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Research Report

Projections from the hypothalamic paraventricular nucleusand the nucleus of the solitary tract to prechoroidal neurons inthe superior salivatory nucleus: Pathways controlling rodentchoroidal blood flow ☆

Chunyan Lia,⁎, Malinda E.C. Fitzgeralda,b,c, Mark S. LeDouxa,d, Suzhen Gongd,Patrick Ryane, Nobel Del Mara, Anton Reinera,b

aDepartment of Anatomy and Neurobiology, University of Tennessee, 855 Monroe Ave., Memphis, TN 38163, USAbDepartment of Ophthalmology, University of Tennessee, 855 Monroe Ave., Memphis, TN 38163, USAcDepartment of Biology, Christian Brothers University, Memphis, TN, USAdDepartment of Neurology, University of Tennessee, 855 Monroe Ave., Memphis, TN 38163, USAeDepartment of Molecular Sciences, University of Tennessee, 855 Monroe Ave., Memphis, TN 38163, USA

A R T I C L E I N F O

0006-8993/$ – see front matter. Published bydoi:10.1016/j.brainres.2010.08.065

A B S T R A C T

Article history:Accepted 20 August 2010Available online 27 August 2010

Using intrachoroidal injection of the transneuronal retrograde tracer pseudorabies virus(PRV) in rats, we previously localized preganglionic neurons in the superior salivatorynucleus (SSN) that regulate choroidal blood flow (ChBF) via projections to thepterygopalatine ganglion (PPG). In the present study, we used higher-order transneuronalretrograde labeling following intrachoroidal PRV injection to identify central neuronal cellgroups involved in parasympathetic regulation of ChBF via input to the SSN. Theseprominently included the hypothalamic paraventricular nucleus (PVN) and the nucleus ofthe solitary tract (NTS), both of which are responsive to systemic BP and are involved insystemic sympathetic vasoconstriction. Conventional pathway tracing methods were thenused to determine if the PVN and/or NTS project directly to the choroidal subdivision of theSSN. Following retrograde tracer injection into SSN (biotinylated dextran amine 3K orFluorogold), labeled perikarya were found in PVN and NTS. Injection of the anterogradetracer, biotinylated dextran amine 10K (BDA10K), into PVN or NTS resulted in denselypacked BDA10K+terminals in prechoroidal SSN (as defined by its enrichment in nitric oxidesynthase-containing perikarya). Double-label studies showed these inputs ended directly onprechoroidal nitric oxide synthase-containing neurons of SSN. Our study thus establishesthat PVN and NTS project directly to the part of SSN involved in parasympatheticvasodilatory control of the choroid via the PPG. These results suggest that control of ChBFmay be linked to systemic blood pressure and central control of the systemic vasculature.

Published by Elsevier B.V.

Keywords:Biotinylated dextran amine (BDA)Pseudorabies virus (PRV)Fluorogold (FG)

Elsevier B.V.

☆ Supported by the University of Tennessee Neuroscience Institute (CL), Department of Ophthalmology unrestricted grant fromResearchto Prevent Blindness (MECF), Benign Essential Blepharospasm Research Foundation Inc. (ML), NIH-EY-12232 (ML), NIH-EY-05298 (AR), andNIH/NEI-5P30EY013080 (D. Johnson).⁎ Corresponding author. Department of Anatomy & Neurobiology, University of Tennessee Health Science Center, 855 Monroe Ave.,

Memphis, TN 38163, USA. Fax: +1 901 448 7193.E-mail address: chli@uthsc.edu (C. Li).Abbreviations: 6, abducens nucleus; 10, dorsal motor nucleus of vagus; 12, hypoglossal nucleus; 3V, third ventricle; 7n, facial nerve; 8cn,

cochlear root of 8n; 8n, vestibulocochlear nerve; 8vn, vestibular root of 8n; A5, A5 noradrenaline cells; Acs7, accessory facial nucleus; AD,anterodorsal thalamic nucleus; AH, anterior hypothalamus; Amb, ambiguus nucleus; AP, area postrema; Arc, arcuate nucleus; AV,anteroventral thalamic nucleus; AVPe, anteroventral periventricular nucleus; BAOT, bed nucleus of accessory olfactory tract; BDA,biotinylated dextran amine; BDA3K, biotinylated dextran amine 3000 kDa MW; BDA10K, biotinylated dextran amine 10,000 kDa MW; BLA,anterior part of basolateral amygdaloid nucleus; BMA, anterior part of basomedial amygdaloid nucleus; BP, blood pressure; BST, bednucleus of stria terminalis; C3, C3 adrenaline cells; CA3, field CA3 of hippocampus; cc, corpus callosum; Ce, central amygdaloid nucleus;cg, cingulum; ChAT, choline acetyltransferase; ChBF, choroidal blood flow; CLSM, confocal laser scanning microscope; CM, central medialthalamic nucleus; CPu, caudate putamen; CxA, cortex–amygdala transition zone; cu, cuneate fasciculus; Cu, cuneate nucleus; DAB,diaminobenzidine tetrahydrochloride; das, dorsal acoustic stria; DC, dorsal cochlear nucleus; DH, dorsomedial hypothalamus; DV,dorsoventrally; ec, external capsule; ECu, external cuneate nucleus; En, endopiriform cortex; f, fornix; FG, Fluorogold; fi, fimbria ofhippocampus; g7, genu of facial nerve; Gi, gigantocellular reticular nucleus; GiA, alpha part of gigantocellular reticular nucleus; Gr, gracilenucleus; HDB, nucleus of horizontal limb of diagonal band; IAM, interanteromedial thalamic nucleus; ic, internal capsule; icp, inferiorcerebellar peduncle; IM, intercalated nucleus of amygdala; IO, inferior olive; ISN, inferior salivatory nucleus; IRt, intermediate reticularnucleus; LA, lateroanterior hypothalamus; LC, locus coeruleus; LD, laterodorsal thalamic nucleus; LGP, lateral globus pallidus; LH, lateralhypothalamus; LPGi, lateral paragigantocellular nucleus; LPO, lateral preoptic area; LRt, lateral reticular nucleus; LSO, lateral superiorolive; m7, facial nucleus; MD, mediodorsal thalamic nucleus; Me, medial amygdaloid nucleus; ml, medial lemniscus; mlf, mediallongitudinal fasciculus; MPO, medial preoptic nucleus; mt, mammillothalamic tract; MVe, medial vestibular nucleus; NO, nitric oxide;NOS, nitric oxide synthase; NTS, nucleus of solitary tract; opt, optic tract; ox, optic chiasm; P5, peritrigeminal zone; Pa6, para-abducensnucleus; Pr5, principal trigeminal nucleus; PAG, periaqueductal central gray; PAP, peroxidase–antiperoxidase; PBL, lateral parabrachialnucleus; pd, predorsal bundle; PDTg, posterodorsal tegmental nucleus; Pir, piriform cortex; PK15, porcine kidney cells; PMn, paramedianraphe nucleus; PnC, caudal pontine reticular nucleus; PnV, ventral pontine reticular nucleus; PPG, pterygopalatine ganglion; PPy,parapyramidal nucleus; Pr, prepositus nucleus; PRV, pseudorabies virus; PT, paratenial thalamic nucleus; PVA, anterior part ofparaventricular thalamic nucleus; PVA, paraventricular thalamic nucleus; PVN, paraventricular nucleus; py, pyramid; RAmb,retroambiguus nucleus; Re, reunions; Rh, rhomboid thalamic nucleus; RMg, raphe magnus; unc, uncinate fasciculus; ROb, rapheobscurus nucleus; RPa, raphe pallidus nucleus; rs, rubrospinal tract; Rt, reticular thalamic nucleus; RVLM, rostroventral lateral medulla;SCG, superior cervical ganglion; SCN, suprachiasmatic nucleus; scp, superior cerebellar peduncle; SD, Sprague-Dawley; sm, striamedullaris of thalamus; sol, solitary tract; SO, superior olive; SON, supraoptic nucleus; sox, supraoptic decussation; Sp5, spinal trigeminalnucleus; sp5, spinal trigeminal tract; SPO, superior paraolivary nucleus; SSN, superior salivatory nucleus; st, stria terminalis; Sub,submedius thalamic nucleus; ts, tectospinal tract; VA, ventral anterior thalamic nucleus; VDB, nucleus of vertical limb of diagonal band;VeCb, vestibulocerebellar nucleus; VeL, lateral vestibular nucleus; VeM, medial vestibular nucleus; VeS, superior vestibular nucleus; VIP,vasoactive intestinal polypeptide; VL, ventrolateral thalamic nucleus; VM, ventromedial thalamic nucleus; VMH, ventromedialhypothalamic nucleus; VPL, ventral posterolateral thalamic nucleus; VPO, ventral paraolivary nucleus; vsc, ventral spinocerebellartract; ZI, zona incerta

