Nervous System - pdfs.semanticscholar.org filebeen well accepted as osmosensors in the central nervous system, the subfornical organ and organum vasculosum laminae terminalis (OVLT)

Nervous System

Macrophage Migration Inhibitory Factor in theParaventricular Nucleus Plays a Major Role in the

Sympathoexcitatory Response to SaltEduardo Colombari, Debora S.A. Colombari, Hongwei Li, Peng Shi, Ying Dong, Nan Jiang,

Mohan K. Raizada, Colin Sumners, David Murphy, Julian F.R. Paton

Abstract—Central hyperosmotic stimulation (HS) evokes increases in sympathetic nerve activity mediated by activation ofangiotensin type 1 receptors in the hypothalamic paraventricular nucleus (PVN). Macrophage inhibitory migration factor(MIF) is an intracellular inhibitory regulator of angiotensin type 1 receptor–mediated actions of angiotensin II withinneurons of the PVN. MIF mediates its actions via its intrinsic thiol-protein oxidoreductase activity. We demonstrate thatintracerebroventricular injection of hypertonic saline into Sprague-Dawley rats elicits a significant (�112%) increase inMIF mRNA expression in the PVN. Next, we evaluated the effect of viral-mediated expression of either MIF or[C60S]-MIF (which lacks thiol-protein oxidoreductase activity) in the PVN on the sympathoexcitation evoked by HS.We used a decorticate, arterially perfused in situ preparation of male Wistar rats (60 to 80 g). HS was induced by raisingperfusate osmolality from 290 to 380 milliosmoles for 40 seconds. Seven to 10 days before experiments, rats wereinjected bilaterally (500 nL per side) with 0.9% saline (control) or with adenoassociated virus to express MIF,[C60S]-MIF, or enhanced green fluorescent protein in the PVN. HS produced sympathoexcitation in both the 0.9%saline and enhanced green fluorescent protein groups (sympathetic nerve activity increase of �27�4% and �25�4%,respectively; P�0.05), an effect that was not observed in the MIF group (�4�5%). Conversely, the HS-inducedincrease in sympathetic nerve activity was potentiated in the [C60S]-MIF group (�45�6%; P�0.05). We propose thatMIF acting within the PVN is a major counterregulator of HS-induced sympathoexcitation, an effect that depends onthiol-protein oxidoreductase activity. (Hypertension. 2010;56:956-963.)

Key Words: hypothalamus � gene transfer � sympathetic nerve activity � angiotensin type 1 receptors� macrophage migration inhibitory factor

The paraventricular nucleus (PVN) of the hypothalamusplays a crucial role in the regulation of cardiovascular

function1 and body fluid homeostasis.2,3 Studies have shownthat activation of PVN neurons increases blood pressure andsympathetic nerve activity in response to a hyperosmoticchallenge, which is driven by the afferent inputs from thelamina terminalis in the forebrain.4–7 Two key structures havebeen well accepted as osmosensors in the central nervoussystem, the subfornical organ and organum vasculosumlaminae terminalis (OVLT) located along the dorsal andventral portions of the lamina terminalis, respectively. Inrespect to OVLT, it has been shown to possess osmosensi-

tivity.4,5,8–10 Lesion or chemical inhibition of OVLT neuronsabolishes hyperosmolality-induced drinking,11–13 vasopressinrelease,6,11,14 and sympathoexcitation.9 Activated OVLT neu-rons project to PVN neurons via monosynaptic input or via arelay in the median preoptic nucleus.10,15–18

The PVN integrates multiple inputs from forebrain (eg,OVLT)2,10,19 and hindbrain (eg, nucleus tractus solitarii)3

during osmotic perturbations and then modulates vasopressinrelease and sympathetic discharge appropriately. PVNhyperosmolality-induced pressor responses and sympa-thetic activation involve activation of the angiotensin type1 receptor (AT1R)20,21 and distinct descending path-

Received April 19, 2010; first decision May 14, 2010; revision accepted September 9, 2010.From the School of Physiology and Pharmacology, Bristol Heart Institute (E.C., J.F.R.P.), and Henry Wellcome Laboratories for Integrative

Neuroscience and Endocrinology (D.S.A.C., D.M.), University of Bristol, Bristol, United Kingdom; Department of Physiology and Functional Genomics(H.L., P.S., Y.D., N.J., M.K.R., C.S.), University of Florida, Gainesville, Fla; Departamento de Fisiologia e Patologia (D.S.A.C.), Faculdade deOdontologia de Araraquara, Sao Paulo State University, Araraquara, Sao Paulo, Brazil.

E.C. and D.S.A.C. contributed equally to this article.Permanent address for D.S.A.C.: Departamento de Fisiologia e Patologia, Faculdade de Odontologia de Araraquara, Sao Paulo State University, Rua

Humaita, 1680, Araraquara, 14801-903, SP, Brazil.Permanent address for E.C.: Departamento de Fisiologia, Universidade Federal de Sao Paulo/Escola Paulista de Medicina, Rua Botucatu, 862, Sao

Paulo, 04023-060, SP, Brazil.Correspondence to Julian F.R. Paton, School of Physiology and Pharmacology, Bristol Heart Institute, Medical Sciences Building, University of Bristol,

Bristol BS8 1TD, UK. E-mail [email protected]© 2010 American Heart Association, Inc.

Hypertension is available at http://hyper.ahajournals.org DOI: 10.1161/HYPERTENSIONAHA.110.155101

956

by guest on October 18, 2017

http://hyper.ahajournals.org/D

ownloaded from

by guest on O

ctober 18, 2017http://hyper.ahajournals.org/

Dow

nloaded from



ownloaded from

by guest on O


Dow

nloaded from



ownloaded from

by guest on O


Dow

nloaded from



ownloaded from

by guest on O


Dow

nloaded from

http://hyper.ahajournals.org/








ways,22–24 including glutamatergic25 and vasopressinergicpathways.19,26

Considering that hyperosmolality, angiotensin II (Ang II)and AT1R in the PVN exert profound stimulatory influenceson sympathetic outflow and arterial blood pressure, it is,therefore, important to understand the mechanisms within thisnucleus that mediate these responses. Our attempts to under-stand regulatory mechanisms led to the discovery of macro-phage migration inhibitory factor (MIF) as a novel intracel-lular inhibitory regulator of AT1R-mediated actions of Ang IIwithin PVN neurons. We demonstrated that MIF acts intra-cellularly to counterregulate the firing responses evoked byAng II in PVN neurons cultured from normotensive rats.27,28

MIF elicits this inhibitory effect via its intrinsic thiol-proteinoxidoreductase (TPOR) activity that is exerted by a C-A-L-Cmotif that exists at residues 57 to 60 of the MIF molecule.27,28

MIF is expressed in neurons in the PVN of normotensive rats,and intracerebroventricular (ICV) injection of Ang II increasesMIF expression in this cardiovascular control center.28,29 Inaddition, transient viral-mediated transduction of MIF into the

PVN of normotensive rats reduces the pressor response toICV-injected Ang II.28 Collectively, this evidence indicates thatMIF is a counterregulator of Ang II–induced cardiovasculareffects mediated via the PVN in normotensive rats.

