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Neurons in the guinea pig (Cavia porcellus) lateral lumbosacralspinal cord project to the central part of the lateralperiaqueductal gray matter

Rutger Kuipers⁎, Esther Marije KlopDepartment of Anatomy and Embryology, University Medical Center Groningen, University of Groningen, Building 3215, Room 721,Antonius Deusinglaan 1, PO Box 196, 9713 AV Groningen, The Netherlands

A R T I C L E I N F O

⁎ Corresponding author. Fax: +31 50 3632461.E-mail address: r.kuipers@med.umcg.nl (R

0006-8993/$ – see front matter © 2006 Elsevidoi:10.1016/j.brainres.2006.05.039

A B S T R A C T

Article history:Accepted 4 May 2006Available online 19 June 2006

In order to micturate successfully, information from the bladder has to be conveyed to thebrainstem. In most experimental animals, this information is relayed, via the lumbosacralspinal cord, to the periaqueductal gray matter (PAG). Although the rat is the most usedexperimental animal in neurourological research, urodynamic studies show that guinea pigmay be a better small experimental animal because its urodynamic profile is, in contrast tothat of a rat, similar to that of humans. Therefore, the present study, using anterograde andretrograde tracing, was performed to determine whether the lumbosacral spinal cordprojects to the PAG in guinea pig. Results show that neurons in the lateral part of thelumbosacral spinal cord project to the central parts of the PAG. This pathway may conveyinformation about the level of bladder filling to the PAG.

© 2006 Elsevier B.V. All rights reserved.

Keywords:MicturitionBladderVisceroceptionLimbic systemSpinomesencephalic

Abbreviations:DAB, diaminobenzidineEUS, external urethralsphincter muscleIML, intermediolateral cell groupIMM, intermediomedial cell groupPAG, periaqueductal gray matterTMB, tetramethyl benzidineWGA-HRP, wheat germ agglutin-conjugated horseradish peroxidase

1. Introduction

Micturition is thought to be controlled by the centralnervous system through a reflex pathway that involvesareas of the lumbosacral spinal cord, the midbrain and thepons (de Groat, 1998; Holstege, 2005). Anatomical studies inrat and cat have shown that sensory information from the

. Kuipers).

er B.V. All rights reserved

lower urinary tract is likely to be first relayed to the lateralpart of the dorsal horn and to the area of the intermedio-lateral cell column (IML) in the lumbosacral spinal cord(Morgan et al., 1981; Nadelhaft and Booth, 1984). Cells inthis part of the lumbosacral spinal cord in turn project tothe central parts of the lateral midbrain periaqueductalgray matter (PAG; (Blok et al., 1995; Vanderhorst et al., 1996;

.

Fig. 1 – Brightfield photomicrographs (A) and schematicdrawings (B) of the WGA-HRP injection sites in thelumbosacral spinal cord in cases GP16 and GP17. Scale barrepresents 100 μm.

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Ding et al., 1997; Keay et al., 1997; Mouton and Holstege,2000; Klop et al., 2005). The PAG, in turn, projects toBarrington's nucleus (Barrington, 1925) which is located inthe dorsolateral pontine tegmentum (Barrington, 1925; Blokand Holstege, 1994; Kuipers et al., 2006). Barrington'snucleus contains the premotor interneurons that coordinatesimultaneous bladder detrusor contractions and externalurethral spincter (EUS) relaxation. This is accomplishedthrough direct excitatory projections on bladder pregangli-onic motoneurons and on interneurons which inhibit EUSmotoneurons (Loewy et al., 1979; Holstege et al., 1986; Bloket al., 1997a; Blok et al., 1998; Sie et al., 2001).

This micturition reflex pathway in turn is thought to beunder control of the limbic system, either by brain areas thatdirectly project to Barrington's nucleus, such as the hypothal-amus (Valentino et al., 1994; Kuipers et al., 2006) and bednucleus of the stria terminalis (Dong and Swanson, 2005), or byareas that do not project directly to Barrington's nucleus but inwhich micturition can be elicited by electrical stimulationsuch as the amygdala and prefrontal cortex (Gjone andSetekleiv, 1963; Gjone, 1966).