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1. Introduction

The choroid contains the blood supply to the outer retina. Thechoroid and blood vessels supplying the choroid are inner-vated by parasympathetic, sympathetic, and sensory nervefibers that adaptively regulate choroidal blood flow (ChBF)according to retinal need (Bill, 1984; Bill, 1985; Bill, 1991;Cuthbertson et al., 1996; Cuthbertson et al., 1997; Fitzgeraldet al., 1990a; Fitzgerald et al., 1990b; Fitzgerald et al., 1996;Guglielmone and Cantino, 1982; Kirby et al., 1978; Stone et al.,1987). Such adaptive control may be important for maintain-ing the health of retinal photoreceptors and maintainingnormal visual function (Fitzgerald et al., 1990a; Fitzgeraldet al., 1990b; Fitzgerald et al., 2001; Hodos et al., 1998; Potts,1966; Reiner et al., 1983; Shih et al., 1993; Shih et al., 1994). Thepterygopalatine ganglion (PPG) is the major source of para-sympathetic input to the choroid and orbital vessels, and thisinput utilizes two vasodilators: vasoactive intestinal polypep-tide (VIP) and nitric oxide (NO) (Alm et al., 1995; Bill, 1984; Bill,1985; Bill, 1991; Ruskell, 1971a; Stone, 1986; Stone et al., 1987;Uddman et al., 1980a; Yamamoto et al., 1993). These fibers alsoappear to be cholinergic (Johansson and Lundberg, 1981;Lundberg et al., 1981; Lundberg et al., 1982; Suzuki et al., 1990).

The PPG receives its preganglionic input from the superiorsalivatory nucleus (SSN) of the hindbrain via the greaterpetrosal branch of the facial nerve (Contreras et al., 1980; Nget al., 1994; Nicholson and Severin, 1981; Schrödl et al., 2006;Spencer et al., 1990; Tóth et al., 1999). The SSN itself is locateddorsolateral to the rostral part of the facial motor nucleus. TheSSN neurons, which are somewhat intermingled with norad-renergic neurons of the more rostrally situated A5 cell group,are cholinergic. In rats, rabbits and humans, a subset has beenreported to contain nitric oxide synthase (NOS), as well(Cuthbertson et al., 2003; Gai and Blessing, 1996; Zhu et al.,1996; Zhu et al., 1997). The SSN also provides preganglionicinput via the corda tympani nerve to the submandibularganglion (Contreras et al., 1980; Jansen et al., 1992; Ng et al.,1994; Nicholson and Severin, 1981), which sends postgangli-onic fibers to the submandibular and sublingual glands, andthereby regulates blood flow and salivary secretion withinthese glands (Izumi and Karita, 1994). The PPG, in addition toits innervation of choroidal and orbital blood vessels, alsoinnervates the Meibomian glands, the lacrimal gland, theHarderian gland, blood vessels of the nasalmucosa and palate,and cerebral blood vessels (LeDoux et al., 2001; Nakai et al.,1993; Ruskell, 1965; Ruskell, 1971b; Ten Tusscher et al., 1990;

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Uddman et al., 1980b; van der Werf et al., 1996). Thus,functionally diverse types of preganglionic neurons arepresent within the SSN.

The locations within the SSN of the preganglionic neuronscontrolling the salivary glands (Tóth et al., 1999), Meibomianglands (LeDoux et al., 2001), the lacrimal gland (Tóth et al.,1999), and the choroid (Cuthbertson et al., 2003) have been

Fig. 1 – Line drawing of coronal sections at the levels of PVN (bre−13.24 mm and bregma −14.08 mm), with schematics based on tdistribution of PRV+neurons in rat R3-07, in which the PRV injecextensive higher-order labeling in PVN and NTS. Each filled circl

described. While these functionally diverse populations haveslightly differing distributions, overlap among them is none-theless present, especially for those innervating orbitalstructures. The present study investigated the projection oftwo neuronal populations to prechoroidal SSN neurons, whichcan be identified within the SSN partly by their location, andnotably by their enrichment in NOS (Cuthbertson et al., 2003).

gma −2.12 mm), SSN (bregma −11.00 mm), and NTS (bregmahe rat atlas of Paxinos and Watson (1994), showing thetion was well confined to the right choroid and yieldede represents one PRV+neuron.

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Using intrachoroidal injection of the transneuronal retrogradetracer, the Bartha strain of pseudorabies virus (PRV), in adultrats, we observed higher-order labeling of many neurons inthe paraventricular nucleus of the hypothalamus (PVN), andthe nucleus of the solitary tract (NTS), among other regions.The PRV studies could not, however, determine if theseprojections were direct or multisynaptic. The inputs of PVNand NTS to prechoroidal SSN are of particular interest, asthese two nuclei play important roles in cardiovascularregulation. Consequently we confirmed the direct projectionsof these two neuronal cell groups to the SSN by a combinationof retrograde pathway tracing with biotinylated dextranamine 3000 kDa MW (BDA3K) or Fluorogold (FG), and ante-rograde pathway tracing with biotinylated dextran amine10,000 kDa MW (BDA10K) in conjunction with the identifica-tion of prechoroidal SSN by NOS immunolabeling.

2. Results

2.1. Transneuronal retrograde tracer (PRV) injectioninto choroid

Using PRV labeling in brain after intrachoroidal PRV injectionto delineate central pathways controlling ChBF depends

Table 1 – Summary of cases involving retrograde labeling of PV

Rat ID Injectionmethod

Injection site

Part of SSNinjected

Region of spreadbeyond SSN

RBPH38,39, 41

Pressure FG Central SSN Spinal 5, principal 5, nerve 7,motor 7, ISN

RBPH42 Pressure FG LaterodorsalSSN

Motor 5, spinal 5, principal 5,nerve 7, SO, MVe

RBPH37 Pressure FG Ventral SSN A5, SO, principal 5, spinal 5,motor 7, nerve 7

RBPH40 Pressure FG None Nucleus subcoeruleus, motor 5spinal 5, principal 5

RBP64 Pressure BDA Central SSN Spinal 5, motor 7RBP65 Pressure BDA Central SSN Principal 5, spinal 5, motor 7,RBP60 Pressure BDA Lateral SSN Principal 5, spinal 5, motor 7RBP56 Pressure BDA None Spinal 5RBP63 Pressure BDA None Spinal 5RBP46 Iontophoresis

BDACentral SSN None

RBP47 IontophoresisBDA

Central SSN Spinal 5

RBP53, 55 IontophoresisBDA

Central SSN Spinal 5

RBP40, 42 IontophoresisBDA

None Lateral spinal 5

RBP43 IontophoresisBDA

None Spinal 5, rostral nerve 7

RBP45 IontophoresisBDA

None Nerve 7, IRt

−: no retrogradely labeled neurons.+: few retrogradely labeled neurons.++: some retrogradely labeled neurons.+++: many number of retrogradely labeled neurons.++++: extensive retrogradely labeled neurons.a Retrogradely labeled neurons only found in NTS posterior to obex.

