excitatory pathways from the vestibular nuclei to the nts and the pbn and indirect...

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Research Report Excitatory pathways from the vestibular nuclei to the NTS and the PBN and indirect vestibulo-cardiovascular pathway from the vestibular nuclei to the RVLM relayed by the NTS Yi-Ling Cai, Wen-Ling Ma , Jun-Qin Wang, Yi-Qian Li, Min Li Department of Military Hygiene, Faculty of Naval Medicine, Second Military Medical University, Shanghai 200433, PR China ARTICLE INFO ABSTRACT Article history: Accepted 28 August 2008 Available online 13 September 2008 Previous studies have confirmed the existence of vestibulo-sympathetic pathways in the central nervous system. However, the exact pathways and neurotransmitters underlying this reflex are unclear. The present study was undertaken to investigate whether the vestibulo-cardiovascular responses are a result of activated glutamate receptors in the caudal vestibular nucleus. We also attempt to verify the indirect excitatory pathways from the vestibular nucleus (VN) to the rostral ventrolateral medulla (RVLM) using a tracing method combined with a vesicular glutamate transporter (VGluTs) immunofluorescence. In anesthetized rats, unilateral injection of L-glutamate (5 nmol) into the medial vestibular nucleus (MVe) and spinal vestibular nucleus (SpVe) slightly increased the mean arterial pressure (MVe: 93.29 F 11.58 to 96.30 F 11.66, SpVe: 91.72 F 15.20 to 95.48 F 17.16). Local pretreatment with the N-methyl-D-aspartate (NMDA)-receptor antagonist MK-801 (2 nmol) significantly attenuated the pressor effect of L-glutamate injected into the MVe compared to the contralateral self-control. After injection of biotinylated dextran amine (BDA) into the MVe and SpVe, and fluorogold (FG) into the RVLM, some BDA-labeled fibres and terminals in the nucleus of solitary tract (NTS) and the parabrachial nucleus (PBN) were immunoreactive for VGluT1 and VGluT2. Several BDA-labeled fibres were closely apposed to FG-labeled neurons in the NTS. These results suggested that activation of caudal vestibular nucleus neurons could induce pressor response and NMDA receptors might contribute to this response in the MVe. The glutamatergic VN-NTS and VN-PBN pathways might exist, and the projections from the VN to the RVLM relayed by the NTS comprise an indirect vestibulo- cardiovascular pathway in the brain stem. © 2008 Elsevier B.V. All rights reserved. Keywords: Vestibular nucleus Nucleus of the solitary tract Parabrachial nucleus Rostral ventrolateral medullar Vesicular glutamate transporter Immunohistochemistry BRAIN RESEARCH 1240 (2008) 96 104 * Corresponding authors. Fax: +86 29 83283229. E-mail addresses: [email protected] (Y.-L. Cai), [email protected] (W.-L. Ma), [email protected] (M. Li). Abbreviations: VN, vestibular nucleus; NTS, nucleus of the solitary tract; PBN, parabrachial nucleus; RVLM, rostral ventrolateral medullar; NMDA, N-methyl-D-aspartate; VGluT, vesicular glutamate transporter; IR, immunoreactivity; 4V, fourth ventricle; MVe, medial vestibular nucleus; SpVe, spinal vestibular nucleus; Pr, prepositus nucleus; Sp5, spinal trigeminal tract; IRt, intermediate reticular nucleus; dl, dorsolateral subnucleus; im, intermediate subnucleus; is, interstitial subnucleus; lt, lateral subnucleus; me, medial subnucleus; sup, subpostremal subnucleus; tr, tractus solitarius; vt, ventral subnucleus; elPBN, external lateral parabrachial nucleus; BP, blood pressure; MAP, mean arterial blood pressure; aCSF, artificial cerebrospinal fluid; BDA, biotinylated dextran amine; FG, Fluoro-gold 0006-8993/$ see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2008.08.093 available at www.sciencedirect.com www.elsevier.com/locate/brainres

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Page 1: Excitatory pathways from the vestibular nuclei to the NTS and the PBN and indirect vestibulo-cardiovascular pathway from the vestibular nuclei to the RVLM relayed by the NTS

B R A I N R E S E A R C H 1 2 4 0 ( 2 0 0 8 ) 9 6 – 1 0 4

ava i l ab l e a t www.sc i enced i r ec t . com

www.e l sev i e r. com/ loca te /b ra in res

Research Report

Excitatory pathways from the vestibular nuclei to the NTS andthe PBN and indirect vestibulo-cardiovascular pathway fromthe vestibular nuclei to the RVLM relayed by the NTS

Yi-Ling Cai, Wen-Ling Ma⁎, Jun-Qin Wang, Yi-Qian Li, Min Li⁎

Department of Military Hygiene, Faculty of Naval Medicine, Second Military Medical University, Shanghai 200433, PR China