Because the PVN mechanism for hyperosmolality-inducedincreases in sympathetic nervous system activity is Ang II/AT1R–dependent, we investigated the role of MIF and its TPORmoiety in PVN neurons. Our novel findings reported herein indicatethat exposure of rats to a hyperosmotic challenge increases MIFmRNA expression in the PVN. In addition, viral-mediated increasesin MIF levels in PVN neurons produce complete inhibition of theincrease in sympathetic nerve activity elicited by hyperosmolar(high-salt) conditions. This inhibitory action of MIF involves itsTPOR activity. These data identify MIF in the PVN as a majorregulator of salt-induced increases in sympathetic outflow.

Materials and MethodsEthical ApprovalFor the experiments that generated the data shown in Figures 1 and 2,we used a total of 15 male Sprague-Dawley rats (200 to 250 g),

100

120

s/

10.0

d AV

Pre

gion

20

40

60

80

Bregma-1.6 Bregma-1.88 Bregma-2.12

AVP

and

MIF

cel

lsPV

N le

vel

2.5

5.0

7.5r o

f co-

loca

lized

F ce

lls/P

VN s

ubr

0

20A

0.0

PVN subregion

Num

ber

and

MIF

Bregma-1.6 Bregma-1.88 Bregma-2.12

AVP MIF AVP/MIF

Figure 1. Colocalization of AVP and MIF in the PVN. Top, Low-power representative fluorescence images of the PVN showing endoge-nous MIF (green) and AVP (red) immunoreactivities and their colocalization (orange) at 3 different levels (�1.60, �1.88, and �2.12 mmrelative to the bregma). Bars�100 �m. Middle, Representative fluorescence micrographs are a higher-power view (from the insetshown in the top left) showing MIF (green) and AVP (red) immunofluorescence at PVN level �1.60 mm relative to bregma. Examples ofAVP and MIF colocalization are indicated by white arrows. Bar�20 �m. Bottom left, Bar graph showing the number of immunoreactiveAVP and MIF cells counted at each of the 3 PVN levels and also the number of overlapping AVP- and MIF-positive cells. Data aremean�SEM of numbers of AVP-, MIF-, or AVP/MIF-positive cells (n�3 rats). Bottom right, Bar graph showing the colocalization ofimmunoreactive AVP and MIF in various subregions of the PVN at each of the levels investigated. dp indicates dorsal parvocellular; mp,medial parvocellular; pm, posterior magnocellular; vp, ventrolateral parvocellular; lp, lateral parvocellular. Data are mean�SEM of num-bers of AVP/MIF-positive cells (n�3 rats).

Colombari et al Sympathoexcitatory Salt-Sensing Peptide 957



ownloaded from


purchased from Charles River Farms (Wilmington, MA). All of theseprocedures were approved by the institutional animal care and usecommittee of the University of Florida. For the experiments thatgenerated the data shown in Figures 3 to 6, we used a total of 42 maleWistar rats (60 to 120 g; postnatal age: 28 to 38 days), purchasedfrom B&K. All of the procedures conformed to the United KingdomAnimals (Scientific Procedures) Act 1986 and were approved by theUniversity of Bristol Ethical Review Committee.

Adenoassociated Viral Vector 2 ConstructsThree adenoassociated viral vectors (AAVs), AAV2-chicken �-actinpromoter (CBA)-MIF, AAV2-CBA-[C60S]-MIF, and AAV2-CBA-enhanced green fluorescent protein (eGFP), were constructed andprepared exactly as detailed previously.29 These constructs containedexpression cassettes flanked by the rAAV2 terminal repeats. Expres-sions of eGFP, MIF, and [C60S]-MIF were driven by a CBA with ahuman cytomegalovirus enhancer. Vector doses were expressed asgenome copies.

Gene Transfer Into the PVN In VivoJuvenile (28- to 38-day–old) rats were anesthetized with ketamine(60 mg/kg) and medetomidine (250 mg/kg) via intramuscular injec-tion and placed in a stereotaxic frame (David Kopf Instruments). Theskull was leveled between bregma and lambda and bilateral injec-tions (500 nL) of the recombinant AAV2-CBA-MIF (1.0�108

genome copies per microliter), AAV2-CBA-C60S-MIF-eGFP(1.0�108 genome copies per microliter), AAV2-CBA-eGFP(8.3�108 genome copies per microliter), or control solution (0.9%saline) were performed into the PVN using the following stereotaxiccoordinates: 0.8 mm caudal to bregma, 0.3 mm lateral to bregma, and7.2 mm ventral to the dura mater. (Note that, in preliminaryexperiments, the appropriate PVN coordinates for this size of ratwere determined.) Each injection was made over 1 minute. Aftersurgery, anesthesia was reversed with an intramuscular injection ofatipamezole (1 mg/kg). Animals were allowed to recover for 7 to 10days before being studied using the decorticate-perfused in situpreparation.

Decorticate, Unanaesthetized, Arterially PerfusedIn Situ Rat PreparationExperiments were performed at the University of Bristol. MaleWistar rats were prepared as described previously.19,30 Rats wereplaced under deep halothane anesthesia (5%) and assessed by a

failure to respond to a noxious pinch of either a paw or the tail.Anesthetized rats were transected below the diaphragm, then sub-merged in ice-cooled Ringer solution (see below), and the cerebralhemispheres, hippocampus and thalamic areas removed by gentleaspiration. The preoptic area and adjacent septal nuclei and hypo-thalamic areas remained intact. The preparation was skinned, trans-ferred to a recording chamber, and the left phrenic nerve isolated. Adouble-lumen catheter was inserted into the descending aorta. Onelumen was used to delivery perfusate pumped using a roller pump(Watson Marlow 505S). The perfusate was an isosmotic Ringersolution (containing, in mM: NaCl 120.00, NaHCO3 24.00, KCl3.00, CaCl2 2.50, MgSO4 1.25, KH2PO4 1.25, and glucose 10.00)containing an oncotic agent (polyethylene glycol: 1.5%; SigmaUnited Kingdom), gassed with carbogen (95% O2 and 5% CO2),warmed to 32°C (pH 7.3) after carbogenation, and filtered using anylon screen (pore size: 25 �m). After respiratory-related move-ments commenced, a neuromuscular blocker (vecuronium bromide,40 �g � mL�1 Norcuron Organon Teknika) was added to the perfus-ate to mechanically stabilize the preparation. The second lumen ofthe catheter was used to monitor aortic perfusion pressure. Phrenicnerve activity was recorded from its cut central end using a glasssuction bipolar electrode held in a 3D micromanipulator. Rhythmicramping phrenic nerve activity gave a continuous physiologicalindex of preparation viability. Sympathetic nerve activity (SNA) wasrecorded from the thoracic sympathetic chain using a bipolar glasssuction electrode. Signals were AC amplified (Neurolog NL104),band-pass filtered (8 Hz to 3 kHz), rectified, and integrated. Noiselevels were subtracted from all of the SNA recordings by application