importantly on limiting the injection site to the choroid. Wecould judge this was the case if the central neuronal labelingprominently included SSN, with minimal or no labeling infacial, oculomotor or abducens motoneurons, or nucleus ofEdinger–Westphal preganglionic neurons. Minute (0.5 μl)injections (rats RF8, RF11-08, RF12-08) favored this outcome.In addition, short survival times (<55 hours) that limitedcollateral spread of virus (rat RF7) also favored restrictinglabeling to central choroidal circuits. The PRV findings wereport here are based on cases that met these criteria. In suchcases, prominent labeling was observed within a cluster ofneurons in rostromedial SSN, namely that controlling ChBF.The higher-order PRV+neuronal labeling seen in these casesincluded many additional structures, notably PVN, the lateralparabrachial nucleus (PBL), the periaqueductal central gray(PAG), the raphe magnus of the pons, the A5 cell group, andNTS. A full description of the higher-order labeling in brainafter intrachoroidal PRV injection will be provided in a laterpublication. In the present study, we particularly focus on PVNand NTS, because of their demonstrated role in peripheralcardiovascular regulation (Badoer and Merolli, 1998; Ciriello,1983; Godino et al., 2005; Krukoff et al., 1997; Rogers et al., 1993;Spencer et al., 1990). The PRV+neuron distributions withinPVN, SSN, andNTS are shown in Fig. 1 for one rat that survived69 hours (R3-07). Labeled cells were tightly clustered in SSN

N and NTS neurons from SSN using Fluorogold or BDA3K.

Location of retrogradely labeled neurons

IpsilateralPVN

ContralateralPVN

IpsilateralNTS

ContralateralNTS

++++ ++ ++++ ++a

+++ + +++ +

++++ ++ ++++ ++a

, − − − −

+ − ++ −nerve 7 + − ++ −

− − + −− − − −− − − −+ − + −

+ − + −

− − + −

− − − −

− − − −

− − + −

Fig. 2 – Illustration of Fluorogold injection sites targeting SSNby microsyringe pressure injection for six cases(RBPH37–RBPH42). Image A shows the cores of the Fluorogoldinjection sites for RBPH41 and RBPH40. In RBPH41, theinjection site is centered on SSN, while for RBPH40 it wasmore rostral and lateral to that in RBPH41. Image B shows thecores of the Fluorogold injection sites for RBPH39 andRBPH42. Image C shows the cores of the Fluorogold injectionsites for RBPH38 and RBPH37. The location and spread of theinjection sites in RBPH41, RBPH39, and RBPH38 are verysimilar. The injection sites and resulting retrograde labelingfor these cases, plus five control pressure injection cases forSSN and six iontophoretic injection cases for SSN, are alsodescribed in Table 1.

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(Fig. 1B). Within PVN, PRV+neurons were located in theipsilateral dorsolateral posterior PVN, with a few PRV+neuronslocatedcontralaterally (Fig. 1A).WithinNTS, PRV+neuronswereobserved posteriorly and dorsally in ipsilateral NTS (Figs. 1Cand D). Although transneuronal retrograde pathway tracingwith PRV identifies circuits, it cannot establish if the connec-tions among any members of the circuits are direct orpolysynaptic. We thus next carried out conventional pathwaytracing studies to determine if PVN and NTS project directly tochoroidal SSN.

2.2. FG or BDA3K injection into SSN

To determine if PVN and NTS project directly to the region ofSSN containing the choroidal–control preganglionic neurons,we injected either the retrograde tracer FG or the retrogradetracer BDA3K into SSN, either by iontophoresis or pressure

injection with microsyringe. The animals with such injectionsare listed in Table 1.

2.2.1. FG pressure injection into SSNSix rats received FG into SSN by pressure injection. Theinjection sites for these six cases are illustrated in Fig. 2. In fiveof these cases (RBPH37, RBPH38, RBPH39, RBPH41, andRBPH42), the FG injection sites were largely or entirelycentered on SSN. In all five, numerous FG+neurons werefound in PVN and NTS. Images of the injection site (Fig. 3A),and the FG labeling within PVN (Figs. 3B and C) and NTS(Figs. 3D–F) for one representative case (RBPH41) are shown inFig. 3. The injection site includes the choroidal SSN, withspread to the principal and spinal trigeminal nuclei, facialmotor nucleus, facial nerve, and inferior salivatory nucleus(ISN). In this and the other four cases with FG injection intoSSN, FG+neurons were observed in PVN on both sides, withmore ipsilaterally (Figs. 3B and C). Within ipsilateral PVN, thelabeled neurons were most common posteriorly and dorsally.FG+neurons were also found bilaterally within the NTS in thefive cases with FG injection into SSN, with an ipsilateralpredominance as well (Figs. 3D–F). The labeled neurons wereparticularly abundant in ipsilateral NTS at or just rostral to theobex. It is important to note that these SSN injectionsalso retrogradely labeled neurons in other regions containingPRV+neurons after intrachoroidal injection, but we will notdetail these here due to our focus on PVN and NTS. In one case(RBPH40) that served as a control, the FG injection wasrostrolateral to SSN and included the nucleus subcoeruleus,motor trigeminal nucleus, the principal trigeminal nucleus,and the spinal trigeminal nucleus. No FG+neurons wereobserved in PVN or NTS in this case. Thus, neither PVN norNTS project to the nucleus subcoeruleus, motor trigeminalnucleus, the principal trigeminal nucleus, or the spinaltrigeminal nucleus, and the FG spread to them in RBPH37,RBPH38, RBPH39, RBPH41, and RBPH42 does not account forthe FG+neurons in PVN and NTS in these cases.

2.2.2. BDA3K pressure injection into SSNFluorogold is a very avid retrograde tracer, and even seeminglywell-confined injections may yield retrograde labeling due tospread to nuclei adjacent to the region of interest. Thisproblem is especially acute for a complex brain region suchas the level of the SSN. Therefore, we carried out a series ofstudies using a less avid retrograde tracer, namely BDA3K. Fiverats received BDA3k pressure injections targeting SSN, whileeight received BDA3K targeting SSN by iontophoresis to makesmall confined injections (Table 1). In two of the five rats withBDA3K pressure injection targeting SSN (RBP64 and RBP65),the injection site included central SSN, with slight spread tothe sensory trigeminal nuclei (principal trigeminal nucleusand spinal trigeminal nucleus) and facial motor nucleus. Theresults were as observed following FG injection into SSN—retrogradely labeled BDA3K+neurons were observed in thedorsal and lateral parvocellular divisions of ipsilateral PVNand in ipsilateral NTS at and just rostral to the obex. Bycontrast, after a more lateral injection site including lateralSSN, sensory trigeminal nuclei and facial motor nucleus(RBP60), only a few BDA3K+neurons were found in ipsilateralNTS and none in PVN. In cases in which the BDA3K pressure

Fig. 3 – Images of Fluorogold-labeled neurons from a rat that had survived 6 days after pressure injection of Fluorogold into theright SSN (RBPH41), with the labeled neurons detected by DAB peroxidase–antiperoxidase immunohistochemistry. All imagesare of coronal sections. Image A shows the Fluorogold injection site into the right superior salivatory nucleus (SSN). Image Bpresents a low-power view through the caudal hypothalamus on the right side of the brain, showing a scattering of FG+neuronson that side in the paraventricular nucleus (PVN). Image C shows a high power view of the PVN presented in image B, in whichthe labeled neurons can be seen more clearly. Images D and E show low-power views of FG+neurons at two levels of thenucleus of the solitary tract (NTS) near the obex. FG+neurons are scattered throughout NTS at these levels. Image F shows ahigh-power view of theNTS presented in image E. The scale bar in B also provides themagnification for D and E. The scale bar inC also provides the magnification for F.