A R T I C L E I N F O

* Corresponding authors. Fax: +86 29 8328322E-mail addresses: [email protected] (YAbbreviations: VN, vestibular nucleus; NT

medullar; NMDA, N-methyl-D-aspartate; VGluvestibular nucleus; SpVe, spinal vestibular nudl, dorsolateral subnucleus; im, intermediatesubpostremal subnucleus; tr, tractus solitariMAP, mean arterial blood pressure; aCSF, art

0006-8993/$ – see front matter © 2008 Elsevidoi:10.1016/j.brainres.2008.08.093

A B S T R A C T

Article history:Accepted 28 August 2008Available online 13 September 2008

Previous studies have confirmed the existence of vestibulo-sympathetic pathways in thecentral nervous system. However, the exact pathways and neurotransmitters underlyingthis reflex are unclear. The present study was undertaken to investigate whether thevestibulo-cardiovascular responses are a result of activated glutamate receptors in thecaudal vestibular nucleus. We also attempt to verify the indirect excitatory pathways fromthe vestibular nucleus (VN) to the rostral ventrolateral medulla (RVLM) using a tracingmethod combined with a vesicular glutamate transporter (VGluTs) immunofluorescence. Inanesthetized rats, unilateral injection of L-glutamate (5 nmol) into the medial vestibularnucleus (MVe) and spinal vestibular nucleus (SpVe) slightly increased the mean arterialpressure (MVe: 93.29F11.58 to 96.30F11.66, SpVe: 91.72F15.20 to 95.48F17.16). Localpretreatment with the N-methyl-D-aspartate (NMDA)-receptor antagonist MK-801 (2 nmol)significantly attenuated the pressor effect of L-glutamate injected into the MVe compared tothe contralateral self-control. After injection of biotinylated dextran amine (BDA) into theMVe and SpVe, and fluorogold (FG) into the RVLM, some BDA-labeled fibres and terminals inthe nucleus of solitary tract (NTS) and the parabrachial nucleus (PBN) were immunoreactivefor VGluT1 and VGluT2. Several BDA-labeled fibres were closely apposed to FG-labeledneurons in the NTS. These results suggested that activation of caudal vestibular nucleusneurons could induce pressor response and NMDA receptors might contribute to thisresponse in theMVe. The glutamatergic VN-NTS and VN-PBN pathwaysmight exist, and theprojections from the VN to the RVLM relayed by the NTS comprise an indirect vestibulo-cardiovascular pathway in the brain stem.

© 2008 Elsevier B.V. All rights reserved.

Keywords:Vestibular nucleusNucleus of the solitary tractParabrachial nucleusRostral ventrolateral medullarVesicular glutamate transporterImmunohistochemistry

9..-L. Cai), [email protected] (W.-L. Ma), [email protected] (M. Li).S, nucleus of the solitary tract; PBN, parabrachial nucleus; RVLM, rostral ventrolateralT, vesicular glutamate transporter; IR, immunoreactivity; 4V, fourth ventricle; MVe, medialcleus; Pr, prepositus nucleus; Sp5, spinal trigeminal tract; IRt, intermediate reticular nucleus;subnucleus; is, interstitial subnucleus; lt, lateral subnucleus; me, medial subnucleus; sup,

us; vt, ventral subnucleus; elPBN, external lateral parabrachial nucleus; BP, blood pressure;ificial cerebrospinal fluid; BDA, biotinylated dextran amine; FG, Fluoro-gold

er B.V. All rights reserved.

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1. Introduction

Table 1 – The effects of aCSF and L-glutamate unilaterallyinjected into the MVe and SpVe on changes in meanarterial pressure (ΔMAP) in group 1, and pretreatmentwith MK801 on changes in mean arterial pressure evokedby L-glutamate in group 2

ΔMAP (mmHg)

MVe SpVe

Group 1 (n=11)aCSF 0.79±0.97 0.88±1.35L-glutamate 3.01±2.49* 3.76±3.80*

Group 2 (n=10)aCSF–l-glutamate 3.09±3.13 3.22±1.68MK801–L-glutamate 1.10±0.43# 2.72±2.35

Values are means±SD; *P<0.05 vs. aCSF self-controls, #P<0.05 vs.aCSF pretreatment self-controls (paired t-test).

Recent studies have demonstrated that the vestibular systemcontributes to autonomic regulation during alterations in bodyposition in space (Zhu et al., 2007). Electrical or adequatevestibular stimulation elicits an increase in sympathetic nerveactivity in both animal and human subjects (Xu et al., 2002;Kaufmann et al., 2002). A large amount of evidence suggestedthe existence of vestibulo-sympathetic reflex pathwaysindependent of baroreflex ones in various species (Woodringet al., 1997; Radtke et al., 2000; Ray, 2001; Gotoh et al., 2004).However, the exact pathways and neurotransmitters under-lying this reflex in the central nervous system (CNS) areunknown.

