University of Groningen

Neuronal control of micturitionKuipers, Rutger

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Neurons in the guinea pig (cavia porcellus) lateral lumbosacral spinal cord project to the central part

of the lateral periaqueductal gray matter

Rutger Kuipers and Esther-Marije Klop

Brain Research; In Press

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

IntroductionMicturition is thought to be controlled by the central nervous system through a reflex pathway that involves areas of the lumbosacral spinal cord, the midbrain and the pons (de Groat, 1998; Holstege, 2005). Anatomical studies in rat and cat have shown that sensory information from the lower urinary tract is likely to be first relayed to the lateral part of the dorsal horn and to the area of the intermediolateral cell column (IML) in the lumbosacral spinal cord (Morgan et al., 1981; Nadelhaft and Booth, 1984). Cells in this part of the lumbosacral spinal cord in turn project to the central parts of the lateral midbrain periaqueductal gray matter (PAG; Cat: Blok et al., 1995; Vanderhorst et al., 1996; Mouton and Holstege, 2000; Klop et al., 2005, Rat: Ding et al., 1997; Keay et al., 1997). The PAG, in turn, projects to Barrington’s nucleus which is located in the dorsolateral pontine tegmentum (Barrington, 1925; Blok and Holstege, 1994; Kuipers et al., 2006). Barrington’s nucleus contains the premotor interneurons that coordinate

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simultaneous bladder detrusor contractions and external urethral sphincter (EUS) relaxation. This is accomplished through direct excitatory projections on bladder preganglionic motoneurons and on interneurons which inhibit EUS motoneurons (Loewy et al., 1979; Holstege et al., 1986; Blok et al., 1997a; Blok et al., 1998; Sie et al., 2001).This micturition reflex pathway in turn is thought to be under control of the limbic system, either by brain areas that directly project to Barrington’s nucleus, such as the hypothalamus (Valentino et al., 1994; Kuipers et al., 2006) and bed nucleus of the stria terminalis (Dong and Swanson, 2005), or by areas that do not project directly to Barrington’s nucleus but in which micturition can be elicited by electrical stimulation such as the amygdala and prefrontal cortex (Gjone and Setekleiv, 1963; Gjone, 1966). In the micturition reflex pathway, the PAG plays a central role, since it receives the information from the lumbosacral cord essential for bladder sensation. In rat, neurons in the area of the sacral parasympathetic nucleus in the lumbosacral spinal cord also project directly to Barrington’s nucleus (Ding et al., 1997), but in cat these direct projections do not exist (Blok et al., 1995). The importance of the PAG in micturition is further indicated by the fact that the PAG can also elicit micturition by means of its descending projections to Barrington’s nucleus. Micturition can be elicited by electrical stimulation of the PAG in rat and cat (Kabat et al., 1936; Taniguchi et al., 2002). Furthermore, the PAG also seems to be important in the control of micturition in humans: imaging studies have shown that PAG is activated both during filling of the bladder (Athwal et al., 2001) and during micturition (Blok et al., 1997b; Nour et al., 2000) and case studies have reported lesions in the PAG resulting in urinary retention (Yaguchi et al., 2004).Although most neurourological research is performed in rats, there is evidence that urodynamic profiles of rat micturition are markedly different from those of higher species such as cat, dog and human (Sundin and Petersen, 1975; Sackman and Sims, 1990; Walters et al., 2005). Guinea pig micturition profiles, however, are very similar to those of higher species (Van Asselt et al., 1995; Walters et al., 2005). This means that guinea pig may be a better animal model for neurourological research than rat. The problem, however, is that little is known about the neuroanatomical substrates that 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., 2004; Kuipers et al., 2006b). The present study investigates the existence of a projection from the lumbosacral spinal cord to the PAG in guinea pig.

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Methods

Surgical proceduresSurgical procedures, pre- and postoperative care and handling and housing of the animals were approved by the Ethical Committee of the Faculty of Medical Sciences of the University of Groningen, The Netherlands. A total of four adult female guinea pigs (Cavia porcellus, Dunkin-Hartley, Harlan, the Netherlands) weighing 400-900 g, were used. Animals were anesthetized with a combination of xylazine (5 mg/kg i.m.) and ketamine (40 mg/kg i.m.). Buphrenorphine was administered (0.1 mg/kg s.c.) for analgesia. During surgery normal body temperature was maintained using a heating pad.

InjectionsA total of four guinea pigs were used for anterograde and retrograde 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 the lumbosacral spinal cord in order to verify whether anterogradely labeled fibers and terminals could be observed in the PAG. These injections were made under visual guidance using a glass micropipette with a pneumatic picopump (World Precision Instruments PV 830).

GP16

L6

GP17

S1

S2B

GP16 GP17

A

Figure 1. Brightfield photomicrograps (A) and schematic drawings (B) of the WGA-HRP injection sites in the lumbosacral spinal cord in cases GP16 and GP17. Scale bar represents 100μm.