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injection site included the spinal trigeminal nucleus but notSSN (RBP56 and RBP63), no BDA3K+neurons were found inPVN and NTS. Thus, the pressure injection of BDA3K casesindicates that PVN and NTS project to choroidal SSN but not tothe spinal trigeminal nuclei. Also the BDA3K spread to theprincipal trigeminal nucleus as seen in RBP60 did not appearto contribute to the labeled neurons found in ipsilateral NTS,as FG spread to this region did not demonstrate labeledneurons in NTS (RBPH40).

2.2.3. BDA3K iontophoretic injection into SSNIn four of the rats with small iontophoretic injections (RBP46,RBP47, RBP53, and RBP55), the BDA3K injection site includedSSN, with no or minor spread to the sensory trigeminal nuclei(Table 1). The one case with an injection confined to SSN

(RBP46) yielded BDA3K+neurons in ipsilateral posterior,dorsal, and lateral PVN and in NTS at or just rostral to theobex. In the other three cases, BDA3K+neurons were consis-tently observed in ipsilateral NTS, but only one of three hadBDA3K+neurons in PVN (RBP47). These results suggest thatthe NTS input to choroidal SSN is more substantial than thePVN input. In four other rats with iontophoretic injection(RBP40, RBP42, RBP43, and RBP45), the BDA injection sitemissed SSN and instead included the spinal trigeminalnucleus, facial nerve root, and/or intermediate reticularnucleus (IRt). No BDA3K+cells were observed in PVN in anyof these four cases (Table 1). These SSN-miss cases thereforeshow that retrogradely labeled neurons in PVN in our SSNcases did not occur due to iontophoretic injection spread tothe spinal trigeminal nucleus or IRt. For NTS, a few BDA3K+

Table 2 – Summary of cases involving anterograde pathway tracing from PVN to SSN using BDA10K.

Rat ID Injectionmethod

Injection site Location of anterograde labeling within SSN and its vicinity

Part of PVN injected Region of spread beyondPVN

IpsilateralSSN

ContralateralSSN

IpsilateralA5

ContralateralA5

IpsilateralISN

ContralateralISN

RBP39 Iontophoresis PVN, complete A–P, medial 1/2 Slight MPO, medial to AH ++++ ++ +++ ++ − −RBP38 Iontophoresis PVN, complete A–P, medial 1/2 Slight ZI, Re ++++ ++ +++ ++ − −RBP26 Iontophoresis None Left LH − − − − − −RBP27,29,30 Iontophoresis None AH, MPO − − − − − −RBP28 Iontophoresis None Left LH − − − − − −RBP31 Iontophoresis None DH − − − − − −RBP32 Iontophoresis None AH, LA, optic chiasm − − − − − −RBP33 Iontophoresis None MPO; near PVN − − − − − −RBP34 Iontophoresis None Re − − − − − −RBP35 Iontophoresis None ZI and VM; lateral to PVN − − − − − −RBP36 Iontophoresis None SCN − − − − − −RBP25 Pressure PVN, complete A–P, medial 2/3 AH, VMH, DH, Re ++++ ++ +++ ++ + +RBP10 Pressure Post A–P PVN, dorsal slightly Medial thalamus ++ + ++ − + −RBP22 Pressure Ant and Mid A–P PVN, medial

2/3DH, septum, MPO, medialthalamus

++ + ++ + ++ +

RBP23 Pressure Ant and Mid A–P PVN MPO, BST ++ + + + − −RBP17 Pressure Ant and Mid A–P PVN,

dorsolateralMedial thalamus, PT, BST + + − − − −

RBP24 Pressure Mid A–P PVN, dorsal Medial thalamus + − + − − −RBP6 Pressure None BST − − − − − −RBP13 Pressure None AVPe, MPO − − − − − −RBP16 Pressure None BST, ZI, medial thalamus − − − − − −RBP18 Pressure None SON, LPO, HDB, LH, septum − − − − − −RBP19 Pressure None Septum, VDB, MPO, SON, LH − − − − − −RBP20 Pressure None LH, PT, PVA − − − − − −

−: no anterogradely labeled fibers.+: few anterogradely labeled fibers.++: some anterogradely labeled fibers.+++: many number of anterogradely labeled fibers.++++: extensive anterogradely labeled fibers.

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Table 3 – Summary of cases involving anterograde pathway tracing from NTS to SSN using BDA10K.

RatID

Injectionmethod

Injection site Location of anterograde labeling within SSN and its vicinity

Part of NTSinjected

Region ofspread

beyond NTS

IpsilateralSSN

ContralateralSSN

IpsilateralA5

ContralateralA5

IpsilateralISN

ContralateralISN

RBP95 Iontophoresis NTS Entire Slight dorsalIRt

++++ − +++ − ++++ +

RBP94 Iontophoresis NTS Entire Slight dorsalIRt

++++ + +++ − ++++ +

RBP83 Iontophoresis Mid A–P NTSmidventral

Dorsal IRt ++++ + ++ − ++++ +++

RBP91 Iontophoresis Mid A–P NTSmedial

None − − − − − −

RBP92 Iontophoresis Mid A–P NTSmedioventral

Slight IRt − − − − − −

RBP89 Iontophoresis None Lathypoglossal,dorsomedialIRt

− − − − ++ −

RBP72 Iontophoresis None Cu − − − − − −RBP81 Iontophoresis None MVe, Cu − − − − − −RBP88 Iontophoresis None C3,

hypoglossal− − − − − −

RBP90 Iontophoresis None Hypoglossal − − − − − −RBP75 Pressure Mid A–P NTS Dorsal IRt ++++ − ++ − ++++ +RBP77 Pressure Mid A–P NTS

lateralIRt, Cu, RVLM ++++ +++ ++++ ++ ++++ +++

−: no anterogradely labeled fibers.+: few anterogradely labeled fibers.++: some anterogradely labeled fibers.+++: many number of anterogradely labeled fibers.++++: extensive anterogradely labeled fibers.

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neurons were found in ipsilateral NTS in one rat (RBP45),possibly due to injection site spread to IRt, but not in the otherthree control cases. The overall data from our FG and BDA3Kretrograde labeling studies are consistent with the notion thatPVN and NTS neurons project directly to SSN.

2.3. Anterograde BDA10K labeling of PVN input toprechoroidal SSN

Our BDA3k retrograde labeling studies could not, by them-selves, however, establish that the PVN andNTS projections toSSN terminate directly on the choroidal–control preganglionicneurons of SSN. To establish this, we used anterogradeBDA10K labeling from PVN or NTS, either by pressure injectionor iontophoresis. The PVN data are presented in this section(Table 2), and the NTS (Table 3) in the next.

2.3.1. BDA10K iontophoretic injection into PVNThirteen rats received iontophoresis of BDA10K into PVN(Table 2). The injection sites for twelve rats are illustrated inFigs. 4A–D. The injection site of RBP27 is not illustrated as it isnearly the same as the injection sites for RBP29 and RBP30(Table 2). Two cases (RBP39 and RBP38) exhibited relativelyclean hits of the entire anterior-to-posterior medial PVN(Table 2). In both cases, many anterogradely labeled terminalswere observed within SSN on both sides, with more fibersipsilaterally. These terminals appeared to cluster in the regionof the prechoroidal SSN. Images from RBP39 are presented in

Fig. 5, including the injection site (Fig. 5A). To better establishthe relationship of the BDA10K+ terminals to the SSNneurons controlling the choroid, pairs of adjacent sectionswere ABC-labeled for BDA10K and immunolabeled for NOS(Figs. 5C and D), the latter of which we previously showed isenriched in the prechoroidal SSN neurons (Cuthbertson et al.,2003). We found that the location of the PVN afferents (Fig. 5D)and the location of the NOS+neurons in SSN (Figs. 5B and C)were largely coincident. To further confirm this overlap, wedouble-labeled sections through SSN using two-color DAB, sothat we could observe the relationship of the PVN afferentfibers to the NOS+prechoroidal SSN neurons. We observedthat black BDA10K+afferents from PVN were distributedamong the brown NOS+neurons of SSN (Figs. 5E and F). Insome instances, the PVN afferents were observed to bejuxtaposed to NOS+perikarya or dendrites within SSN (Fig. 5F).

