans- Physiol.281,H2328–H2336. hindlimbunloadedrats.BrainRes. integration.Prog.Biophys.Mol.Biol.
mission in isolated solitary tract Li, Y.-F., and Patel, K. P. (2003). 1115,65–74. 84,197–215.
nucleus neurons. Brain Res. 1286, Paraventricular nucleus of the Olsen, R., and Li, G. D. (2011). Swanson,L.W.,andSawchenko,P.E.
75–83. hypothalamusandelevatedsympa- GABA(A) receptors as molecular (1983). Hypothalamic integration:
Kannan, H., Hayashida, Y., and thetic activity in heart failure: the targetsofgeneralanesthetics:iden- organizationoftheparaventricular
Yamashita, H. (1989). Increase alteredinhibitorymechanisms.Acta tification of binding sites provides and supraoptic nuclei. Annu. Rev.
in sympathetic outflow by par- Physiol.Scand.177,17–26. cluestoallostericmodulation.Can. Neurosci.6,269–324.
aventricular nucleus stimulation Li, Y., Zhang, W., and Stern, J. E. J.Anaesth.58,206–215. Villar,M.J.,Ceccatelli, S.,Ronnqvist,
in awake rats. Am.J. Physiol. 256, (2003).Nitricoxideinhibitsthefir- Patel,K.P.(2000).Roleofparaventric- M., and Hokfelt, T. (1994). Nitric
R1325–R1330. ing activity of hypothalamic par- ular nucleus in mediating sympa- oxidesynthaseincreasesinhypotha-
Kannan, H., Niijima, A., and aventricular neurons that inner- theticoutflowinheartfailure.Heart lamic magnocellular neurons
Yamashita, H. (1987). Inhibition vatethemedullaoblongata:roleof Fail.Rev.5,73–86. after salt loading in the rat. An
of renal sympathetic nerve activ- GABA.Neuroscience118,585–601. Patel, K. P.,Li,Y.-F., and Hirooka, Y. immunohistochemical and in situ
ity by electrical stimulation of Machado, B. H. (2001). Neuro- (2001).Roleofnitricoxideincen- hybridizationstudy.BrainRes.644,
the hypothalamic paraventricu- transmission of the cardiovascular tralsympatheticoutflow.Exp.Biol. 273–281.
lar nucleus in anesthetized rats. reflexesinthenucleustractussoli- Med.226,814–824. Wang, Y., Lui, X. F., Cornish, K. G.,
J.Auton.Nerv.Syst.21,83–86. tariiofawakerats.Ann.N.Y.Acad. Patel, K. P., and Zhang, K. (1996). Zucker,I.H.,andPatel,K.P.(2005).
Kantzides, A., and Badoer, E. (2005) Sci.940,179–196. Neurohumoral activation in heart Effects of nNOS antisense in the
nNos-containing neurons in the Martin, D. S., and Haywood, J. R. failure: role of paraventricular paraventricular nucleus on blood
hypothalamusandmedullaproject (1998). Reduced GABA inhibition nucleus. Clin. Exp. Pharmacol. pressureandheartrateinratswith
to the RVLM. Brain Res. 1037, of sympathetic function in renal- Physiol.23,722–726. heart failure.Am.J. Physiol.Heart
25–34. wrapped hypertensive rats. Am. J. Paxinos,G.,andWatson,C.(2007).The Circ.Physiol.288,H205–H213.
Kenney, M. J., Weiss, M. L., Patel, Physiol.Regul.Integr.Comp.Physiol. RatBraininStereotaxicCoordinates. Wible,J.H.Jr.,Luft,F.C.,andDiMicco,
K. P., Wang, Y., and Fels, R. 275,R1523–R1529. SanDiego,CA:AcademicPress. J. A. (1988). Hypothalamic GABA
J. (2001). Paraventricular nucleus Martin,D.S.,Segura,T.,andHaywood, Pyner, S., and Coote, J. H. (2000). suppresses sympathetic outflow to
bicuculline alters frequency com- J. R. (1991). Cardiovascular Identification of branching the cardiovascular system. Am. J.
ponents of symapthetic nerve dis- responses to bicuculline in the paraventricular neurons of the Physiol.Regul.Integr.Comp.Physiol.
chargebursts.Am.J.Physiol.Heart paraventricular nucleus of the rat. hypothalamus that project to the 254,R680–R687.
Circ.Physiol.281,H1233–H1241. Hypertension18,48–55. rostroventrolateral medulla and Zanzinger,J.(1999).Roleofnitricoxide
King, T. L., Heesch, C. M., Clark, C. Martins-Pinge,M.C.,Baraldi-Passy,I., spinal cord. Neuroscience 100, intheneuralcontrol ofcardiovas-
G.,Kline,D.D.,andHasser,E.M. andLopes,O.U.(1997).Excitatory 549–556. cularfunction.Cardiovasc.Res.43,
(2012). Hypoxia activates nucleus effects of nitric oxide within the Roychowdhury, S., Noack, J., 639–649.
tractus solitarii neuronsprojecting rostral ventorlateral medulla of Engelmann, M., Wolf, G., and Zhang, K., and Patel, K. P. (1998).
to the paraventricular nucleus of freelymovingrats.Hypertension30, Horn,T.F.(2006).AMPAreceptor- Effect of nitric oxide within the
the hypothalamus. Am. J. Physiol. 704–707. induced intracellular calcium paraventricular nucleus on renal
Regul. Integr. Comp. Physiol. 302, Mastelari, R. B., de Souza, H. C., response in the paraventricular sympathetic nerve discharge:
R1219–R1232. Lenhard, A., de Aguiar Correa, nucleus is modulated by nitric role of GABA. Am. J. Physiol.
Kvochina, L., Hasser, E. M., and F. M., and Martins-Pinge, M. C. oxide: calcium imaging in a Regul. Integr. Comp. Physiol. 275,
Heesch, C. M. (2009). Pregnancy (2011). Nitric oxide inhibition hypothalamic organotypic cell R728–R734.
decreases GABAergic inhibition of in paraventricular nucleus on culture model. Nitric Oxide 14, Zhang, K., Zucker, I. H., and Patel,
the hypothalamic paraventricular cardiovascular and autonomic 290–299. K. P. (1998). Altered number of
FrontiersinPhysiology|IntegrativePhysiology January2013|Volume3|Article490|10