Lesions of the central vestibular system can produce aprolonged impairment in posture-related cardiovascularresponses while bilateral labyrinthectomy only elicits atransient effect (Jian et al., 1999; Mori et al., 2005). Theseexperiments suggested that the vestibular nucleus (VN) playsa key role in compensatory vestibulo-sympathetic responseduring movement and posture changes. Studies haveindicated that the medial and spinal vestibular nuclei (MVe,SpVe) located caudal to the lateral vestibular nucleusmight bethe bautonomic regionQ of the vestibular nucleus complex(Uchino et al., 1970; Shiba et al., 1996; Xu et al., 2002). Inanesthetized, tracheotomized and spontaneously breathingrats, electrical stimulation of MVe evoked pressor response(Xu et al., 2002). However, whether this pressor responsewas aresult of activated local neurons or fibres of passage remainedunclear. Balaban and Porter reported that neurons in thecaudal MVe and SpVe projected directly to nucleus of thesolitary tract (NTS) and other autonomic regions, such as thecaudal part of parabrachial nucleus (PBN), nucleus ambiguousand dorsal motor nucleus of the vague (Balaban and Porter,1998). Lesion studies showed that the SpVe and adjacent MVeand the rostral ventrolateral medullary reticular formationwere essential for vestibulo-sympathetic reflex during otolithstimulation in cats (Yates andMiller, 1994; Yates et al., 1995). Itis well known that the rostral ventrolateral medullar (RVLM)plays a critical role in the sympathetic tone regulation andblood pressure (BP) control, and this area contains tonicallyactive presympathetic neurons innervating sympatheticpreganglionic neurons of the intermediolateral cell columnmonosynaptically (Guyenet, 2006). Although no studies haveshown that the vestibular nuclei provide direct projection tothe RVLM, physiological studies have confirmed that vestib-ular related neurons in the RVLM receive indirect vestibularinputs (Dampney et al., 1987; Yates et al., 1991).

In our previous study, we found that phosphate-activatedglutaminase (PAG)-immunoreactive VN neurons constitutedexcitatory glutamatergic VN-NTS and VN-PBN transmissionpathways and these pathways were involved in vestibulo-autonomic reflexes during vestibular stimulation (Cai et al.,2007). In addition, there are anatomical and functionalevidences for direct excitatory projections from the NTS andPBN to the RVLM in rats (Aicher et al., 1996; Mauad andMachado, 1998; Len and Chan, 1999). Therefore, it is possiblethat the indirect pathways from the VN to the RVLM may berelayed by the NTS and PBN. Furthermore, if the VN-NTS-

RVLM and (or) VN-PBN-RVLM pathways exist, it remainsunclear whether glutamate acts as an excitatory neurotrans-mitter in these projections or not. The present study isundertaken to investigate whether focal administration ofexcitatory amino acid into the caudal VN can elicit bloodpressure response. We also attempt to investigate the indirectexcitatory VN-RVLM pathways using retrograde and antero-grade tracing method combined with vesicular glutamatetransporters (VGluTs) immunofluorescence and confocal laserscanning microscopy.

2. Results

2.1. BP responses to L-glutamate injection into the MVeand SpVe

In group 1, unilateral injection of L-glutamate (5 nmol, n=11)into the MVe and SpVe slightly increased mean arterialpressure (MAP) (MVe: from 93.29F11.58 to 96.30F11.66,SpVe: from 91.72F15.20 to 95.48F17.16, P<0.01). In a self-control test, injection of the same volume of aCSF into thesame region did not alter basal BP. The changes inMAP evokedby aCSF and L-glutamate injection into MVe and SpVe wereshown in Table 1. In group 2, pretreatment with aCSF had noeffect on the pressor responses caused by microinjection of L-glutamate in the same region of the MVe and SpVe (aCSFpretreatment self-control) (Figs. 1A, B). Local injection withNMDA receptor antagonist MK801 into the controlateral MVeand SpVe had no effect on blood pressure baseline(94.65F17.68 mm Hg vs. 94.05F17.85 mm Hg, 92.35F17.68mm Hg vs. 92.15F16.75 mm Hg), but significantly attenuatedthe pressor effect of L-glutamate injected into the same regionof MVe afterward compared to the contralateral aCSF pre-treatment self-control (Fig. 1C). The changes in MAP evoked byL-glutamate injection into MVe and SpVe of animals pre-treated with aCSF on one side (aCSF-L-glutamate) andpretreated with MK801 on another side (MK801-L-glutamate)were shown in Table 1.

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Fig. 1 – Representative tracings showing the effect ofL-glutamate (5 nmol)microinjection intoMVe (A) and SpVe (B)on blood pressure, and the effect of prior injection of MK-801(20 nmol) into MVe on changes in blood pressure evoked byL-glutamate. (A) The pressor action evoked by L-glutamateinjection into the unilateral MVe. (B) The pressor actionevoked by L-glutamate injection into the unilateral SpVe. (C)The effect of prior injection of MK-801 (2 nmol) into the MVeon changes in blood pressure evoked by L-glutamate.

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2.2. BDA and FG injection sites

In group 3, BDA was injected into the caudal VN region (MVeand SpVe) yielding pressor response to L-glutamatemicroinjection (n=10). Eight of these animals received FGmicroinjection into the RVLM following chemical identifica-tion. The cases, with the BDA injection sites extended into therostral part of NTS underlying the caudal VN and/or with theFG diffused into adjacent structures including ambiguousnucleus and lateral paragigantocellular nucleus, wereexcluded from further analysis (n=2).