*1.25

1.50

1.75s

rRN

Ani

ts)

0.50

0.75

1.00

MIF

mR

NA

/18s

(arb

itrar

y un

0.00

0.25M

Figure 2. Hyperosmotic stimulation induces MIF mRNA expres-sion in the PVN via an AT1R-dependent process. Rats wereinjected ICV with either 2 �L of losartan (Los; 2.1 nmol/�L) or 2�L of isoosmotic (0.9%) saline. Fifteen minutes later, half of therats from each group were injected ICV with 2 �L of isoosmotic(0.9%) saline, and the others were injected ICV with 2 �L of 2.0-mol/L NaCl. Three hours later brains were removed for analysisof MIF mRNA in the PVN. Data are mean�SEM of MIF mRNAlevels normalized against 18S rRNA (n�5 to 10 per treatmentcondition). *P�0.05 (Student t test).

Figure 3. Increased MIF expression in the PVN decreasesHS-evoked sympathoexcitation. Representative tracings show-ing changes in raw and integrated SNA during isosmotic (290mosmol/kg � water�1) and hyperosmotic stimuli (HS; 380mosmol/kg � water�1; 40 seconds) in rats that had receivedbilateral injections of AAV2-CBA-eGFP, AAV2-CBA-MIF, orAAV2-CBA-[C60S]-MIF into the PVN 10 days earlier.

958 Hypertension November 2010



ownloaded from


of lidocaine to the chain, which was subtracted from the integratedsignal during analysis.

Osmotic StimulusThe osmolalities of iso-osmotic and hyperosmotic solutions were,respectively, 290 and 380 milliosmol (mosmol; kilograms � water�1), asmeasured by a freezing point depression osmometer (Camlab,Roebling Micro-osmometer). Hyperosmotic ionic Ringer solutionwas prepared by adjusting the final concentration of NaCl. The insitu preparation was perfused with isosmotic Ringer solution, and theosmotic stimulus was performed by perfusing the preparation for 40seconds with hyperosmotic Ringer solution, contained in a separatereservoir and connected to the perfusion system via a 3-way tap.

ICV Injections of Saline and Analysis ofMIF mRNARats were anesthetized with a mixture of 4% isoflurane in pure O2(1 L/min) and placed in a Kopf stereotaxic frame. Anesthesia wasmaintained using an O2/isoflurane (2%) mixture delivered through aspecialized nose cone for the duration of the injection procedure.Rats were injected into the right lateral cerebroventricle (ICV) witheither 2 �L of losartan (2.1 nmoL/�L) or isoosmotic (0.9%) saline,followed 15 minutes later by ICV injections of 2 �L of isoosmotic(0.9%) saline or 2.0 mol/L of NaCl at an infusion rate of 1 �L/min.All of the stereotaxic ICV injection procedures were as detailedpreviously.28 An analgesic agent (buprenorphine; 0.05 mg/kg SC)was administered to the rats before waking. Three hours later, ratswere euthanized, brains were removed, and the PVN was isolatedfrom each as detailed previously.31 Endogenous levels of MIFmRNA in the PVN were analyzed by real-time RT-PCR, as detailedpreviously.29 MIF mRNA data were normalized to 18S rRNA.

Immunocytochemical Detection of EndogenousMIF in PVN Vasopressinergic NeuronsPlease see http://hyper.ahajournals.org for the online Data Supple-ment for text and Figures S2 and S3.

Immunohistochemistry for Arginine Vasopressin,Neuron-Specific Nuclear Protein, and VirallyExpressed MIFPlease see the online Data Supplement for text and Figures S2 and S3(http://hyper.ahajournals.org).

Data AnalysisAll of the values are expressed as the mean�SEM. One-wayANOVA followed by Student-Newman-Keuls post hoc or Student ttest were used to assess differences between individual means.Differences were taken as significant at P�0.05.

ResultsColocalization of MIF and Arginine Vasopressin inthe PVNIn a previous study, we showed that endogenous MIF stainingin the PVN of normotensive rats was localized to neuronsprimarily with lesser amounts in glia.29 Because of theimportance of vasopressin and vasopressinergic neurons incardiovascular control, we wished to determine whetherendogenous MIF resided within this neuronal phenotype. Thelower power fluorescence micrographs shown in Figure 1(top) depict MIF (green) and arginine vasopressin (AVP)(red) immunofluorescence at 3 different levels of the PVN(�1.60 mm, �1.88 mm, and �2.12 mm relative to thebregma) and also where MIF and AVP overlap. The fluores-cence micrographs shown in Figure 1 (middle) are a higherpower view (from the inset shown at the top left) depicting

MIF (green) and AVP (red) immunofluorescence at PVNlevel �1.60 mm relative to bregma. MIF and AVP colocal-izations are shown in orange. Quantification of the colocal-ization of MIF and AVP in different parts of the PVN isshown in the bar graphs in Figure 1. The data in the left bargraph show the number of immunoreactive AVP cellscounted at each of the 3 PVN levels and also the number thatoverlap with MIF-positive cells. The data in the right bargraph show the colocalization of AVP and MIF in varioussubregions of the PVN at each of the levels investigated.Collectively, these data indicate that the degree of colocal-ization is low within each of the PVN levels, and when itoccurs it is in both magnocellular and parvocellular regions ofthe PVN.

Induction of MIF mRNA Expression in the PVNby Central Hyperosmotic StimulationBecause the PVN mechanism for hyperosmolality-inducedincreases in sympathetic nervous system activity is AngII/AT1R–dependent and MIF is a regulator of Ang II/AT1Ractions in the PVN,27,28 we investigated whether hyperos-motic challenge could induce changes in endogenous MIFmRNA levels at this hypothalamic nucleus. The data inFigure 2 indicate that rats injected ICV with hypertonic NaCl(2 �L of 2.0 mol/L) displayed significantly greater levels ofMIF mRNA in the PVN when compared with rats thatunderwent ICV injections of 0.9% saline. This stimulatoryeffect of hypertonic saline on MIF mRNA levels in the PVNwas abolished by pretreatment of rats with losartan (2.1nmol/�L; ICV; Figure 2).