Although the injection site following iontophoresis (dia-meter<1 mm) was very small, BDA10K spread to areasadjacent to PVN could not be avoided (Table 2). The spilloverareas observed in RBP39 involved the medial preoptic nucleus(MPO) and the small area medial to anterior hypothalamus(AH). The injection site spread outside of PVN observed inRBP38 included nucleus reuniens (Re) and zona incerta (ZI).Eleven rats with BDA10K injected by iontophoresis into cellgroups surrounding, but not including, PVN served as controls(Figs. 4A–D and Table 2). In none of these control rats wereBDA10K+fibers found in SSN. The injection sites in some rats(RBP26, RBP27, RBP28, RBP32, RBP33, and RBP36) included

Fig. 4 – Illustration of cases in which PVN was targeted byiontophoresis or microsyringe pressure. Image A shows theinjection site of a successful case RBP39 and those of threecontrol cases (RBP28, RBP34, and RBP35). Image B shows theinjection site of another successful case RBP38 and that ofone control case RBP26. Image C shows the injection sites oftwo control cases, RBP29 and RBP30, at a more rostral level.Image D shows the injection sites of five control cases. Of thefive control cases, RBP23 was a pressure injection case, whilethe remaining four were iontophoretic cases. Image E showsthe injection sites for two cases with pressure injection. Theinjection sites and resulting anterograde labeling for thesecases, and additional caseswith injections targeting PVN, arealso described in Table 2.

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structures ventral to PVN such as lateral hypothalamus (LH),AH, MPO, and suprachiasmatic nucleus (SCN), and in otherrats (RBP29 and RBP30) structures rostral to PVN such as MPOand AH. The absence of BDA10K fibers in SSN from these casessupported the notion that the terminals observed in RBP39were from PVN, but not fromMPO or AH or their vicinity. In yetadditional rats (RBP31 and RBP34), the BDA10K injection siteinvolved structures dorsal to PVN such as Re and dorsomedialhypothalamus (DH), and in one rat (RBP35) structures lateral toPVN such as ZI and ventromedial thalamus (VM). The absenceof BDA10K fibers in SSN from these cases supported the notionthat the terminals observed in RBP38 were from PVN but notfrom Re or ZI or their vicinity.

2.3.2. BDA10K pressure injection into PVNInitially, we also tried pressure injection of BDA10K (0.1–0.2 μl)into PVN on 12 rats. The results were consistent with thefindings from iontophoretic injection presented in the pre-ceding section. The pressure injection with microsyringeyielded a larger injection site (diameter>3 mm), and conse-quently more spillover outside PVN (Table 2). In the case ofRBP25, the injection site was located mainly in right PVN withspillover ventrally (Fig. 4E). Many anterogradely labeled fiberswere observed within SSN bilaterally, with more intenselylabeled fibers found in the ipsilateral SSN (right side). Fiveother rats (RBP10, RBP17, RBP22, RBP23, and RBP24) wereconsidered as partially successful cases, since the injectionsite, while not centered on right PVN, did include it.Anterogradely labeled fibers were more common in SSNipsilaterally for three of these cases, RBP10, RBP22, andRBP23, than in RBP17 and RBP24. In RBP10 (Fig. 4E), theBDA10K injection site included a very small part of dorsolat-eral PVN, while the major injection site was in medialthalamus. In RBP23, the injection site was more anterior(Fig. 4D), and similar injections sites were observed in RBP22and RBP17. Six cases with pressure injection of BDA10K servedas control cases (RBP6, RBP13, RBP16, RBP18, RBP19, andRBP20), in which the injection sites did not include PVN. Noanterograde labeling was seen in SSN in any of these cases(Table 2). These six control cases were valuable as theysuggested that the areas surrounding PVN do not projectdirectly to SSN. These control results were consistent with theiontophoretic findings, yet more informative, as the injectionsites were bigger.

In summary, the findings from our anterograde pathwaytracing study with BDA10K injection into PVN, either byiontophoresis or pressure injection, strongly supported thenotion that PVNneurons, especially those locateddorsolaterallyand posteriorly, project directly to prechoroidal SSN neurons.

2.4. Anterograde BDA10K labeling of NTS input toprechoroidal SSN

Ten rats received iontophoretic injections of BDA10K targetingNTS, while two received pressure injections targeting NTS.These are discussed separately below (Table 3).

2.4.1. BDA10K iontophoretic injection into NTSThe injection sites for the iontophoretic cases are illustrated inFigs. 6A–D. In two of these rats, the injections were confined to

Fig. 5 – Images of BDA10K+terminals from a rat that had survived 13 days after an iontophoretic delivery of BDA10K into theright PVN (RBP39), with the labeling detected by the ABC procedure with nickel intensified DAB. All images are of coronalsections. Image A shows the BDA10K injection site into the right PVN. Image B presents a camera lucida drawing of the brainstem at the level of the facial motor nucleus (7) and SSN. The dots represent NOS+neurons. Images C and D show SSN inadjacent sections in which prechoroidal SSN neurons have been immunostained for NOS (C) and BDA10K+terminals from PVNneurons visualized by the ABC method (D), respectively. Note that the location of NOS+neurons within SSN overlaps with thelocation of BDA10K+terminals from PVN neurons. Image E shows a high-power view of a section with two-color DAB doublelabeling to highlight the overlap between NOS+neurons (visualized by a brown DAB reaction) and BDA10K+terminals(visualized by a black nickel intensified DAB reaction). Image F is an enlargement of part of image E to show more clearly thatBDA10K+terminals are juxtaposed to NOS+neurons. The scale bar in C provides the magnification for C and D.

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the right NTS, with small spillover to dorsal IRt (RBP95 andRBP94). In both, anterogradely labeled fibers were observed inthe ipsilateral SSN and within the region dorsal to the SSN. Toestablish the relationship of the terminals within SSN to SSNneurons controlling the choroid, we used immunofluorescencedouble labeling to simultaneously visualize both the BDA10K+NTS input and NOS+prechoroidal SSN neurons in the samesections from the two cases with the BDA10K injection largelyconfined toNTS (Figs. 6A andB). In Fig. 7, images fromRBP95 arepresented, including the injection site and the confocal laserscanningmicroscope (CLSM) images of the double labeling. TheCLSM images confirmed the comingling of the NTS afferents

and NOS+neurons within the SSN (Figs. 7B–D). Moreover, inmany instances, BDA10K+terminalswere observed immediate-ly adjacent to the NOS+soma and dendrites of SSN neurons(Fig. 7D). TheBDA10K+terminalswereunlikely tocome fromthespillover into dorsal IRt, since in our PRV transneuronal labelingstudies, we did not observed PRV+neurons in dorsal IRt (Fig. 1).

Based on iontophoretic BDA10K injections into differentparts of NTS, our results suggest that input to prechoroidal SSNappears to arise from lateral NTS at and rostral to the obex. Forexample, in two cases (RBP91 and RBP92), the injection siteswere confined tomedial NTS at the obex. Anterogradely labeledfibers were not found in SSN in either case. By contrast, when

Fig. 6 – Illustration of central injection sites for the BDA10Kinjection cases targeting NTS by iontophoresis ormicrosyringe. Image A shows the injection site of asuccessful case (RBP95) and that of a control case (RBP92).Image B shows the injection site of another successful case(RBP94) and those of two control cases. Image C shows theinjection sites of one partially successful case (RBP83) and acontrol case (RBP30). Image D shows the injection sites forthree control cases at a more rostral level. Image E show theinjection sites for two cases with pressure injection. Theinjection sites and resulting anterograde labeling for thesecases are also described in Table 3.

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the BDA10K injection site included lateral NTS at the obex level,as in RBP94 and RBP95 (noted above) and RBP83, anterogradelylabeled fibers were abundant in prechoroidal SSN ipsilaterally.In five other rats (RBP72, RBP81, RBP88, RBP89, and RBP90), theiontophoretic injections did not include NTS but did includeneuronal groups aroundNTS (Table 3).Noanterogradely labeledfibers were found in SSN in any of these five cases.