Fig. 2 – Fluorescence photomicrographs showing BDA injection s(B) of one representative rat. 4V: fourth ventricle; MVe:medial vesof solitary tract; Pr: prepositus nucleus; RVLM: rostral ventrolatereticular nucleus.

Injection sites where BDA labeled with FITC presented agranular core of densely packed neurons surrounded by anarea inwhich labeled neuronswere sparse. The center of theseinjection sites were located in the caudal MVe (approximately2.4 mm rostral to the obex, 0.6 mm lateral to the midline, and0.5 mm below the dorsal surface of the medulla) and SpVe(approximately 2.3 mm rostral to the obex, 1.4 mm lateral tothe midline, and 0.3 to 0.6 mm below the dorsal surface of themedulla). Because of the big size and superficial location of theMVe and SpVe, the diffusion area of the BDA injection sitesinevitably varied from case to case.

FG injection sites consisted of a homogeneous, compactlight core surrounded by a halo. The center of these injectionsites was located in the RVLM approximately 2.7mm rostral tothe obex, 1.8 mm lateral to the midline, and 3.0 mm below thedorsal surface of the medulla. All the injection sites (both BDAand FG) were generally ovoid, with the diameter of the centralcore typically measuring 200–400 lm in the mediolateraldirection and extending for a similar distance rostrocaudallyacross transverse sections. Fig. 2 shows the injection site ofone representative rat of the accepted cases.

2.3. Double labeling of BDA and VGluTs

2.3.1. VGluT1 immunoreactivity (IR) of VN projecting fibresand terminals in the NTSAmong accepted cases, similar density and distribution ofanterograde labeled fibres and terminals were observed in theNTS and PBN. Anterogradely labeled fibres and terminals wereobserved bilaterally in the caudal NTS with ipsilateralpredominance covering a rostro-caudal extent of approxi-mately 400 lm. A relatively high density of thick fibres andterminals were found in the region between cuneate nucleusand NTS adjacent to the border of dorsolateral subnucleus. Inventral subnuclei, a moderate density of fibres and terminalswere seen. The medial, intermediate, and interstitial sub-nuclei contained fewer fibres and terminals. The lateralsubnuclei contained very few fibres and terminals (Fig. 3A1).In addition, a very high density of BDA-labeled fibres andterminals were seen in the beta subnucleus of ipsilateralinferior olive and controlateral VN, and a low density of fibresrandomly scattered in the lateral reticular nucleus of themedulla oblongata. BDA-labeled fibers in these areas showed

ites in the MVe and SpVe (A) and FG injection site in the RVLMtibular nucleus; SpVe: spinal vestibular nucleus; NTS: nucleusral medullar; Sp5: spinal trigeminal tract; IRt: intermediate

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sharp contour and/or beaded appearance. Meanwhile, VGluT1and VGluT2 immunoreactive fibers and terminals wereobserved throughout the caudal–rostral extent of the NTS inaccordance with findings of a previous immunohistochemicalstudy (Lin et al., 2004).VGluT1-IR exhibits a bouton or fiber-likepattern fluorescein staining, suggestive of neuronal terminalsor processes (Fig. 3A2). By using confocal microscopy, we wereable to detect several VGluT1-positive BDA-labeled (VGluT1/BDA-LI) bouton-like varicosities and/or fibers in the caudalportion of dorsolateral and ventral subnuclei (Figs. 3A3, C, D).No VGluT2 and BDA double-labeled fibers and terminals werefound in the whole extent of NTS.

2.3.2. VGluT2 immunoreactivity of VN projecting fibres andterminals in the PBNIn the PBN, anterogradely labeled fibres and terminals werepresented bilaterally in the caudal region of external lateralparabrachial (elPBN), dorsal lateral parabrachial, centrallateral parabrachial, and externalmedial parabrachial nucleuswith a rostro-caudal extent of about 500 lm. Meanwhile, wealso observed a high density of VGluT2-containing nerve fibersand terminals in the right and left elPBN (Fig. 3B1).

Under confocal microscopy, we observed some VGluT2 andBDA double-labeled axonal varicosities and associated smallround structures in the external part of lateral parabrachialnucleus (approximately from 9.1 to 9. 3 mm caudal to the

Fig. 3 – Confocal microscope images showing the appearance of aterminals in the caudal NTS, and anterogradely labeled (B1) and Vinjection into the MVe and SpVe. The merged images are shownimmunohistochemical staining for BDA (green) andVGluT1 (red) seat high magnification. Arrowheads indicate BDA-labeled fibres anterminals, and double arrowheads indicate BDA/VGluT1, 2 doublemedial; im: intermediate; is: interstitial; lt: lateral; elPBN: external lC, D and E.

Bregma) (Fig. 3B3). Under high magnification images obtainedby confocal laser microscopy, these VGluT2-IR fibers andterminals showed varicosities about 2–5 lm thick in diameter,suggestive of synaptic buttons in the PBN (Fig. 3E). No VGluT1and BDA double-labeled fibers and terminals were found inthe whole extent of the entire nucleus.