In the micturition reflex pathway, the PAG plays acentral role, since it receives the information from thelumbosacral cord essential for bladder sensation. In rat,neurons in the area of the sacral parasympathetic nucleusin the lumbosacral spinal cord also project directly toBarrington's nucleus (Ding et al., 1997), but in cat thesedirect projections do not exist (Blok et al., 1995). Theimportance of the PAG in micturition is further indicated bythe fact that the PAG can also elicit micturition by meansof its descending projections to Barrington's nucleus (Blokand Holstege, 1994; Kuipers et al., 2006). Micturition can beelicited by electrical stimulation of the PAG in rat and cat(Kabat et al., 1936; Taniguchi et al., 2002). Furthermore, thePAG also seems to be important in the control ofmicturition in humans: imaging studies have shown thatPAG is activated both during filling of the bladder (Athwalet al., 2001) and during micturition (Blok et al., 1997b; Nouret al., 2000) and case studies have reported lesions in thePAG resulting in urinary retention (Yaguchi et al., 2004).

Although most neurourological research is performed inrats, there is evidence that urodynamic profiles of ratmicturition are markedly different from those of higherspecies such as cat, dog and human (Sundin and Petersen,1975; Sackman and Sims, 1990; Walters et al., 2005). Duringrat micturition, the EUS contracts rhythmically during thebladder detrusor contractions that empty the bladder. Incontrast, during cat, dog and human micturition, there isno EUS activity during bladder contraction but onlybetween bladder contractions. This means that in rat theEUS facilitates micturition, while in higher species the EUSfunctions as a sphincter that contributes to urethral closingpressure to maintain continence. Guinea pig micturitionprofiles, however, are very similar to those of higherspecies (Van Asselt et al., 1995; Walters et al., 2005). Thismeans that guinea pig may be a better animal model forneurourological research than rat. The problem, however, isthat little is known about the neuroanatomical substratesthat control micturition in guinea pig. In previous reports,we provided data on the location of motoneurons involved

in micturition and of Barrington's nucleus in the guinea pig(Kuipers et al., submitted for publication; Kuipers et al.,2004). The present study investigates the existence of aprojection from the lumbosacral spinal cord to the PAG inguinea pig.

2. Results

2.1. Anterograde tracing experiments

In two cases, wheat germ agglutin-conjugated horseradishperoxidase (WGA-HRP) injections were placed in thelumbosacral spinal cord (Fig. 1) in order to study ante-rograde labeling in the PAG. In both cases, large injectionssites were found centered in the S1 spinal segment thatincluded the ventral and dorsal horns bilaterally (Fig. 1). Incase GP16, the injection extended into the left dorsal andventral horn of the L6 and S2 segments. In case GP17, theinjection was slightly larger and included the ventral anddorsal horn of the L6 and S2 segments bilaterally.

In both cases (GP16 and GP17), a similar pattern ofanterograde labeling was observed in the PAG bilaterally(Figs. 2 and 3), with more labeled terminals in the casewith the larger injection site (GP17). In the rostral half ofthe lateral PAG, anterograde labeling was found mainly inthe central parts of the lateral PAG adjoining the ependy-mal cell layer bordering the aqueduct (Fig. 2, GP16: a–f,GP17 a–e). In the caudal half of the PAG, anterogradelabeling was also observed in the central parts of the lateral

Fig. 2 – Schematic drawings of anterograde (lines) and retrograde (dots) WGA-HRP labeling in the PAG after injections in thelumbosacral spinal cord in cases GP16 and GP17.

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PAG but here the labeling was observed dorsolaterally andventrolaterally to the aqueduct in the central part of thelateral PAG while in between these two areas no labelingwas found (Fig. 2, GP16: g–h, GP17: f–h). In case GP17, someanterograde labeling was also observed in the peripheralparts of the lateral PAG and in the dorsomedial subdivisionof the PAG (Fig. 2). In both cases, anterograde labelingoutside the PAG was found in the nucleus of the solitarytract, the ventrolateral medulla and in the medial andlateral parabrachial nuclei.