Increased Expression of MIF in the PVN Bluntsthe HS-Induced SympathoexcitationAs an initial step, we performed studies to confirm thedependency of HS-induced increases in SNA on AT1Ractivation in the in situ preparation. Baseline SNA wasrecorded in rats perfused intra-arterially with isosmotic solu-tion (290 mosmol/kg � water�1). After this, rats exposed toHS by perfusion for 40 seconds with a hyperosmotic solution(380 mosmol/kg � water�1) demonstrated a 23�3% increasein SNA. Addition of the AT1R antagonist losartan(20 �mol/L) to the perfusate for 10 minutes severely reducedthe HS-induced sympathoexcitation to 5�5% (P�0.002).Considering that the HS-induced sympathoexcitation dependson the PVN19 and is AT1R mediated20 (please see Figure S1at http://hyper.ahajournals.org), hyperosmotic challenge in-creases MIF expression in the PVN (Figure 2), and MIFcounterregulates AT1R actions with the PVN,27,28 we deter-mined whether increased expression of MIF in this hypotha-lamic nucleus would modify the increase in SNA producedby acute HS. The left and right PVNs of rats were microin-jected with 0.9% saline, AAV2-CBA-MIF, or AAV2-CBA-eGFP, as described in the Methods section. AAV2-CBA-MIFand AAV2-CBA-eGFP produce expression of MIF andeGFP, respectively, in the PVN within 7 days. Seven to 10days after the PVN injections, the decorticate-perfused in siturat preparation was made. Baseline SNA in each group wasestablished after intra-arterial perfusion of isosmotic Ringersolution (290 mosmol/kg � water�1). Intra-arterial infusion of




ownloaded from


hyperosmotic solution (Ringer solution at 380 mosmol/kg � water�1) for 40 seconds elicited sympathoexcitation inboth the 0.9% saline and eGFP groups (increases of 27�4%and 25�4%, respectively; Figures 3 and 4). In contrast, HSfor 40 seconds did not produce sympathoexcitation in the ratsoverexpressing MIF in the PVN (increased SNA of 4�5%;Figures 3 and 4A; P�0.008 versus saline). The area under thecurve and the duration of the sympathoexcitation werereduced in the MIF-treated rats compared with the 0.9%saline (respectively, P�0.012 and P�0.001) and the eGFPgroups (P�0.019 and P�0.001; Figure 4B and 4C). WhenMIF injections were located outside of the PVN (0.5 mmdorsal; see Figure 5A), there was no inhibition of the

HS-induced sympathoexcitation, and a response similar tocontrol was observed (data not shown), indicating a specificrole of the PVN in this response. Finally, the sympathoexcit-atory response to stimulating the peripheral chemoreflex(NaCN: 0.03%; 25 to 75 �L) was similar between control andMIF-expressing rats indicating relative specificity of MIF inthe PVN for HS-induced sympathoexcitation (data notshown).

The inhibitory effects of MIF on Ang II actions in the PVNare mediated by its intrinsic TPOR moiety.27,29 Because thesympathetic response to hyperosmotic stimulation was AT1Rdependent, we next tested whether the inhibitory effect ofMIF in the PVN on HS-induced sympathoexcitation was also

B

MIF sites which blocked thesympathoexcitation

-0.8 mm 200 µm

MIF sites which did notblock sympathoexcitationC60s-MIF sites which potentiated thesympathoexcitation

eGFP sites

3V PaMP

PaMM

Pe

PaV 3V

PaV

Pe

PaMP

PaDC PaLM

Arc

PaDCPaMP

PaLM

PaV

3V Pe

Arc

PaDC PaMP

PaLM

PaV

3VPe

ArcMArcL

ArcD

A

Figure 5. Localization of MIF transductionin the PVN. A, The center of the injectionsites for eGFP, MIF, and C60S-MIF withinthe PVN are indicated. Spread of trans-duction was between 400 to 500 �m indiameter. Injections of MIF that fell outsideof the PVN were not effective in blockingthe hyperosmolality-induced sympathoex-citation. Section levels are relative to thebregma. Abbreviations: PaDC indicatesparaventricular hypothalamic nucleus, dor-sal cap; PaLM, paraventricular hypotha-lamic nucleus, lateral magnocellular part;PaMP, paraventricular hypothalamicnucleus, medial parvicellular part; PaV,paraventricular hypothalamic nucleus, ven-tral part; PaMM, paraventricular hypotha-lamic nucleus, medial magnocellular part;Pe, periventricular hypothalamic nucleus;Arc, arcuate hypothalamic nucleus; 3V,third ventricle. B, A representative exam-ple of MIF immunostaining in PVN inducedby AAV2-CBA-MIF in vivo gene transfer.

% c

hang

e in

SN

A

0

10

20

30

40

50

60

*

**

control0

1000

2000

3000

4000

5000

6000

*

**

SN

A D

urat

ion

(s)

0

20

40

60

80

100

120

140

*

**

A B C

eGFP MIF C60S-MIF control eGFP MIF C60S-MIF control eGFP MIF C60S-MIF

SN

A A

rea

(µV

x s

)

Figure 4. HS-evoked sympathoexcitation is reduced by MIF and augmented by [C60S]-MIF expression in the PVN. Sympathoexcitationwas measured as the percentage increase (peak response) from baseline over the 40-second period of HS (A), the total increase abovebaseline (B), and the duration of the response (C). Values in bar graphs are mean�SEM from rats that had received bilateral injectionsof saline, AAV2-CBA-eGFP, AAV2-CBA-MIF, or AAV2-CBA-[C60S]-MIF into the PVN 10 days earlier. (*�P�0.05, 1-way ANOVA; A:F(3,20)�11.432; B: F(3,20)�24.891; C: F(3,20)�77.750; n�6 rats per group).




ownloaded from


attributed to its TPOR activity. An additional group of ratsreceived injections of AAV2-CBA-[C60S]-MIF bilaterallyinto the PVN for expression of [C60S]-MIF as before.29

[C60S]-MIF, in which the cysteine at position 60 is replacedwith serine, lacks TPOR activity.32 In contrast to MIF, theHS-induced sympathoexcitation was potentiated (45�6%) inrats that expressed [C60S]-MIF (Figures 3 and 4A; P�0.023versus saline). The area under the curve and the duration ofthe sympathoexcitation were both augmented significantly inthe [C60S]-MIF group (Figure 4B and 4C; P�0.001 versussaline).