2.4. Appositions between BDA-labeled terminals andFG-labeled neurons in the NTS

The distribution pattern of retrograde labeled neurons in theNTS and PBN was similar among the accepted cases. Withinthe NTS, FG-labeled neurons were mainly seen ipsilaterally inthe subpostremal, medial, and intermediate subnuclei of thecaudal NTS approximately from 13.8–14.0 caudal to theBregma, and in the intermediate and ventral subnuclei from13.2–13.8 caudal to the Bregma (Figs. 4A2, B2). In the PBN,retrograde labeled neurons were distributed in the externallateral, dorsal lateral, central lateral and KF subnuclei withipsilateral predominance. Since both VGluTs/BDA-LI fibresand FG-labeling neurons were present in some subnuclei ofthe NTS and PBN, we performed triple labeling for VGluTs,BDA and FG to examine their relationship. Under the confocallaser scanning microscope, some BDA-labeled terminals and/or single fibers were seen to be apposed to the somata ofretrograde labeled neurons in the medial, intermediate and

nterogradely labeled (A1) and VGluT1-IR (A2) fibres andGluT2-IR (B2) fibres and terminals in the caudal PBN after BDAin A3 and B3 at low magnification. (C, D) Doubleen in thedorsolateral (C) and ventral (D) subnucleus of theNTSd terminals, arrows indicate the VGluT1, 2-IR fibres and-labeled fibres and terminals. dl: dorsolateral; vt: ventral; me:ateral parabrachial; Bars: 100μminA1–A3andB1–B3; 25μmin

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Fig. 4 – Confocal microscope images showing the appearance of BDA-labeled (A1, B1 and C1) fibres and terminals andFG-labeled neurons (A2, B2, C2) in the caudal NTS (A1, A2, B1, B2) and PBN (C1, C2) after BDA injection into the MVe and SpVeand FG injection into the RVLM in the same rat. The superimposed images are shown in A3, B3 and C3 at low magnification.(D–F) Double immunohistochemical staining for BDA-labeled fibres and terminals (green) and FG- (red) labeled neurons seen inthe medial (D) dorsolateral (E) and ventral (F) subnucleus of the NTS at high magnification. Arrowheads indicate BDA-labeledfibres and terminals, arrows indicate the FG-labeled neurons, and double arrowheads indicate BDA-labeled fibres andterminals closely opposed (white arrows) to FG-labeled neurons. sup: subpostremal; me: medial; im: intermediate; vt: ventral;elPBN: external lateral parabrachial; Bars: 100 μm in A1–A3, B1–B3 and C1–C3; 25 μm in D–F.

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ventral lateral subnucleus of the NTS following FG injectioninto RVLM (Figs. 4A3, B3, D, E, F). These neurons, apposed byBDA-labeled fibers and/or terminals, scattered throughout thecaudal NTS with ipsilateral predominance. It was found thatfibres or terminals, apposed to the FG-labeled neurons, werenot labeled with VGluTs. In addition, no BDA-labeledterminals were seen to be closely contacted with FG-labeledneurons in the PBN after FG injection into RVLM (Fig. 4C).

3. Discussion

In the present study we found that: (1) microinjection of L-glutamate into the MVe or SpVe increased the blood pressurein anesthetized rats, while the pressor response was

attenuated by pretreatment with NMDA receptor antagonist(MK801) only injected in the MVe; (2) some VN-NTS projectingfibres and terminals co-localized with VGluT1, while someVN-PBN projecting fibres and terminals co-existed withVGluT2; and (3) several VN-NTS projecting fibres were closelyapposed to NTS-RVLM projecting neurons in the NTS.

Previous studies pointed out that the vestibular systemcontributed to pressor response in conscious rats duringexposure to gravitational changes (Matsuda et al., 2004;Tanaka et al., 2006). In baroreceptor-denervated animals,vestibular-elicited alterations in sympathetic nerve activitycan produce changes in blood pressure and blood flow tospecific vascular beds (Woodring et al., 1997; Kerman et al.,2000a,b). In humans, linear acceleration of the body, caloricstimulation of the ear, small backward drops of the head or