2.2. Retrograde tracing experiments

To determine the location of the neurons projecting fromthe lumbosacral spinal cord to the central PAG, WGA-HRPinjections were made that included the central parts of thelateral PAG. In case GP39, the relatively small injection site

included most of the parts of the PAG in which labeling wasobserved in the anterograde tracing study (Fig. 4). Theinjection site extended into the ventrolateral part of thePAG but not outside the borders of the PAG. In case GP41, arelatively large injection site involved the central PAG, butalso extended ventrally into the dorsal part of the mesen-cephalic medial tegmentum, the mesencephalic nucleusraphé and the nucleus Edinger-Westphal (Fig. 4). Someleakage of tracer was observed in the superficial and deeperlayers of the superior colliculus (Fig. 4).

In both cases, a similar pattern of retrogradely labeledneurons was observed in the lumbosacral spinal cord, butin the case with the larger injection (GP41) more retro-gradely labeled neurons were found. A group of retrograde-ly labeled neurons was present in the lateral part of thegray matter at spinal cord segments L6–S3 bilaterally (Figs.5 and 6). This group of neurons was most prominent at the

Fig. 3 – Polarized darkfield photomicrograph of anterogradelabeling in the PAG in case GP17. Note the dense anterogradelabeling in the central parts of the PAG. Scale bar represents50 μm.

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segments S2, but extended into the caudal part of the L6segments and the rostral part of S3 (Fig. 5). Most of thecells were observed in the lateral parts of laminae V–VII butlabeled neurons were also found in the lateral part oflamina I and in lamina X. Finally, a small number of largeneurons were found in the medial laminae VI/VII and inthe medial part of lamina X at segments L6–S3 (Fig. 5). Veryfew retrogradely labeled cells were observed in the L5spinal cord segment (Fig. 5).

These results show that the projection from the lumbosa-cral spinal cord to the central PAG originatesmainly froma cellgroup that is located in the lateral part of laminae VI/VII and inthe lateral part of lamina I.

Fig. 4 – Schematic drawings (A) and polarized dark- and brightfieldin cases GP39 (B) and GP41(C). Scale bar represents 100 μm.

3. Discussion

3.1. Methodological issues

It has been suggested that WGA-HRP, in contrast to fluores-cent tracers, would not be a suitable retrograde tracer to studyascending spinal cord neurons because it would underesti-mate the number of neurons (Craig et al., 1989). However, closeexamination of the data presented in this study reveals thatthe conclusion cannot be drawn from the data. In this study,tracing results from a ‘HRP’ group are compared with resultsfrom a ‘fluorescent’ group. However, for unknown reasons, the‘HRP’ group contains tracing results from both WGA-HRP andfree HRP experiments. From the data, it appears that free HRPas a retrograde tracer underestimates the number of neuronsfound, while the number of cells found with WGA-HRP issimilar to numbers found with fluorescent tracers.

Another possible problem with using WGA-HRP as aretrograde tracer is that WGA-HRP is known to be taken upby fibers of passage. However, because the retrograde tracinginjections in the PAG in this study do not extend in any of theascending tracts originating from the spinal cord, it is highlyunlikely that uptake by fibers of passage has led to labeledneurons in the lumbosacral spinal cord.

3.2. Other issues

The projection from the lateral part of the lumbosacral spinalcord to the central lateral PAG that this study has demon-strated in guinea pig is similar to spino-PAG projections fromthe lumbosacral cord in rat and cat (Blok et al., 1995;Vanderhorst et al., 1996; Keay et al., 1997; Mouton andHolstege, 2000; Klop et al., 2005). It is likely that this cellgroup relays information from the lower urinary tract to the

photomicrographs of theWGA-HRP injection sites in the PAG

Fig. 5 – Schematic drawings showing the distribution of retrogradely labeled neurons in the spinal cord segments L5–S3 afterWGA-HRP injections in the PAG in cases GP39 and GP41. Note the cluster of labeled neurons bilaterally in the lateral part of thespinal cord.