Localization of MIF Expression in the PVNSchematic reconstruction of injection sites at different rostral-caudal levels of the PVN are shown in Figure 5A. As can beseen, the sites of injection of AAV2-CBA-MIF, AAV2-CBA-[C60S]-MIF, and AAV2-CBA-eGFP were, in most part,within the PVN (maximal spread was between 400 to 500 �min diameter). We never saw transduction of circumventricularorgans or ependymal cells. We recognize the difficulty ofdistinguishing between endogenous and virally expressedMIF, which is a limitation of our study. However, we diduse a lower concentration of antibody to detect virally

induced MIF that rarely detected endogenous protein(1:500 versus 1:200 dilution, respectively; please see FigureS2 at http://hyper.ahajournals.org). Figure 5 also shows sitesof AAV2-CBA-MIF injection outside the PVN, which wereineffective. Figure 5B shows a fluorescence micrographdepicting bilateral expression of MIF immunoreactivity in thePVN, typically obtained after AAV2-CBA-MIF injection.Virally expressed MIF is largely found in neurons, as dem-onstrated by its coexpression with the specific neuronalmarker neuron-specific nuclear protein (NeuN) (Figure 6A).Interestingly, the neuronal populations in which MIF istransduced in the PVN include vasopressinergic neurons(Figure 6B).

DiscussionThe major findings of this study are as follows: (1) MIF ispresent in PVN vasopressinergic neurons; (2) hyperosmoticchallenge increases MIF expression in the PVN, which isAT1R dependent; (3) increased MIF expression within thePVN of normotensive rats abolishes the sympathoexcitationproduced by hyperosmotic stimulation; and (4) the actions ofMIF appear to be mediated by its intrinsic TPOR activity.Collectively, these data provide the first indication that MIF,

200 µµµµm

2

MIF

NeuN

merge

MIF

AVP

merge

500 µm

50 µm

500 µm

50 µm

BA

Figure 6. AAV2-CBA-MIF–induced expression of MIF in AVP neurons in the PVN. Wistar rats were injected bilaterally into the PVN withAAV2-CBA-MIF, as detailed in the Methods section. A, Micrographs of MIF and NeuN immunostaining from the same field of the PVN10 days after microinjection of AAV2-CBA-MIF. Merge: MIF�NeuN. Yellow arrows indicate coexpression of MIF and NeuN. B, Repre-sentative fluorescence micrographs of showing MIF and AVP immunostaining from the same field of the PVN 10 days after microinjec-tion of AAV2-CBA-MIF. Merge: MIF�AVP. Yellow arrows indicate coexpression of MIF and AVP.




ownloaded from


present within neurons in the PVN, serves as a negativeregulator of HS-induced sympathoexcitation. The demonstra-tion that HS conditions also produce an increase in MIFmRNA expression in the PVN opens up the possibility thatMIF is a major central nervous system regulator of sympa-thetic outflow in conditions of high salt load.

The osmotic stimulus used in our in situ experiments ishigher than that used previously (see Reference 19). Here weused 380 mosmol/kg � water�1. This produced a robust andreproducible sympathoexcitation that was important for as-sessing accurately the magnitude by which MIF expression inPVN could sequester this evoked response. We acknowl-edge that the stimulus is high. However, this level ofosmolality is unlikely to be seen by the preparationbecause of the transient nature of the stimulus and signif-icant dilution that occurs within the perfusion circuit andpreparation; this is discussed further in the online DataSupplement at http://hyper.ahajournals.org.

The mechanisms for hyperosmolality-induced increases inSNA from the PVN are Ang II/AT1R dependent,28 and thenegative regulatory action of MIF quenches Ang II/AT1R-stimulated increases in neuronal discharge and elevations inarterial pressure.28 Findings from the latter study were con-sistent with the earlier in vitro observation that the AngII-increased firing response of PVN neurons is blunted byMIF, which is likely to be activated by continual AT1Rstimulation.28 Thus, an Ang II activity-dependent feedbackmechanism may exist to protect against overstimulation byAng II, which we presume operates in vivo. However, crucialfurther experiments should include determining whether HS-induced induction of endogenous MIF in the PVN can beprevented by AT1R blockade and provide tempering overHS-induced sympathoexcitation. Also, it now becomes im-portant to determine the role that MIF plays in the PVN forchronic elevations of sympathetic nerve activity and arterialpressure observed during dehydration, for example.

We acknowledge that our study does not allow us todifferentiate whether the MIF mechanism is sodium orosmotically sensitive. However, if endogenous MIF doesserve as a “salt-sensitive” endogenous regulator of HS-induced sympathoexcitation, then one primary question con-cerns the nature of the mechanisms that mediatehyperosmolality-induced increases in MIF expression in thePVN. Osmolality-induced changes in SNA are mediatedthrough osmosensitive neurons in the OVLT and subfornicalorgan,3 which subsequently transmit information concerningthe osmotic status of the animal via efferent pathways to thePVN.2,19 Ang II, released either from these efferent pathwaysor generated locally within the PVN itself increases, viapresynaptic and/or postsynaptic AT1R,33,34 the activity ofPVN presympathetic neurons projecting to the intermediolat-eral nucleus or rostral ventrolateral medulla.1 Our current dataconfirm that the HS-induced sympathoexcitation is AT1Rdependent. Considering that ICV injection of Ang II increasesMIF expression in the PVN of normotensive rats,29 it islikely that the HS-induced MIF expression in the PVN isalso Ang II/AT1R dependent. However, overexpression ofMIF does not affect basal blood pressure in both normoten-sive (this study) and spontaneously hypertensive rats,29 sug-

gesting a specific functional role related to salt-mediatedsympathoexcitation.

The inhibitory action of MIF on HS-induced sympathoex-citation appears to involve its TPOR activity. This is becauseincreased expression of the mutant protein [C60S]-MIF in thePVN failed to blunt the HS-induced increased in SNA(Figures 3 and 4). However, the [C60S]-MIF rats displayedan unexpected higher increase in sympathoexcitation inresponse to HS challenge compared with the eGFP group(Figures 3 and 4). The reasons for this action of [C60S]-MIFare not obvious but may involve a dominant-negative actionof this mutant protein over endogenous MIF within the PVN;however, this remains speculative. If so, this would argue thatPVN parvocellular neurons are under tonic control by MIF,which would be entirely consistent with the specific role as amediator of increased sympathetic activity in response to saltloading.