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off-vertical-axis rotation elicits alterations in sympatheticnerve firing by modulation of vestibular nerve activity (Cui etal., 1997; Yates et al., 1999; Radtke et al., 2000; Kaufmann et al.,2002). In the present study, cardiovascular response was alsoelicited by L-glutamate injection into caudal vestibular sub-nucleus in anesthetized rats. Nevertheless, the amplitude ofthe pressor response in our study wasmuch smaller than thatin the conscious rats induced by hyper- or hypogravitystimulation (Matsuda et al., 2004; Tanaka et al., 2006). Thereasons might be: (1) the number of neurons in the VNactivated by L-glutamate injectionwas limited by the diffusionarea of the chemical and the activity of these neurons mightalso be influenced by anesthesia; (2) in unanesthetizedanimals, several factors, such as stress, visual and proprio-ceptive sense, might have an additive effect on cardiovascularresponse during exposure to gravitational changes; and (3)there might also be an additive interaction for arterialpressure when the vestibulo-sympathetic and skeletal musclereflexes are engaged simultaneously in conscious animalsduring alterations in body position in space (Ray, 2001).Meanwhile, lesions of the vestibular nuclei could inducepersistent cardiovascular deficits during orthostaticchallenges (Mori et al., 2005). It is considered that vestibulo-sympathetic reflex pathways are independent from musclereflex and baroreflex pathways (Ray, 2001; Gotoh et al., 2004).Thus, it is suggested that activation of vestibular nucleusneurons by L-glutamate injections can induce cardiovascularresponse, which might be due to the activation of pathwaysbetween the vestibular system and other areas of the centralnervous system participating in cardiovascular modulation.

MK801 was chosen in the present study as an antagonistfor NMDA receptors based on its specificity in blocking thisreceptor (Wong et al., 1986). In the present study, localpretreatment with MK801 attenuated the pressor responsecaused by L-glutamate microinjection in the MVe, implyingthat NMDA receptor might be involved in the cardiovascularresponse to L-glutamate injections in the MVe. Anatomicaland physiological data showed that glutamatergic neuro-transmission occurred between primary vestibular afferentsand second-order vestibular nuclear neurons, indicating thatglutamate might be a major excitatory neurotransmitter ofprimary vestibular afferents (Raymond et al., 1988; Takahashiet al., 1994; Straka et al., 1996). Meanwhile, NMDA receptorsare abundantly localized in vestibular nuclear neurons andmost of NMDA receptors expressing VN neurons are activatedby otolith stimulation (Chen et al., 2003). Since the otolithcomponent of a vestibular organ is the receptor for gravityforce, it is reasonable to speculate that NMDA receptors in theMVe are involved in the modulation of cardiovascularresponse during hypo- or hypergravity stimulation. Besides,in the present study MK801 did not influence the pressorresponse of L-glutamate microinjection in the SpVe, suggest-ing that other types of glutamate receptors in the vestibularnucleus, such as a-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA), kainate (KA) and metabotropicglutamate receptors, might be involved in the cardiovascularregulation in this subnucleus (Chen et al., 2000). Thus, thecontribution of the non-NMDA receptors and metabotropicglutamate receptors and their interaction in vestibulo-cardiovascular regulation await further investigation.

It is well known that the PBN and NTS play important rolesin the central regulation of cardiovascular functions. Electricalor chemical stimulation of the ventrolateral regions andKÎelliker–Fuse (KF) subnucleus of the PBN complex signifi-cantly suppressed the reflex bradycardia via glutamatergicpathways from the PBN to the RVLM. Injections of L-glutamateinto the NTS in anesthetized rats produced hypotension andbradycardia similar to baroreflex activation (Talman et al.,1980). However, injections of L-glutamate into the NTS inawake rats usually produce pressor responses andbradycardia similar to peripheral chemoreceptor activation(Olivan et al., 2001). In our previous study, we observed thatFos immunolabeled neurons were present in the caudal VN,NTS, PBN and RVLM after Ferris-wheel rotation in rats (datanot shown).These observations are indicative of possibleindirect pathways relayed by the NTS and/or the PBN andthe contribution to vestibulo-cardiovascular response. In thepresent study, we did observe that some VN-NTS anterogradelabeled fibres and terminals were closely apposed to RVLM-VNretrograde labeled neurons in the NTS. These evidencessuggested that the VN-NTS-RVLM pathway might contributeto pressor response induced by L-glutamate injection intocaudal VN in this study. However, a rigorous electrophysiolo-gical exam should be carried out to give a full proof to ouropinion. Besides, previous studies showed that vestibulo-sympathetic reflexes were patterned according to both theanatomical location and innervation target of a particularsympathetic nerve, and could lead to distinct changes in localblood flow (Kerman et al., 2000a,b; Ray, 2001). Thus, it ispossible that aside from the excitatory vestibulo-sympatheticpathways theremay also exist inhibitory ones in themedullar.For example, the caudal ventrolateral medullar (CVLM), whichconveys inhibitory projections to the RVLM, plays animportant role in the regulation of cardiovascular activitiessuch as mediating arterial baroreceptor baroreflex (Dampney,1994). However, the role of CVLM in vestibulo-cardiovascularreflex has not been established.

In our previous study, we identified glutamatergicpathways from the VN to the NTS and the PBN usingphosphate-activated glutaminase (PAG) as a marker forneurons (Cai et al., 2007). In this study, we found that someVN-NTS and VN-PBN projecting fibres and terminals wereimmunoreactive for VGluT1 and VGluT2, respectively,suggesting that VN-NTS and VN-PBN excitatory pathwaysmight exist. Recent studies indicated that expression of VGluTisoforms appeared to correlate with the probability ofglutamate release and the potential for synaptic plasticity,which is generally low at synapses in the hippocampus andparallel fiber synapses in the cerebellum (which use VGluT1)and high at climbing fiber synapses in the cerebellum (whichuse VGluT2) (Fremeau et al., 2004). Thus it is suggested that theVN-NTS and VN-PBN fibres labeled with different VGluTisoformsmight be different in synaptic properties. In addition,we also found that no VN-NTS projecting fibres and terminals,which were apposed to the RVLM-VN retrograde labeledneurons, were labeled with VGluT immunostaining,suggesting that the indirect VN-RVLM pathways relayed bythe NTS might not use glutamate as transmitter. Furtherinvestigation is warranted to identify the neurotransmittersinvolved in this indirect pathway in the future.