2.4.2. BDA10K pressure injection into NTSTwo rats received BDA10K pressure injections via microsyr-inge targeting NTS. The injection in one case (RBP75) wascentered on NTS, and in the second (RBP77) included lateralNTS. Both RBP75 and RBP77 possessed densely packed fineBDA10K+fibers in SSN and its surrounding area, including A5,ISN, PBL, the vestibular nucleus, and the principal trigeminalnucleus. Scattered thick labeled fibers were also seen amongthe fine BDA10K+fibers. These results are consistent with ouriontophoretic studies.

In summary, our anterograde labeling studieswith BDA10Kinjection into NTS either by iontophoresis or pressure stronglysupport the notion that NTS neurons, especially those locateddorsolaterally at or rostral to obex, project directly and heavilyto prechoroidal SSN neurons.

3. Discussion

The ventral part of the SSN contains the preganglionicneurons projecting to the PPG via the greater petrosal nerve(Contreras et al., 1980; Jansen et al., 1992; Ng et al., 1994;Nicholson and Severin, 1981; Spencer et al., 1990; Tóth et al.,1999). The PPG itself, however, innervates diverse cranialtargets, including the lacrimal gland, the Meibomian glands,the orbital conjunctiva, choroidal blood vessels, the cerebralvasculature, and the nasal and palatal mucosa (Nakai et al.,1993; Ruskell, 1965; Ruskell, 1971b; Schrödl et al., 2006; TenTusscher et al., 1990; Uddman et al., 1980b; van der Werf et al.,1996). Prior studies using PRV injected into different peripheralPPG targets have suggested that the SSN populations respon-sible for innervation of the different PPG targets show over-lapping, but somewhat segregated, distributions. For example,SSN neurons controlling lacrimal gland are centered slightlycaudal to those responsible for Meibomian gland control(LeDoux et al., 2001; Tóth et al., 1999). A prior study of ours(Cuthbertson et al., 2003) indicated that preganglionic neuronscontrolling PPG choroidal neurons in rat reside withinventromedial SSN, and that NOS is a marker for prechoroidalSSN neurons. The possibility remains, nonetheless, thatchoroidal preganglionic SSN neurons may control more thanone of the target structures of the PPG. For example, given thesimilar metabolic and regulatory needs of the brain and eye(Bill, 1984; Bill, 1985), the same SSN neurons may mediatevasodilation of brain and choroidal vessels.

Prior studieshaveshownthat thePPGneurons innervating thechoroid contain NOS, VIP and choline acetyltransferase (ChAT)(Cuthbertsonet al., 2003; Stoneet al., 1987;Yamamotoet al., 1993).The same PPG neurons innervating the choroid or additional PPGneurons may innervate orbital vessels feeding into the choroidand thereby exert a further influence on ChBF (Stone et al., 1987).The presence of prechoroidal neurons in the SSN is consistentwith prior studies showing that facial nerve or SSN activationyields choroidal vasodilation, which appears to be mediatedmainly by NOS and VIP released from PPG terminals in thechoroid (Bill and Sperber, 1990; Steinle et al., 2000). It is importantto note that, in addition to the parasympathetic vasodilatoryinfluence on the choroidmediated by the PPG, the choroid is alsoregulated by vasodilatory sensory fibers from the ophthalmicnerve and vasoconstrictory sympathetic fibers from the superior

Fig. 7 – Images of BDA10K+terminals from a rat that survived 11 days after an iontophoretic injection of BDA10K into the rightNTS (RBP95). All images are of coronal sections. Image A shows the BDA10K injection site in the right NTS at the level of theobex. Image B shows an intermediate magnification CLSM image of NOS+neurons (green) and BDA10K+terminals (red) at thelevel of the facial somatomotor nucleus and SSN, showing the extensive overlap of NTS terminals with the NOS+neurons ofprechoroidal SSN. Image C shows a low-power view of NOS+neurons and BDA10K+fibers and boutons within SSN by using arendered z-stack obtained by CLSM. Densely distributed BDA10K+fibers are juxtaposed to the NOS+neurons. Image D ishigh-power view centering on one of the NOS+neurons shown in C. Note thatmany BDA10K+boutons can be seen surroundingthe dendrites and perikarya of this NOS+neuron, presumably forming direct synaptic contacts.

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cervical ganglion (Bill, 1984; Bill, 1985; Bill, 1991; Guglielmone andCantino, 1982; Kirby et al., 1978; Stone et al., 1987).

The higher-order labeling obtained here with PRV providesinsight into the central circuitry and mechanisms underlyingcontrol of ChBF. In particular, higher-order labeling wasobserved in dorsolateral posterior PVN and in caudolateralNTS at the level of the obex in cases with PRV injections wellconfined to the choroid. Consistent with our findings, Spenceret al. observed PRV+neurons in PVN and NTS after directinjection of virus into the PPG (Spencer et al., 1990). Thehigher-order labeling in NTS and PVN following short survivalwas of interest, since it suggested both project directly tochoroidal neurons of SSN. Prior studies have, in fact, shownthat the PVN and NTS are sources of input to the SSN(Agassandian et al., 2002; Hosoya et al., 1984; Hosoya et al.,1990). We confirmed that the subdivisions of the PVN and NTScontaining PRV+neurons after intrachoroidal PRV injectionscould be retrogradely labeled from SSN by Fluorogold andBDA3K. Thus, the dorsolateral caudal PVN and the caudolat-eral dorsal NTS at the level of the obex project to the SSNregion that contains the choroidal preganglionic neurons.Given that SSN neurons possess glutamate receptors, and SSNhas been shown to receive central glutamatergic inputs, it islikely the PVN and NTS inputs are excitatory (Lin et al., 2003).

The PVN region of the diencephalon is known to beresponsive to systemic blood pressure (BP) changes and mayexert a vasodilatory influence on cerebral blood flow (Badoer

and Merolli, 1998; Godino et al., 2005; Guyenet, 2006; Krukoffet al., 1997; Spencer et al., 1990). The caudolateral part of theNTSprojecting to the SSN is known to receive aortic baroreceptorinput via the vagus nerve and respond to systemic BPfluctuation (Ciriello, 1983; Guyenet, 2006; Rogers et al., 1993).The NTS also exerts a vasodilatory influence on cerebral bloodflow (Agassandian et al., 2003; Nakai and Ogino, 1984). Inaddition, the NTS projects directly and polysynaptically via thePBL to thePVN (Calarescuetal., 1984;GoldsteinandKopin, 1990).We have observed that activation of the PBL, NTS or the PVNincreases ChBF in the ipsilateral eye (Fitzgerald et al., 2010).Since the present results show that both the PVN and the NTSproject to the choroidal subdivision of SSN, it is likely that theChBF increase occurring with their activation is in large partmediated by their input to SSN prechoroidal neurons. Given theapparent role of the PVN and the NTS in mediating peripheralvascular responses to fluctuations in systemic BP, this suggeststhe possibility that these inputsmight regulate ChBF, in part, asa function of systemic BP. Prior studies have in fact shown thatChBF does compensate for fluctuations in systemic BP andremain stable over a range of 35% above and below basal BP inrats, rabbits, and pigeons (Kiel and Shepherd, 1992; Reiner et al.,2003; Reiner et al., 2010).

The similarities in systemic and ocular blood flow controlcircuitries reinforce the possibility that control of the twooperates in parallel—with the systemic sympathetic controlserving to restore BP in the face of any episodic declines in BP

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and the parasympathetic control of ChBF serving to maintainhigh ChBF during bouts of low systemic BP. Consistent with theprotective role that such parasympathetic ocular vascularcontrol might play, severing PPG input to the cerebral vascula-ture intensifies the cerebral damage occurringwith an ischemicevent (Kano et al., 1991; Koketsu et al., 1992). Further studies areneeded to determine if some of the same PVN andNTS neuronsare involved in regulation of both cerebral blood flow and ChBF.