The finding that a vasopressinergic descending pathwayfrom PVN to intermediolateral nucleus contributes to sym-pathoexcitation elicited by hyperosmolality19 suggests that apossible location of MIF’s inhibitory actions in the PVNinclude the vasopressinergic neurons. Indeed, our data (Fig-ure 1) illustrate that PVN vasopressinergic neurons expressMIF endogenously. Moreover, Figure 6B demonstrates thatthe AAV2-CBA-MIF–injected rats display MIF transductioninto AVP neurons in the PVN. Thus, it is likely that theinhibitory actions of MIF in the PVN on HS-induced sym-pathoexcitation involve, in part, the vasopressinergic neurons.However, whereas the CBA promoter that is used in thepresent study produces MIF expression primarily withinneurons in the PVN,29 it does not discriminate betweendifferent neuronal phenotypes. Further development of pro-moters that would allow for transduction of MIF into specificneuron populations (eg, AVP) would help to tease outwhether the actions of MIF in controlling sympathoexcitationin response to HS are limited to a specific neuronal pheno-type, such as glutamatergic PVN neurons known to beactivated by osmotic stimuli that project to the rostralventrolateral medulla.26 Also, we cannot comment onwhether AT1Rs involved are localized to the vasopressinergicneurons or nonvasopressinergic interneurons within PVN.35

PerspectivesOur new findings indicate that MIF operates as a novelinhibitor within the PVN of osmolality-induced increases insympathetic outflow and substantiates the developing notionthat MIF is an important regulator of Ang II-AT1R–inducedexcitability within the PVN, especially to salt loading. Fur-ther phenotyping of the MIF containing neurons in the PVNin terms of their neurochemical content and connectivity isnow needed. Subsequently, chronic studies to assess whetherMIF expression in the PVN can prevent the hypertensionassociated with salt loading and dehydration in consciousanimals would further validate the importance of this protein.Indeed, whether MIF plays a role in controlling other formsof hypertension, such as stress-related hypertension, wouldadd further credence as to its wider applicability to hyperten-sion. It also needs to be established where else in the brainMIF may exist to keep sympathetic nerve activity in check:




ownloaded from


the nucleus tractus solitarii and rostral ventrolateral medullaare both likely sites. The significant finding described hereinthat the salt-induced sympathetic overdrive depended on theTPOR activity of MIF leads way to proof-of-principle studiestesting whether the AAV2-CBA-C60S-MIF-eGFP vector canprevent hypertension development in a salt-sensitive ratmodel such as the Dahl. Accordingly, we propose that MIFmight be exploitable as a novel target to control (salt-related)hypertension in humans.

Sources of FundingE.C. was supported by Conselho Nacional de DesenvolvimentoCientifico e Tecnologico, Coordenaçao de Aperfeiçoamento dePessoal de Nivel Superior, and Faculdade de Medicina ABC fromBrazil and a Benjamin Meaker Fellowship awarded from theUniversity of Bristol. D.S.A.C. was supported by Conselho Nacionalde Desenvolvimento Cientifico e Tecnologico and a Wellcome TrustValue in People Award (University of Bristol). This work wassupported by the British Heart Foundation and National Institutes ofHealth grants 1R01HL-076803 and HL33610. J.F.R.P. was in receiptof a Royal Society Wolfson Research Merit Award.

DisclosuresNone.

References1. Guyenet PG. The sympathetic control of blood pressure. Nat Rev

Neurosci. 2006;7:335–346.2. Toney GM, Chen QH, Cato MJ, Stocker SD. Central osmotic regulation

of sympathetic nerve activity. Acta Physiol Scand. 2003;177:43–55.3. Bourque CW. Central mechanisms of osmosensation and systemic osmo-

regulation. Nat Rev Neurosci. 2008;9:519–531.4. Johnson AK, Gross PM. Sensory circumventricular organs and brain

homeostatic pathways. FASEB J. 1993;7:678–686.5. McKinley MJ, Allen AM, May CN, McAllen RM, Oldfield BJ, Sly D,

Mendelsohn FA. Neural pathways from the lamina terminalis influencingcardiovascular and body fluid homeostasis. Clin Exp Pharmacol Physiol.2001;28:990–992.

6. McKinley MJ, Johnson AK. The physiological regulation of thirst andfluid intake. News Physiol Sci. 2004;19:1–6.

7. Stocker SD, Osborn JL, Carmichael SP. Forebrain osmotic regulation ofthe sympathetic nervous system. Clin Exp Pharmacol Physiol. 2008;35:695–700.

8. Ciura S, Bourque CW. Transient receptor potential vanilloid 1 is requiredfor intrinsic osmoreception in organum vasculosum lamina terminalisneurons and for normal thirst responses to systemic hyperosmolality.J Neurosci. 2006;26:9069–9075.

9. Shi P, Stocker SD, Toney GM. Organum vasculosum laminae terminaliscontributes to increased sympathetic nerve activity induced by centralhyperosmolality. Am J Physiol Regul Integr Comp Physiol. 2007;293:R2279–R2289.

10. Shi P, Martinez MA, Calderon AS, Chen Q, Cunningham JT, Toney GM.Intra-carotid hyperosmotic stimulation increases fos staining in forebrainorganum vasculosum laminae terminalis neurones that project to thehypothalamic paraventricular nucleus. J Physiol. 2008;586:5231–5245.

11. Thrasher TN, Keil LC, Ramsay DJ. Lesions of the organum vasculosumof the lamina terminalis (OVLT) attenuate osmotically-induced drinkingand vasopressin secretion in the dog. Endocrinology. 1982;110:1837–1839.

12. Ho JM, Zierath DK, Savos AV, Femiano DJ, Bassett JE, McKinley MJ,Fitts DA. Differential effects of intravenous hyperosmotic solutes ondrinking latency and c-fos expression in the circumventricular organs andhypothalamus of the rat. Am J Physiol Regul Integr Comp Physiol.2007;292:R1690–R1698.

13. McKinley MJ, Denton DA, Leksell LG, Mouw DR, Scoggins BA, SmithMH, Weisinger RS, Wright RD. Osmoregulatory thirst in sheep is dis-rupted by ablation of the anterior wall of the optic recess. Brain Res.1982;236:210–215.

14. Morris M, Rocha MJ, Sim LJ, Johnson AK, Callahan MF. Dissociationbetween vasopressin and oxytocin mRNA and peptide secretion afterAV3V lesions. Am J Physiol. 1994;267:R1640–R1645.

15. Saper CB, Levisohn D. Afferent connections of the median preopticnucleus in the rat: anatomical evidence for a cardiovascular integrativemechanism in the anteroventral third ventricular (AV3V) region. BrainRes. 1983;288:21–31.

16. Camacho A, Phillips MI. Horseradish peroxidase study in rat of the neuralconnections of the organum vasculosum of the lamina terminalis.Neurosci Lett. 1981;25:201–204.

17. Stocker SD, Toney GM. Vagal afferent input alters the discharge ofosmotic and ANG II-responsive median preoptic neurons projecting tothe hypothalamic paraventricular nucleus. Brain Res. 2007;1131:118–128.

18. Stocker SD, Hunwick KJ, Toney GM. Hypothalamic paraventricularnucleus differentially supports lumbar and renal sympathetic outflow inwater-deprived rats. J Physiol. 2005;563:249–263.

19. Antunes VR, Yao ST, Pickering AE, Murphy D, Paton JF. A spinalvasopressinergic mechanism mediates hyperosmolality-induced sympa-thoexcitation. J Physiol. 2006;576:569–583.

20. Chen QH, Toney GM. AT(1)-receptor blockade in the hypothalamic PVNreduces central hyperosmolality-induced renal sympathoexcitation. Am JPhysiol Regul Integr Comp Physiol. 2001;281:R1844–R1853.