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PAG, because of the fact that the lateral part of thelumbosacral spinal cord is the location where pudendal andpelvic afferents reach the spinal cord (Morgan et al., 1981;Ueyama et al., 1984; Nadelhaft and Booth, 1984; McKenna andNadelhaft, 1986) and parasympathetic bladder motoneuronsand their dendrites are located (Morgan et al., 1979; Nadelhaftand Booth, 1984; Kuipers et al., 2004). Studies which haveexamined C-fos expression in the lumbosacral spinal cordafter different kinds of stimulation of the bladder have shown

Fig. 6 – Polarized darkfield photomicrograph of retrogradelyWGA-HRP labeled neurons lateral in the spinal cord at levelS2 afterWGA-HRP injection in the PAG in case GP41. Scale barrepresents 100 μm.

that neurons in this lateral region in the rat are activated bydistension of the bladder, while cold stimulation or chemicalirritation of the bladder activates neurons in themedial part oflamina I of the dorsal horn (Birder and de Groat, 1992; Jiangand Hermanson, 2004). Furthermore, cells in the lateral regionof the lumbosacral spinal cord have been shown to express C-fos after isometric micturition in the cat (Grill et al., 1998).These results, together with the neuroanatomical studieswhich show that cells in the lateral lumbosacral spinal cordproject to the PAG, suggest that sensory information about thedegree of bladder distension is relayed via the laterallumbosacral cell group to the PAG (Blok et al., 1995; Vander-horst et al., 1996; Keay et al., 1997; Mouton and Holstege, 2000;Klop et al., 2005). It should be pointed out that sensoryinformation from the organs involved in reproduction mayalso be relayed to the PAG by means of the projectiondemonstrated in this study. This is indicated by the fact thatthe urogenital reflex results in increased C-fos expression inthe lateral cell group in the lumbosacral spinal cord (Marson etal., 2003). Another argument in favor of a viscerosensoryfunction for the cells in the lateral lumbosacral spinal cord isthe fact that the cells in the same lateral lumbosacral regionproject to other nuclei associated with viscerosensory relay,such as the parabrachial nuclei (Panneton and Burton, 1985)and the hypothalamus (Katter et al., 1991), but not to themainstructure associated with somatosensory relay: the thalamus(Klop et al., 2005).

The idea that afferent information is relayed to the PAG andthat thispathway formstheafferent limbof thespinobulbospinal

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micturition reflex pathway is a well established concept inneurourology (de Groat, 1998; Holstege, 2005) and neuroimagingstudies have shown that the PAG is involved in both the sensory(Athwal et al., 2001) and motor (Blok et al., 1997b) aspects ofmicturition in humans as well. This study shows that alumbosacral-PAG projection that forms the afferent limb of themicturition reflex also exists in guinea pig. This result togetherwith earlier results (Kuipers et al., submitted for publication;Kuipers et al., 2004), adds to the idea that guinea pig may be agood small animal model for neurourological research based onneuroanatomical grounds, in addition to the fact that guinea pigseems a good animalmodel on urodynamic grounds (Van Asseltet al., 1995; Walters et al., 2005).

4. Experimental procedures

4.1. Surgical procedures

Surgical procedures, pre- and postoperative care and handlingand housing of the animals were approved by the EthicalCommittee of the Faculty of Medical Sciences of the Universityof Groningen, The Netherlands. A total of four adult femaleguinea pigs (Cavia porcellus, Dunkin-Hartley, Harlan, theNetherlands) weighing 400–900 g, were used. Animals wereanesthetizedwith a combination of xylazine (5mg/kg i.m.) andketamine (40 mg/kg i.m.). Buphrenorphine was administered(0.1 mg/kg s.c.) for analgesia. During surgery, normal bodytemperature was maintained using a heating pad.

4.2. Injections

A total of four guinea pigs were used for anterograde andretrograde tracing experiments using WGA-HRP. In two cases(GP16, GP17), after laminectomy, approximately 100 nl of 2.5%WGA-HRP (Sigma) in saline was injected bilaterally into thelumbosacral spinal cord in order to verify whether antero-gradely labeled fibers and terminals could be observed in thePAG. These injections were made under visual guidance usinga glass micropipette with a pneumatic picopump (WorldPrecision Instruments PV 830).