4. Experimental procedures

4.1. Subjects and approaches

Sixty-six adult male Sprague-Dawley rats (SD rats; 300–500 g;from Harlan, Indianapolis, IN) were used (10 for PRV intochoroid, 13 for BDA3K into SSN, 6 for Fluorogold into SSN, 25for BDA10K into PVN, and 12 for BDA10K into NTS). Allexperiments were undertaken in compliance with the ARVOstatement on the Use of Animals in Ophthalmic and VisionResearch and with NIH and institutional guidelines. This studyfocused on the projections of the PVN and NTS to the choroidalcontrol neurons of SSN, using a two-step approach. In the firststep, the participation of PVN and NTS in circuitry projecting toprechoroidal SSN was established by injections of the trans-neuronal retrograde tracer PRV into choroid. In the second step,we assessed whether PVN and NTS project directly to prechor-oidal SSN neurons, using conventional retrograde and ante-rograde pathway-tracing methods. In the retrograde tracerstudies, BDA3K or FG was injected unilaterally into SSN todetermine if PVN and NTS were among the nuclei projectingdirectly to SSN. The anterograde-tracing studies involvedinjection of BDA10K into PVN or NTS, and examination ofprechoroidal SSN for the presence of resulting anterogradelylabeled BDA10K+fibers in SSN in general and upon NOS+prechoroidal SSN neurons, in particular.

4.2. Pseudorabies virus transneuronal retrogradepathway tracing

To identify SSN neurons involved in the control of ChBF, aretrograde transneuronal tracer, theBarthastrainofpseudorabiesvirus (PRV), was injected into the right choroid. Viruses such asPRV are valuable for delineating central circuits since, unlike aconventional retrograde tracer, they are transported transneur-onally (i.e., across synapses), and provide robust labeling inrecipient neurons due to virus replication (LeDoux et al., 2001).PRV injectionswereperformedwithaHamiltonsyringewitha30-gauge needle, and rats receiving intrachoroidal injection of PRVhad received prior bilateral resections of the superior cervicalganglia (SCG) to prevent retrograde transneuronal labeling alongsympathetic circuitry (LeDoux et al., 2001). Since PRV does nottypically show transganglionic transport via sensory ganglia(Jansen et al., 1992; LeDoux et al., 2001; Rotto-Percelay et al.,1992), there was no reason to transect the ophthalmic nerve toprevent transport of PRV to the brain via trigeminal circuitry. Forthe intrachoroidal injections, rats were anesthetized with anintraperitoneal injection of 0.1 ml/100 g of a ketamine/xylazinemixture (87/13mg/kg), and the right superior–temporal choroidwas injected with 0.5–2.0 μl of PRV (3×108 plaque-forming units/

ml or 1.1×109 plaque-forming units/ml), as described previously(Cuthbertson et al., 2003). The animals were allowed to survivebetween 52 and 87.5 hours after virus injection. Brains from theserats were fixed by transcardial perfusion, and the PRV wasdetected by immunolabeling. Fig. 1 presents one illustrative case(R3-07, with a survival of 69 hours) from the 10 rats that receivedintrachoroidal PRV. Schematics adapted from Paxinos andWatson (1994) were used. Some of these rats had been used todetect prechoroidal SSN neurons in a prior study (Cuthbertson etal., 2003).

One concern associated with this method is the extent oftracer spread beyond choroid after injection with microsyringe.It is very difficult to detect PRV itself within the choroidalinjection site as the virus is taken up, and the virus at theinjection site as a result undetectable after a few days ofsurvival. Confinement of the injection site to choroid is revealedby the pattern of labeling within the brain, since spread toextraocular or periorbital facial muscles is revealed by labelingwithin brain in oculomotor, trochlear, abducens, or facialmotoneurons, while intravitreal spread is revealed by labelingin the nucleus of Edinger–Westphal of the oculomotor complex.In addition, in a prior study, we injected Fluorogold into thechoroid to label PPGneurons (Cuthbertsonet al., 2003) and foundthat the intrachoroidal injections were largely restricted to thechoroid. Any slight leakage into the musculature surroundingthe temporal side of the eye did not yield labeling ofmotoneurons in brain nor did spread to the Harderian glandoccur and cause Fluorogold labeling in the PPG. Therefore,intrachoroidal PRV injections could be carried out withoutundetected confounding spread beyond the choroid.

4.3. Retrograde tracer injection into SSN

Fluorogold (FG, 10%, 0.03 μl–0.05 μl) was delivered into SSN bymicrosyringe in six rats (RBPH37–RBPH42). Pressure injectionswere performed using a 0.5-μl syringe with 25-gauge needle(Hamilton, #86250). Rats were anesthetized by ketamine/xylazine, with supplemental doses during surgery to maintainanesthesia (asmonitored bywhiskermovement). Body temper-aturewasmaintained at 37 °C, and the rats were positioned in astereotaxic device. Based on the rat brain stereotaxic atlas ofPaxinos and Watson (1994), the target coordinates of right SSNwere 10.6 mm behind bregma, 2.2 mm lateral to the midline,and7.6 mmto8.0 mmbelowthebrainsurface.Two injectionsoftracer separated by 0.2 mm (DV) weremade in SSN via pressureto increase the chance of hitting target area. The animals withFG injections were allowed to survive 5–7 days.

We also used BDA3K tomakemore confined injections intoSSN by pressure or iontophoresis. A solution of 10% BDA3K(Molecular Probes, BDA3000kD MW) was prepared with 0.1 Msodium–citrate–HCl buffer (pH 3). Injection of 0.06–0.1 μl of 10%BDA3Kwasmade into the right SSN in five rats using the samepressure injection procedure as for Fluorogold. Eight addition-al rats received iontophoretic injections of BDA3K into theright SSN. Micropipettes for iontophoresis were pulled fromglass tubing (World Precision Instrument, 1B150F-4) with a PE-2 microelectrode puller (Narashige), and micropipettes with atip diameter of 50–100 μm used for iontophoresis. Micropip-etteswere filledwith 7–8 μl of 10%BDA3K, and a platinumwirewas inserted. A Grass stimulator (Model S88) was used to

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deliver constant current pulses (+ current, 7 s on, 7 s off, 5 μA,20–60 min) via a stimulation isolation unit (Grass Instrument,PSIU-6) (Reiner et al., 2000). The animals with BDA3K injec-tions were allowed to survive 10–14 days.

4.4. Anterograde tracer injection into PVN

Twelve rats received pressure injections of 0.1–0.2 μl of 5%BDA10K (Molecular Probes, BDA 10,000 kDa MW) diluted with0.1 M sodium phosphate buffer into the right PVN via a 0.5-μlsyringe with 25-gauge needle. Since the injection sites made bymicrosyringepressure injection invariably spreadbeyondPVN, 13additional SD rats received smaller injections of 5% BDA10K intothe right PVN via iontophoresis (+ current, 7 s on, 7 s off, 5 μA, 10–30min). Stereotaxic methods were as noted above. The targetcoordinates for PVN were -−1.9 mm caudal to the bregma,−0.4 mm lateral to the midline, and −7.3 mm to 7.6mm fromthe brain surface (Paxinos and Watson, 1994). BDA10K was theninjected at two sites separated by 0.1–0.2 mm dorsoventrally forboth the pressure and iontophoretic injections. The animalswithBDA10K injections in PVN were allowed to survive 10 to 14 days.

4.5. Anterograde tracer injection into NTS

Two rats received pressure injection of 0.06 μl of 5% BDA10K in0.1 M sodium phosphate buffer into the right NTS via a 0.5-μlmicrosyringe with 25-gauge needle. Nine additional ratsreceived iontophoretic injections of 15% BDA10K in 0.01 Msodium phosphate buffer into the right NTS via amicropipettewith a 20- to 50-μm tip diameter. The rats were anesthetizedand placed in a stereotaxic device, and their body temperaturewas maintained at 37 °C as described above. The coordinatesfor NTS (Paxinos and Watson, 1994) were −13.6 mm behindbregma, −1.1 mm lateral to bregma, and 5.1–5.9 mm below thebrain surface. Animals with BDA10K injection into NTS wereallowed to survive 7 to 11 days.