21. Freeman KL, Brooks VL. AT(1) and glutamatergic receptors in paraven-tricular nucleus support blood pressure during water deprivation. Am JPhysiol Regul Integr Comp Physiol. 2007;292:R1675–R1682.

22. Coote JH, Yang Z, Pyner S, Deering J. Control of sympathetic outflowsby the hypothalamic paraventricular nucleus. Clin Exp PharmacolPhysiol. 1998;25:461–463.

23. Badoer E. Hypothalamic paraventricular nucleus and cardiovascular reg-ulation. Clin Exp Pharmacol Physiol. 2001;28:95–99.

24. Saper CB, Loewy AD, Swanson LW, Cowan WM. Direct hypothalamo-autonomic connections. Brain Res. 1976;117:305–312.

25. Yang Z, Bertram D, Coote JH. The role of glutamate and vasopressin inthe excitation of RVL neurones by paraventricular neurones. Brain Res.2001;908:99–103.

26. Stocker SD, Simmons JR, Stornetta RL, Toney GM, Guyenet PG. Waterdeprivation activates a glutamatergic projection from the hypothalamicparaventricular nucleus to the rostral ventrolateral medulla. J CompNeurol. 2006;494:673–685.

27. Sun C, Li H, Leng L, Raizada MK, Bucala R, Sumners C. Macrophagemigration inhibitory factor: an intracellular inhibitor of angiotensinII-induced increases in neuronal activity. J Neurosci. 2004;24:9944–9952.

28. Li H, Gao Y, Freire CD, Raizada MK, Toney GM, Sumners C. Macro-phage migration inhibitory factor in the PVN attenuates the centralpressor and dipsogenic actions of angiotensin II. FASEB J. 2006;20:1748–1750.

29. Li H, Gao Y, Qi Y, Katovich MJ, Jiang N, Braseth LN, Scheuer DA, ShiP, Sumners C. Macrophage migration inhibitory factor in hypothalamicparaventricular nucleus neurons decreases blood pressure in sponta-neously hypertensive rats. FASEB J. 2008;22:3175–3185.

30. Paton JF. A working heart-brainstem preparation of the mouse.J Neurosci Methods. 1996;65:63–68.

31. Jiang N, Shi P, Li H, Lu S, Braseth L, Cuadra AE, Raizada MK, SumnersC. Phosphate-activated glutaminase-containing neurons in the rat para-ventricular nucleus express angiotensin type 1 receptors. Hypertension.2009;54:845–851.

32. Kleemann R, Kapurniotu A, Frank RW, Gessner A, Mischke R, FliegerO, Juttner S, Brunner H, Bernhagen J. Disulfide analysis reveals a role formacrophage migration inhibitory factor (MIF) as thiol-protein oxi-doreductase. J Mol Biol. 1998;280:85–102.

33. Li DP, Chen SR, Pan HL. Angiotensin II stimulates spinally projectingparaventricular neurons through presynaptic disinhibition. J Neurosci.2003;23:5041–5049.

34. Cato MJ, Toney GM. Angiotensin II excites paraventricular nucleusneurons that innervate the rostral ventrolateral medulla: an in vitropatch-clamp study in brain slices. J Neurophysiol. 2005;93:403–413.

35. Qadri F, Edling O, Wolf A, Gohlke P, Culman J, Unger T. Release ofangiotensin in the paraventricular nucleus in response to hyperosmoticstimulation in conscious rats: a microdialysis study. Brain Res. 1994;637:45–49.




ownloaded from


Mohan K. Raizada, Colin Sumners, David Murphy and Julian F.R. PatonEduardo Colombari, Debora S.A. Colombari, Hongwei Li, Peng Shi, Ying Dong, Nan Jiang,

Role in the Sympathoexcitatory Response to SaltMacrophage Migration Inhibitory Factor in the Paraventricular Nucleus Plays a Major

Print ISSN: 0194-911X. Online ISSN: 1524-4563 Copyright © 2010 American Heart Association, Inc. All rights reserved.

is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Hypertension doi: 10.1161/HYPERTENSIONAHA.110.1551012010;56:956-963; originally published online October 11, 2010;Hypertension.

http://hyper.ahajournals.org/content/56/5/956World Wide Web at:

The online version of this article, along with updated information and services, is located on the

http://hyper.ahajournals.org/content/suppl/2010/10/08/HYPERTENSIONAHA.110.155101.DC1Data Supplement (unedited) at:

http://hyper.ahajournals.org//subscriptions/

is online at: Hypertension Information about subscribing to Subscriptions:

http://www.lww.com/reprints Information about reprints can be found online at: Reprints:

document. Permissions and Rights Question and Answer this process is available in the

click Request Permissions in the middle column of the Web page under Services. Further information aboutOffice. Once the online version of the published article for which permission is being requested is located,

can be obtained via RightsLink, a service of the Copyright Clearance Center, not the EditorialHypertensionin Requests for permissions to reproduce figures, tables, or portions of articles originally publishedPermissions:



ownloaded from

http://hyper.ahajournals.org/content/56/5/956

http://hyper.ahajournals.org/content/suppl/2010/10/08/HYPERTENSIONAHA.110.155101.DC1

http://www.ahajournals.org/site/rights/

http://www.lww.com/reprints

http://hyper.ahajournals.org//subscriptions/


Supplemental File: Macrophage migration inhibitory factor (MIF) in the paraventricular nucleus plays a

major role in the sympathoexcitatory response to salt

Eduardo Colombari1,*, Debora S.A. Colombari2,*, Hongwei Li3, Peng Shi3, Ying Dong3, Nan Jiang3, Mohan K. Raizada3, Colin Sumners3, David Murphy2, Julian F.R. Paton1