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4. Experimental procedures

4.1. General procedures

Adult male Sprague–Dawley (SD) rats weighing 250–300 g wereused. All animal protocols and procedures described wereapproved by the Animal Use and Care Committee for Researchand Education of the Second Military Medical University(Shanghai, PR China). The animals were housed under a 12-hlight:12-h dark cycle with free access to food andwater. Effortswere made to minimize the number of animals used and theirsuffering.

Blood pressure (BP) recording and microinjections wereperformed in anesthetized rats. In detail, animals wereinitially anesthetized with sodium pentobarbital (40 mg/kg,i.p.). A catheter was inserted into the right femoral artery andconnected to a pressure transducer (Chongqin RM6280C) tomeasure blood pressure (BP) directly. BP was sequentiallymeasured and displayed on a channel of a recording system(software: RM6280C 3.3) by a computer continuously. Thelevel of anesthesia was verified during the following surgeryby evaluating vibrissa movement, corneal reflex andresponse to surgical stimuli. After anesthesia, animals wereplaced on a stereotaxic frame (Narishige, Japan). The skinand muscles of the neck were incised and dissected along themidline dorsally to expose the atlanto-occipital membrane.Then, the partial occipital craniotomy was performed andthe cerebellum was pushed forward to expose the underlyingfourth ventricle. A black eye shield was used to prevent anyvisual cues during the whole experimental process. Bodytemperature was maintained at about 37 -C with a heatingpad and an infrared lamp.

4.2. L-glutamate and MK801 microinjection into the MVeand SpVe

4.2.1. Group 1Eleven animals received a microinjection of 0.1 ll 50 mM L-glutamate dissolved in artificial cerebrospinal fluid (aCSF, inmM: 133.3 NaCl, 3.4 KCl, 1.3 CaCl2, 1.2 MgCl2, 0.6 NaH2PO4,32.0 NaHCO3, and 3.4 glucose, with pH adjusted to 7.4) into therightMVe (2.2–2.7mm rostral to the obex, 0.6–0.8mm lateral tothe midline, and 0.3 to 0.6 mm below the dorsal surface of themedulla) and the SpVe (2.2–2.7 mm rostral to the obex, 1.2–1.6 nbsp;mm lateral to the midline, and 0.3 to 0.6 mm belowthe dorsal surface of the medulla) through a glass micropipette(tip caliber 50–100lm)attached toaHamiltonmicrosyringe (1 ll).Microinjection of 0.1 ll aCSF 5 min before L-glutamateinjection at the same region of MVe and SpVe served as thevehicle control. The injections were made over a period of 5 s.The micropipette remained at the microinjection site until theobservations at that site were completed.

4.2.2. Group 2In another 10 animals, 0.1 ll 20 mM MK801 dissolved in aCSFwas injected into the MVe on one side of the brain stem 5 minbefore L-glutamate injection into the same region. After atleast 60 min, MK801 was injected into the SpVe of the sameside and L-glutamate was also injected into the same region

5 min after. Self-control experiment was performed on thecontralateral side of the brain stem following the sameprocedures with the MK801 replaced by aCSF. An interval ofat least 60 min between two injections of L-glutamate wasrespected for the action of L-glutamate might disappear.Finally, 50 nl of 2k pontamine sky blue solution was injectedto mark the sites for histology identification.

4.3. Tracer injections

Tracers were injected at the sites in the caudal VN and RVLMfrom which pressor responses were elicited by L-glutamatemicroinjection in 12 rats (group 3). Briefly, 10 min after pressorresponse was induced in the right MVe and SpVe, themicropipette was withdrawn and 0.1 ll solution of 10kbiotinylated dextran amine (BDA; MW 10,000; MolecularProbes, Eugene, OR) dissolved in 0.01 M phosphate buffer (pH7.2) was injected into the same sites through another glassmicropipette inserted at the same distance down the sametrack. The syringe was left in place for an additional 10 minbefore withdrawal to reduce the efflux up the track. Tenminutes after BDA injections, chemical identification of theRVLM (2.6–2.8mm rostral to the obex, 1.6–2.0mm lateral to themidline, and 3.0 to 3.2 mm below the dorsal surface of themedulla) was accomplished by obtaining a pressor responseelicited by 2 nmol L-glutamate injection, and similarprocedures were employed to inject 0.04 ll 4k Fluoro-gold(FG; Fluorochrome, Denver, CD) water solution at the samesite. Subsequent to these injections, the opening in the skullwas closed with dental cement, and the overlying neckmuscles and skin were sutured. At last, the right femoralartery was ligated, the catheter was withdrawn, and theoverlying skin was sutured.