In order to identify which neurons within the lumboscaralspinal cord project to the PAG, in two other cases (GP39, GP41),injections with approximately 50 nl of 2.5% WGA-HRP weremade in the PAG. These injections were made stereotaxicallyusing a glassmicropipette with a pneumatic picopump (WorldPrecision Instruments PV 830). Stereoxac coordinates weredetermined using a stereotaxic atlas of the guinea pig brain(Rapisarda and Bacchelli, 1977).

4.3. Perfusion and histological procedures

After a survival period of 72 h, the animals were deeplyanesthetized with an overdose (5 ml) of pentobarbital (6%solution). Subsequently, the animals were perfused transcar-dially with 800 ml of heparinized saline followed by 800 ml of0.1 M phosphate-buffered fixative containing 2% glutaralde-hyde, 1% paraformaldehyde and 4% sucrose. Brain, brainstemand spinal cord were removed, postfixed for 2 h in the samefixative and cryoprotected by overnight storage in 0.1 M

phosphate buffered 25% sucrose at 4 °C. The next day,forebrain, brainstem and spinal cord segmentswere separatedwith transverse cuts and tissue was frozen in an isopentanebath (−55 °C).

Serial 40 μm frozen transverse sections of brainstem andlumbosacral spinal cord segments were cut using a cryostat.Every second section was incubated according to the tetra-methyl benzidine (TMB) method (Mesulam, 1978; Gibson et al.,1984). All sections were mounted on chromalum-gelatinecoated slides, dried, dehydrated in graded alcohols, cleared inxylene and coverslippedwith Permountmountingmedium. Inorder to define the extent of the injection site, an extra seriesof sections containing the injection site was incubated withdiaminobenzidine (DAB).

4.4. Mapping of WGA-HRP anterograde and retrogradelabeling

Sections, stained with the DAB method, containing theinjections sites were photographed using a Leica DC500camera connected to a Zeiss stereomicroscope using bothdark and brightfield illumination. Schematic drawings ofthe injection sites were made using a drawing tubeconnected to the same stereomicroscope.

After WGA-HRP injections in the lumbosacral spinalcord, brainstem sections were screened for the presence ofanterogradely labeled fibers and terminals in the PAG.Schematic drawings of the anterogradely labeled fibers andterminals in every eighth section containing the PAG weremade using a drawing tube connected to a Zeiss Axioplanmicroscope with darkfield polarized illumination.

After WGA-HRP injections in the PAG, the lumbosacralspinal cord was screened for the presence of retrogradelylabeled neurons. Plottings drawings of retrogradely labeledneurons in the lumbosacral sections were made using aNeurolucida System (MicroBrightField Inc., Colchester, USA)connected to a Zeiss Axioplan microscope with darkfieldpolarized illumination. In these drawings, retrogradely labeledneurons of every second section were plotted in one standardschematic section for each spinal cord segment.

To describe the laminar location of the spinal neuronsprojecting to the PAG, in all drawings the laminaewere depicted,as was a line dividing laminae VI and VII into a medial and alateral part. This line was set at half the distance between thelateral borderof laminaXandthe lateral borderof thegraymatter(see Fig. 5). We decided to differentiate between the lateral andmedial parts of laminae VI–VII because preliminary resultsindicated that large differences existed between these areas inthe numbers of PAG projecting neurons.

Photomicrographs of the relevant WGA-HRP sections weretaken using a Leica DC500 digital camera, connected to a LeicaDM500 microscope with darkfield polarized illumination,using Leica Qwin software. Minor adjustments in brightnessand contrast were made using Adobe Photoshop.

Acknowledgments

The authors wish to thank Mrs. E. Eggens-Meijer and Ms.A. Algra for their valuable histotechnical assistance. This

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research was sponsored by a grant from Pfizer Global Re-search and Development. Sandwich, United Kingdom.

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