Methods The following protocols were based on those used before (see supplemental references 1-4 below). Immunocytochemical detection of endogenous MIF in PVN vasopressinergic neurons Adult rats were anesthetized and euthanized following protocols approved by the University of Florida Institutional Animal Care and Use Committee. Brains were perfused transcardially in situ with 0.9% saline containing 4% formaldehyde and then cryoprotected in 30% sucrose for 1 week. The prepared brains were trimmed and embedded in optimal cutting temperature (OCT) compound (Sakura Finetek USA, Torrance, CA). Forebrain blocks were stored at 80ºC until they were sectioned on a Leica CM 1850 cryostat. Fixed frozen sections (15 µm) were cut from the PVN and air dried overnight at room temperature. Slides were washed for 5 minutes to remove residual OCT. Sections were blocked in 5% goat serum for 30 mins and then incubated overnight at 4C in a mixture of 1:200 rabbit anti-MIF (Torrey Pines Biolabs, Inc, Houston, TX, USA) and 1:250 Guinea pig anti- AVP (Peninsula laboratories LLC, San Carlos, CA, USA). Slides were washed in buffer 3 times for 10 minutes and incubated for one hour at room temperature in combined secondary antibodies. (Alexa Fluor 594 goat anti- guinea pig and Alexa Fluor 488 goat anti-rabbit IgG, both at a 1:1000 dilution, Invitrogen, Carlsbad, CA). The sections were mounted in Vectashield and imaged using an Olympus BX41 fluorescence microscope. For the analyses of colocalization of MIF and AVP immunoreactivities, we focused on 3 levels of the PVN as described by Stocker et al., 2004. Relative to the bregma, these levels were -1.60mm, -1.88mm and -2.12mm. In addition, we utilized the same nomenclature as Stocker et al. to describe the subregions of the PVN at each of these levels: dp = dorsal parvocellular; mp = medial parvocellular; pm = posterior magnocellular; vp = ventrolateral parvocellular; lp = lateral parvocellular. Numbers of prominent green (MIF), red (AVP) and orange (MIF/AVP co-localized) cells in each subregion at each of the above 3 levels were counted. The data presented in Figure 1 are mean ± SEM of numbers of AVP or AVP/MIF positive cells.

Immunohistochemistry for AVP, NeuN and virally expressed MIF After the experiments with the perfused in situ preparation, the brains were removed and left for 2 days in 4% paraformaldehyde in 0.1 M PBS at 4º C, followed by another 2 days in the cryoprotectant solution of 20% sucrose and 4% paraformaldehyde in 0.1 M PBS at 4°C. The brains were then rapidly frozen over liquid nitrogen and coronal sections (30 μm) cut of the PVN using a cryostat (Leica Cryocut CM1900, Switzerland). The free-floating sections were collected in 24-well tissue culture plates containing PBS prior to being processed for immunohistochemical detection of MIF, vasopressin and/or NeuN as follows. Free-floating rat hypothalamic sections were incubated for 15 minutes in a blocking solution comprised of 10%

normal goat serum (NGS, Sigma, St. Louis, MO) and 0.3% Triton X-100 (Sigma) in 0.1 M PBS followed by rinses in PBS (3 × 10 minutes). One set of sections were incubated in rabbit anti-rat MIF primary antibody (1:500; Torrey Pines Biolabs) with mouse monoclonal anti-NeuN antibody (1:1000, from Chemicon International). A further set of sections were incubated with rabbit anti-rat MIF and monoclonal mouse anti-neurophysin II (1:200; vasopressin-derived; PS41) in PBS containing 1% NGS and 0.3% Triton X-100 for 24 hours at 4°C (anti-neurophysin II was kindly provided by Prof. H. Gainer, National Institutes of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, MD) as performed previously 1‐3. After the primary antibody incubation the sections were rinsed in PBS (3 × 10 minutes) prior to 1-hour incubation in biotinylated goat anti-rabbit (1:500, Vector Laboratories), followed by another rinse in PBS (3 x 10 minutes). The sections were then incubated for 1 hour with Strept-avidin Alexa Fluor 488 conjugate and Alexa Fluor 594 goat anti-mouse (both 1:500, Molecular Probes). Following further rinses in PBS (3 × 5 minutes) sections were mounted onto slides in 0.5% gelatin and allowed to air-dry for 10-15 minutes before being cover slipped using an antifade fluorescent mounting solution (VectorShield, Vector Laboratories). The sections were visualized on a fluorescence microscope (Leica DM IRB with C-Plan optics; Leica, Germany) using the appropriate filter. The extent of virus spread were carefully examined and quantified. We observed that the dorsal-ventral, medio-lateral and rostral-caudal extent of the injection covers a sphere of approximately 400-500 μm diameter. Discussion Extended Discussion on the osmotic stimulus used in our in situ experiments: We used 380 mOsmol kg water-1 as this produced a robust and reproducible sympathoexcitation that was important for assessing accurately the magnitude by which MIF expression in PVN could sequester this evoked response. We acknowledge that the stimulus is high. However, this level of osmolality is unlikely to be seen by the preparation. There is significant dilution of the hypertonic perfusate prior to entering the preparation. On making the switch there will be significant mixing of the hypertonic solution with the isotonic solution already in the perfusion circuit. The latter is around a metre in length and contains two ~18 ml bubble traps and a filter holder with an additional volume of ~15 ml; all the latter will contain isoosmotic perfusate providing dilution. The switch from the isoosmotic to the hypertonic perfusates is also transient so achieving osmotic equilibrium is unlikely. On switching back to isoosmotic perfusate the hyperosmotic solution will again be diluted. Further dilution will occur within the circulation and tissues of the preparation. Gven the transient nature of the stimulus and its significant dilution, we do not believe that the brain is exposed to 380 mOsmol (kg water-1). Moreover, the sympathetic hyperactivity observed was antagonised by losartan, suggesting that the response was specific and mediated by physiological mechanisms that sense high salt.

References 1. Ben-Barak Y, Russell JT, Whitnall MH, Ozato K, Gainer H. Neurophysin in the hypothalamo-neurohypophysial system. I. Production and characterization of monoclonal antibodies. J Neurosci. 1985;5:81-97. 2. Gouraud SS, Heesom K, Yao ST, Qiu J, Paton JF, Murphy D. Dehydration-induced proteome changes in the rat hypothalamo-neurohypophyseal system. Endocrinology. 2007;148:3041-3052. 3. Stocker SD, Cunningham JT, Toney GM. Water deprivation increases Fos immunoreactivity in PVN autonomic neurons with projections to the spinal cord and rostral ventrolateral medulla. Am J Physiol Regul Integr Comp Physiol. 2004;287:R1172-R1183. 4. Whitnall MH, Key S, Ben-Barak Y, Ozato K, Gainer H. Neurophysin in the hypothalamo-neurohypophysial system. II. Immunocytochemical studies of the ontogeny of oxytocinergic and vasopressinergic neurons. J Neurosci. 1985;5:98-109.

% c

hang

e in

SN

A

0

5

10

15

20

25

30

*

control Losartan(20 μM)

Online supplemental Figure S1

Online supplemental Figure 1 legend:

Sympathoexcitation evoked by HS is mediated by AT1R. Bar graphs are means ± SEM of the change in SNA produced by HS in the absence or presence of losartan (20 μM). (* = P<0.05, Paired t test; n=6).

Online Supplemental Figure S2

Online Supplemental Figure S3 Immunocytochemical preabsorption control for MIF staining MIF staining ‐ AAV2‐CBA‐MIF induced MIF expression

MIF Staining: MIF antibody pre‐absorbed with rMIF

(10 μM)

Documents

Nervous System - pdfs.semanticscholar.org filebeen well accepted as osmosensors in the central nervous system, the subfornical organ and organum vasculosum laminae terminalis (OVLT)