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In order to identify which neurons within the lumboscaral spinal cord project to the PAG, in two other cases (GP39, GP41), injections with approximately 50 nl. of 2.5% WGA-HRP were made in the PAG. These injections were made stereotaxically using a glass micropipette with a pneumatic picopump (World Precision Instruments PV 830). Stereotaxic coordinates were determined using a stereotaxic atlas of the guinea pig brain (Rapisarda and Bacchelli, 1977).

Perfusion and histological proceduresAfter a survival period of 72 h, the animals were deeply anesthetized with an overdose (5 ml) of pentobarbital (6% solution). Subsequently, the animals were perfused transcardially with 800 ml of heparinized saline followed by 800 ml of 0.1 M phosphate buffered fixative containing 2% glutaraldehyde, 1% paraformaldehyde and 4% sucrose. Brain, brainstem and spinal cord were removed, postfixed for 2 hrs in the same fixative and cryoprotected by overnight storage in 0.1 M phosphate buffered 25% sucrose at 4°C. The next day, forebrain, brainstem and spinal cord segments were separated with transverse cuts and tissue was frozen in an isopentane bath (-55˚C). Serial 40µm frozen transverse sections of brainstem and lumbosacral spinal cord segments were cut using a cryostat. Every second section was incubated according to the tetramethyl benzidine (TMB) method (Mesulam, 1978; Gibson et al., 1984). All sections were mounted on chromalum-gelatine coated slides, dried, dehydrated in graded alcohols, cleared in xylene and coverslipped with Permount mounting medium. In order to define the extent of the injection site an extra series of sections containing the injection site was incubated with diaminobenzidine (DAB).

Mapping of WGA-HRP anterograde and retrograde labelingSections, stained with the DAB method, containing the injections sites were photographed using a Leica DC500 camera connected to a Zeiss stereomicroscope using both dark and brightfield illumination. Schematic drawings of the injection sites were made using a drawing tube connected to the same stereomicroscope.After WGA-HRP injections in the lumbosacral spinal cord, brainstem sections were screened for the presence of anterogradely labeled fibers and terminals in the PAG. Schematic drawings of the anterogradely labeled fibers and terminals in every eighth section containing the PAG were made using a drawing tube connected to a Zeiss Axioplan microscope with darkfield polarized illumination. After WGA-HRP injections in the PAG, the lumbosacral spinal cord was screened for the presence of retrogradely labeled neurons. Plottings drawings

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GP 16

GP 17

cd

e

g

hgfe

dcba

hf

ba

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

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Figure 3. Polarized darkfield photomicrograph of anterograde labeling in the PAG in case GP17. Note the dense anterograde labeling in the central parts of the PAG. Scale bar represents 50μm.

of retrogradely labeled neurons in the lumbosacral sections were made using a Neurolucida System (MicroBrightField Inc.,Colchester, USA) connected to a Zeiss Axioplan microscope with darkfield polarized illumination. In these drawings, retrogradely labeled neurons of every second section were plotted in one standard schematic section for each spinal cord segment. To describe the laminar location of the spinal neurons projecting to the PAG, in all drawings the laminae were depicted, as was a line dividing laminae VI and VII into a medial and a lateral part. This line was set at half the distance between the lateral border of lamina X and the lateral border of the gray matter (see figure 5). We decided to differentiate between the lateral and medial parts of laminae VI–VII because preliminary results indicated that large differences existed between these areas in the numbers of PAG projecting neurons.Photomicrographs of the relevant WGA-HRP sections were taken using a Leica DC500 digital camera, connected to a Leica DM500 microscope with darkfield polarized illumination, using Leica Qwin software. Minor adjustments in brightness and contrast were made using Adobe Photoshop.

Results

Anterograde tracing experimentsIn two cases wheat germ agglutin-conjugated horseradish peroxidase(WGA-HRP) injections were placed in the lumbosacral spinal cord (figure 1) in order to study anterograde labeling in the PAG. In both cases large injections sites were found centered in the S1 spinal segment that included the ventral and dorsal horns bilaterally (figure 1). In case GP16 the injection extended into the left dorsal and ventral horn of the L6 and S2 segments. In case GP17 the injection

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A

B C

GP39

GP41

Figure 4. Schematic drawings (A) and polarized dark- and brightfield photomicrographs of the WGA-HRP injection sites in the PAG in cases GP39 (B) and GP41(C). Scale bar represents 100μm.

was slightly larger and included the ventral and dorsal horn of the L6 and S2 segments bilaterally.In both cases (GP16 and GP17) a similar pattern of anterograde labeling was observed in the PAG bilaterally (figures 2; 3), with more labeling in the case with the larger injection site (GP17). In the rostral half of the lateral PAG, anterograde labeling was found mainly in the central parts of the lateral PAG adjoining the ependymal cell layer bordering the aqueduct (figure 2, GP16: a-f, GP17 a-e). In the caudal half of the PAG, anterograde labeling was also observed in the central parts of the lateral PAG but here the labeling was observed dorsolaterally and ventrolaterally to the aqueduct in the central part of the lateral PAG while in between these two areas no labeling was found (figure 2, GP16: g-h, GP17: f-h). In case GP17 some anterograde labeling was also observed in the peripheral parts of the lateral PAG and in the dorsomedial subdivision of the PAG (figure 2).