4.6. Histological tissue preparation

As described previously (Cuthbertson et al., 2003), rats wereanesthetized andperfused transcardiallywith sodiumphosphatebuffered saline and400–500ml of 4%paraformaldehydepreparedin 0.1 M sodium phosphate buffer (pH7.4) with 0.1 M lysine and0.01 M sodium periodate (PLP fixative). Brains were removed andcryoprotected at 4 °C in 20% sucrose/10% glycerol/0.138% sodiumazide in 0.1 M sodiumphosphate buffer. Brainswere then frozen,and coronal sections cut at 40 μm with a sliding microtome. Thefree-floating sectionswere stored at 4 °C in a 0.02% sodiumazide/0.02% imidazole in 0.1 M PB until processed for detection of PRV,FG, BDA3K, or BDA10K, as described below.

4.7. Single-label immunohistochemistry

Immunohistochemical single labeling was carried out asdescribed previously to detect the PRV and FG tracers or theNOS+neurons within SSN (Cuthbertson et al., 2003; LeDouxet al., 2001). For PRV detection, the primary antibody was ahighly sensitive and specific goat anti-PRV (LeDoux et al., 2001;Cuthbertson et al., 2003), diluted 1:15,000–1:100,000 with 0.1 Mphosphate buffer/0.3% Triton X-100/0.001% sodium azide (PB/

Tx/Az)+5% normal horse serum. The anti-PRV antibody spec-ificity was demonstrated by viral immunostaining in cultureand in virus-infected brain. In the case of in vitro tests ofspecificity, PRV was incubated with PK15 (porcine kidney) cellsat 4 °C for 1 hour, to producemonolayer culturesof PRV-infectedPK15 cells. The cultures were subsequently washed three timeswith PBS and fixed in 2% paraformaldehyde. A 1:10,000 dilutionof antibody followed by 1:10,000 dilution of fluorescein iso-thiocyanate (FITC) anti-goat antibody resulted instrongstainingof virus particles on the cell surface. For in vivo tests of antibodyspecificity, brain tissue fromnormal uninfected rats or fromratswith intrachoroidal or intra-Meibomian gland injection of PRV>60 hours previously was used. Robust staining of virus-infected neurons with postimmunization goat anti-PRV antise-rum was observed at concentrations ranging from 1:10,000 to1:100,000. Uninfected brains did not stain with the anti-PRVantibody at these concentrations (LeDoux et al., 2001; Cuthbert-son et al., 2003). Moreover, no immunostaining was observed invirus-infected brain tissue treated with preimmune serum.

For FG detection, the primary antibody was rabbit anti-FG(from Fluorochrome, LLC), diluted at 1:2000 with the samediluent). This antibody has been widely used for detecting FG(Hayward and Von Reitzenstein, 2002; Kingsbury et al., 2005),and it produces no labeling in brains that have not been injectedwith Fluorogold.

For NOS, a rabbit anti-NOS1 (neuronal NOS, R-20, Santa CruzBiotechnology, Inc., Santa Cruz, CA), diluted 1:2000 to 1:4000with the same diluent, was used. The antibody specificity hasbeen verified byWestern blot analysis by themanufacturer, andthe distribution of perikaryal NOS staining it produces inrodents matches the known distribution of NOS+perikarya inrodent brain as seen in the Allen Brain Atlas (http://mouse.brain-map.org/welcome.do).

Our immunolabeling procedures have been described indetail elsewhere (Reiner et al., 1991; Cuthbertson et al., 2003). Inbrief, sections were incubated in primary antibody overnight atroom temperature. Sections for PRV detection were beforehandpretreatedwith 0.5%H2O2 to quench endogeneous peroxidases,followed by 1% nonfat dry milk to reduce nonspecific back-ground.Afterprimaryantibody incubation, sectionswere rinsedand incubated in donkey antiserum against goat IgG in the caseof PRV visualization, or donkey antiserum directed againstrabbit IgG in the case of FG or neuronal NOS detection. Thesections were subsequently rinsed and incubated in goatperoxidase–antiperoxidase (Gt PAP, diluted at 1:1000 with PB/Tx; goat PAP from Jackson ImmunoResearch Laboratories) orrabbit peroxidase–antiperoxidase (Rb PAP, diluted at 1:1000;rabbit PAP from Jackson ImmunoResearch Laboratories). Thelabeling was visualized using diaminobenzidine tetrahy-drochloride (DAB) in a 0.05 M sodium cacodylate–0.05 M imidi-zole buffer (pH 7.2–7.4). The sectionswere then rinsed,mountedair-dried, dehydrated, and coverslippedwithPermount® (FisherScientific, Pittsburgh, PA). The sections were examined with anOlympus BHS microscope with standard transmitted light ordifferential interference contrast optics.

4.8. ABC procedure to visualize BDA

Sections were rinsed, followed by pretreatment with 0.5% H2O2

and 0.3%Triton X-100 PB (PB/Tx) (Reiner et al., 2000). The sections

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were then rinsed and incubated in ABC solution preparing withtheVectastainABCElite kit (Vector Laboratories, Burlingame,CA).BDA3K or BDA10K was visualized as a black reaction productusing nickel intensification of the chromagen, DAB (Sigma). Thesections were then rinsed, mounted, air-dried, dehydrated,coverslipped, and examined with an Olympus BHS microscope.

4.9. Two-color DAB double-label immunohistochemistry

Our previous studies showed that prechoroidal SSN neuronslargely correspond to NOS+neurons of the SSN (Cuthbertsonet al., 2003). This information was used to judge the location ofBDA10K+fibers from the PVN or NTS in relation to prechoroidalSSN neurons. Immunohistochemical two-color DAB doublelabeling was used to visualize both the PVN or NTS inputs andthe NOS+prechoroidal neurons of SSN. For these double-labelstudies, brainsections fromratswithBDA10K injection into PVNor NTS were first labeled black by the ABC procedure usingnickel-intensified DAB as described above. The tissue was thenrinsed extensively, and incubated overnight in rabbit anti-NOS1(neuronalNOS). Tissuewas thensequentially incubatedat roomtemperature for 1 hour in a donkey anti-rabbit secondaryantibody and rabbit PAP. The NOS immunolabeling wasvisualized with a brown DAB procedure. The sections weresubsequently mounted, air-dried, dehydrated, and cover-slipped. They were examined with a BHS Olympusmicroscope.

4.10. Fluorescent double-label immunohistochemistry

We also used immunofluorescence double labeling to visualizethe relationship of BDA10K+fibers originating from NTS toNOS+prechoroidal SSN neurons. The sections were rinsed andincubated in rabbit anti-NOS1 (1:800) overnight. After rinsing,the tissue was incubated in donkey anti rabbit Alexa 488 (1:100;Molecular Probes) and streptavidin Alexa 594 (1:100; MolecularProbes) for 3 hours. The sectionswere thenmountedon gelatin-coated slides, air-dried, and coverslipped in glycerol phosphatebuffer (9:1). Sections were viewed, and images were capturedusing a Nikon C1 confocal laser-scanning microscope. Single-image planes as well as Z-stacks were captured for viewing,analysis, and illustration.

Acknowledgments

Special thanks to Dr. Yunping Deng for valuable advice ontechnical matters and Ting Wong, Marion Joni, AminahHenderson, Phoungdinh Nguyen, Amanda Walters, Dr.Rebeca-Ann Weinstock, Dr. Raven Babcock, Amanda Valencia,Dr. Seth Jones, Julia Jones, Karen Hanks, and Christy Loggins fortheir excellent technical assistance.

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