Criteria for further analyses were as follows: (1) pressorresponsewas induced by L-glutamate injection into the caudalVN region (MVe and SpVe) and RVLM before tracer injection;(2) preparations with no sign of dye or with very largemarkingat the injection site which showed wide spread tracerdiffusion or left a track along the pipette path were discarded.

4.4. Tissue preparation

Seven days after surgery, animals of group 3were anesthetizedwith an overdose of sodium pentobarbital (100 mg/kg, i.p.) andperfused transcardially with 100 ml chilled (4 -C) phosphate-buffered saline (PBS) for 20 min, followed by perfusion withchilled 400 ml 0.1 mol/L phosphate buffer (PB, pH 7.4)containing 4k paraformaldehyde for 20 min. Brains wereremoved, postfixed with 4k paraformaldehyde 4 -C for 1 h.They were then placed in 0.1 mol/L PB containing 30k sucroseat 4 -C for 2 days and cut into five serials of 20 lm-thick sectionsthroughout the brain stem.

4.5. Immunohistochemistry

In one series of sections, BDA and FG labeling was visualizedusing fluorescentmethod. The sectionswerewashed in 0.01MPBS (PH; 7.4) then incubated in 0.01 M PBS (pH 7.4)+0.3k TritonX-100 for 1 h at room temperature. Aftermultiple rinses in PBS,the samples were exposed to 1:200 streptavidin–FITC (Sigma,

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St. Louis, MO) for 4 h at 4 -C. After washing in PBS again, thesesections were mounted onto a gelatin-coated glass slide forobservation under a fluorescence microscope (BX-60; Olym-pus) with appropriate filters for FITC (excitation wavelength:490 nm, emission wavelength: 520 nm) and FG (excitationwavelength: 340 nm, emission wavelength: 430 nm). BDA andFG injection sites in the VN and RVLM were visualized.

Tissue sections, with acceptable microinjection sitesmeeting the following criteria and explicit BDA-labeled fibersand FG-labeled neurons, were further stained for VGluT1 andVGluT2. Preparations which had no sign of dye at the VNinjection sites or in which microinjections produced a largemarking extending to the ventral and/or lateral border of thenuclei were not analyzed. Sections showingwide spread tracerdiffusion or left a track along the pipette path at the RVLMinjection sites were also discarded.

In each accepted preparations, another two series ofsections were used to triple immunoflourescent staining ofVGluT1/BDA or VGluT2/BDA with FG retrograde labeledneurons in PBN and NTS. These sections were washed in0.01 M PBS (PH; 7.4) then incubated in 1:1000 guinea-pig anti-VGluT1 IgG (Chemicon, Temecula, CA) (second series) or1:1000 mouse anti-VGluT2 IgG (Chemicon) (third series) for48 h at 4 -C. After washing in PBS, the sections were rinsed inPBS, then incubated in 1:200 streptavidin–FITC (Sigma, St.Louis, MO) and 1:200 Cy3-labeled goat anti-guinea-pig IgG(Jackson,West Groove, PA) or Cy3-labeled goat anti-mouse IgG(Jackson) for 4 h at room temperature. After washing thesections were mounted onto a gelatin-coated glass slide forobservation under a fluorescence microscope (BX-60; Olym-pus) with appropriate filters for FG (excitation wavelength:340 nbsp;nm, emission wavelength: 430 nm), FITC (excitationwavelength: 490 nm, emission wavelength: 520 nm), and Cy3(excitation wavelength: 554 nm, emissionwavelength: 568 nm).Then, the sections exhibiting positive staining were examinedunder a laser confocal scanning microscope (TCS SP2, Leica,Germany) by using laser beams 570 nm for Cy3, 488 nbsp;nmfor FITC, and 633 nm for FG. For optimal visualization of therelationship between FG and FITC-labeled elements, imagesfrom these channels were assigned the pseudocolor red forFG and green for FITC and were superimposed. The fourthseries of sections were processed without the primary anti-body to rule out non-specific immunostaining in thesesections.

4.6. Control experiments

Some animals served as control group were sham-operatedwith FG replaced by distilled water or BDA replaced by 0.01 Mphosphate buffer (n=5 respectively). To test the specificity ofBDA and FG labeling, these animals were subjected to thesame injection protocol as experimental animals. When theprimary antibodies (VGluT1 and VGluT2) were omitted orreplaced with normal sera, no corresponding immunoreactivelabeling was found.

4.7. Statistical analysis

BP was expressed as mean arterial pressure (MAP) andpresented asmeanFS.D. The paired t-test was used to compare

two mean values of different variables (BP or MAP) obtainedfrom the same animal. Differences were considered significantat P<0.05.

Acknowledgments

The authors are grateful for the confocal microscopephotographic help of Mrs. Ying Tang. This project wassupported by the Military Medical Foundation (WL, Ma) ofthe Second Military Medical University.

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