Retrograde tracing experimentsTo determine the location of the neurons projecting from the lumbosacral spinal cord to the central PAG, WGA-HRP injections were made that included the central parts of the lateral PAG. In case GP39, the relatively small injection site included most of the parts of the PAG in which labeling was observed in the anterograde tracing study (figure 4). The injection site extended into the ventrolateral part of the PAG but not outside the borders of the PAG. In case GP41, a relatively

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L5

L6

S1S2

S3

X

VI/VIIlat

VIII

VI/VIImed

III/IV

I/II

V

L5

L6

S1S2

S3

GP39

GP41

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

large injection site involved the central PAG, but also extended ventrally into the dorsal part of the mesencephalic medial tegmentum, the mesencephalic nucleus raphé and the nucleus Edinger-Westphal (figure 4). Some leakage of tracer was observed in the superficial and deeper layers of the superior colliculus (figure 4). In both cases a similar pattern of retrogradely labeled neurons was observed in the lumbosacral spinal cord, but in the case with the larger injection (GP41) more retrogradely labeled neurons were found. A group of retrogradely labeled neurons was present in the lateral part of the gray matter at spinal cord segments L6-S3 bilaterally (figures 5; 6). This groups of neurons was most prominent at the segments S2, but extended into the caudal part of the L6 segments and the rostral part of S3 (figure 5). Most of the cells were observed in the lateral parts of laminae V-VII but labeled neurons were also found in the lateral part of lamina I and in lamina X. Finally, a small number of large neurons were found in the medial laminae VI/VII and in the medial part of lamina X at segments L6-S3

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Figure 6. Polarized darkfield photomicrograph of retrogradely WGA-HRP labeled neurons lateral in the spinal cord at level S2 after WGA-HRP injection in the PAG in case GP41. Scale bar represents 100μm.

(figure 5). Very few retrogradely labeled cells were observed in the L5 spinal cord segment (figure 5). These results show that the projection from the lumbosacral spinal cord to the central PAG originates mainly from a cell group that is located in the lateral part of laminae VI/VII and in the lateral part of lamina I.

DiscussionThe projection from the lateral part of the lumbosacral spinal cord to the central lateral PAG that this study has demonstrated in guinea pig is similar to spino-PAG projections from the lumbosacral cord in rat and cat (Blok et al., 1995; Vanderhorst et al., 1996; Keay et al., 1997; Mouton and Holstege, 2000; Klop et al., 2005). It is likely that this cellgroup relays information from the lower urinary tract to the PAG, because of the fact that the lateral part of the lumbosacral spinal cord is the location where pudendal and pelvic afferents reach the spinal cord (Morgan et al., 1981; Ueyama et al., 1984; Nadelhaft and Booth, 1984; McKenna and Nadelhaft, 1986) and parasympathetic bladder motoneurons and their dendrites are located (Morgan et al., 1979; Nadelhaft and Booth, 1984; Kuipers et al., 2004). Studies which have examined C-fos expression in the lumbosacral spinal cord after different kinds of stimulation of the bladder have shown that neurons in this lateral region in the rat are activated by distension of the bladder, while cold stimulation or chemical irritation of the bladder activates neurons in the medial part of lamina I of the dorsal horn (Birder and de Groat, 1992; Jiang and Hermanson, 2004). Furthermore, cells in the lateral region of 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 studies which show that cells in the lateral lumbosacral spinal cord project to the PAG,

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suggest that sensory information about the degree of bladder distension is relayed via the lateral lumbosacral cellgroup to the PAG (Blok et al., 1995; Vanderhorst et al., 1996; Keay et al., 1997; Mouton and Holstege, 2000; Klop et al., 2005). Another argument in favor of a viscerosensory function for the cells in the lateral lumbosacral spinal cord is the fact that the cells in the same lateral lumbosacral region project 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 the main structure associated with somatosensory relay: the thalamus (Klop et al., 2005).The idea that afferent information is relayed to the PAG and that this pathway forms the afferent limb of the spinobulbospinal micturition reflex pathway is a well established concept in neurourology (de Groat, 1998; Holstege, 2005) and neuroimaging studies have shown that the PAG is involved in both the sensory (Athwal et al., 2001) and motor (Blok et al., 1997b) aspects of micturition in humans as well. This study shows that a lumbosacral-PAG projection that forms the afferent limb of the micturition reflex also exists in guinea pig. This result together with earlier results (Kuipers et al., 2004; Kuipers et al., 2006b), adds to the idea that guinea pig may be a good small animal model for neurourological research based on neuroanatomical grounds, in addition to the fact that guinea pig seems a good animal model on urodynamic grounds (Van Asselt et al., 1995; Walters et al., 2005).

AcknowledgementsThis research was sponsored by a grant from Pfizer Global Research and Development, Sandwich, UK.