Sleep Research Online 1(3): 102-108, 1998 http://www.sro.org/1998/Fort/102/ Printed in the USA. All rights reserved.

1096-214X 1998 WebSciences

Anatomical Demonstration of a Medullary Enkephalinergic Pathway Potentially Implicated in the Oro-Facial Muscle Atonia of Paradoxical Sleep in the Cat
Patrice Fort, Claire Rampon, Damien Gervasoni, Christelle Peyron and Pierre-Herv Luppi
Neurobiologie des tats de Sommeil et d'veil, INSERM U480, Universit Claude Bernard, Lyon 69373, France
The present study was aimed to compare in detail the distribution within the rostral ventromedial medulla of MethioninEnkephalin-immunoreactive neurons with efferent projections to the facial or trigeminal motor nuclei, using a double immunostaining technique in colchicine-treated cats. Following cholera toxin B subunit injections in the facial or trigeminal motor nuclei, we found that respectively 55% and 65% of the medium to large-sized retrogradely labeled cells in the lateral part of the nucleus reticularis magnocellularis were Methionin-Enkephalin-positive. For both motor nuclei, the double-labeled neurons had similar morphology and size and were located exactly in the same area. They could therefore belong to the same population of reticular enkephalinergic neurons. Based on these and previous anatomical and electrophysiological data, we propose that these enkephalin-containing neurons could participate in the hyperpolarization of brainstem and spinal somatic motoneurons during paradoxical sleep. CURRENT CLAIM: This study describes a new enkephalinergic pathway potentially involved in the atonia of oro-facial muscles during paradoxical sleep.

The state of paradoxical sleep (PS) is characterized by the simultaneous appearance of desynchronized EEG activity and the atonia of the anti-gravitic muscles including those of the oro-facial sphere (Jouvet, 1962). By means of intracellular recordings in unanesthetized head-restrained cats, combined with the local iontophoretic application of strychnine (a glycinergic antagonist), pharmacological evidence has been provided that the loss of muscular tone is mainly due to a PSspecific tonic hyperpolarization of cranial (including facial, trigeminal and hypoglossal) and spinal motoneurons by glycine (Glenn et al., 1978; Morales and Chase, 1978; Chandler et al., 1980; Nakamura et al., 1978; Chase et al., 1980, 1989; Morales et al., 1987; Soja et al., 1987a, 1987b, 1991; review in Chase and Morales, 1990; Yamuy et al., 1998). Recently, it has been further shown in the cat that the PSspecific glycinergic inhibition of motoneurons is potentiated by opioids (Xi et al., 1996). Indeed, microiontophoretic application during PS of morphine, an opiate receptor agonist, increased the amplitude of the inhibitory post-synaptic potentials (IPSP) evoked in lumbar motoneurons by electrical stimulation of the medullary reticular formation. Moreover, Naloxone, a non-selective opiate receptor antagonist, reduced the IPSP's amplitude. Of great interest regarding these data, we reported that Methionin-Enkephalin (M-Enk) afferent projections to the cat facial nucleus (FN) originate from the caudal raphe nuclei and the nucleus paragigantocellularis lateralis. We also showed that the M-Enk inputs to the cat trigeminal motor nucleus (TMN)

arise from the rostral ventromedial medullary reticular formation (nuclei reticularis magnocellularis and gigantocellularis and nucleus paragigantocellularis lateralis) and the caudal raphe nuclei (Fort et al., 1989, 1990). Among these structures, it seems unlikely that the enkephalinergic projections from the caudal raphe nuclei to the FN and TMN play a role in the oro-facial atonia of PS. Indeed, enkephalin is colocalized with serotonin in these neurons (Hunt and Lovick, 1982; Lger et al., 1986; Fort et al., 1989, 1990) and a number of studies indicate that serotonin has a facilitary rather than an inhibitory action on motoneurons (McCall and Aghajanian, 1979; Jacobs and Fornal, 1993). In contrast, enkephalinergic projections from the rostral ventromedial medullary reticular formation to the FN and TMN could play a role in the atonia of the oro-facial musculature during PS in as much as this region, more precisely the nucleus reticularis magnocellularis (Mc), has been proposed to contain neurons responsible for the hyperpolarization of motoneurons during PS (Magoun and Rhines, 1946; Pompeiano, 1967; Sakai et al., 1979, 1981; Nakamura, 1986; Lai and Siegel, 1988; Yamuy et al., 1993). To test this hypothesis, it was necessary to precisely reexamine the localization of the M-Enk neurons in the rostral ventromedial medullary reticular formation projecting to the TMN and FN. Indeed, in our previous studies, we localized these neurons projecting to the FN and the TMN in different nuclei respectively the paragigantocellularis lateralis nucleus and the nucleus reticularis magnocellularis. Further, we did not provide detailed localization and counting of the double-

Correspondence: Dr. Patrice Fort, Neurobiologie des tats de Sommeil et d'veil, INSERM U480, Facult de Mdecine, Universit Claude Bernard, 8 avenue Rockefeller, 69373, cedex 08, Lyon, France, Tel: 33-4-78-77-71-23, Fax: 33-4-78-77-71-72 , E-mail: fort@sommeil.univ-lyon1.fr.

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Figure 1. Photomicrograph of a frontal section that was processed for immunohistochemical staining of CTb (DAB-Nickel, black punctate staining) and M-Enk (DAB, brown coloration), showing singly M-Enk+ and double-labeled cells in the lateral part of the Mc, medial to the facial nucleus, following a tracer injection in the TMN. Bar: 30 m

labeled cells. Therefore, to localize and compare the distribution of M-Enk-immunoreactive neurons within the rostral ventromedial medulla projecting to the FN or TMN, we combined retrograde tracing with cholera toxin B (CTb) subunit and M-Enk immunohistochemistry in colchicinetreated cats.

METHODS
Tracer injection and perfusion-fixation procedure For the retrograde-tracing experiments, 17 adult cats of both sexes weighing 2.5-4.0 kg were used (n = 9 for the FN and n = 8 for the TMN). Under profound anesthesia with pentobarbital (25 mg/kg, i.v.), 0.1 l of 1% CTb (List Biological Laboratories) was injected stereotaxically into the right TMN or FN with a 5-l Hamilton syringe. Twenty-four hours after the tracer injection, two guide cannulae were implanted above the lateral and fourth ventricles for the colchicine treatment (200 g in 20 l of 0.9% NaCl for each ventricle). Forty-eight hours later, the cats were deeply anesthetized and perfused through the ascending aorta with 1 liter of Ringer's lactate solution, followed by 2.5 liters of an ice-cold fixative in 0.1M PB (pH 7.4) containing 4% paraformaldehyde (PF), 0.1% glutaraldehyde and 0.2% picric acid (PA). After overnight post-fixation, the brains were put in a 30% sucrose solution at 4C for 48 hours.

Double-immunostaining procedure Immunohistochemical detection of CTb was carried out by sequential incubations of free-floating coronal sections (20 m thick). They were first submitted to a long incubation over 3-4 days at 4C with a goat CTb antiserum (1:40,000, List Biological Laboratories, in PB saline with 0.3% Triton X-100 and 0.1% azide, PBST-A). They were then incubated for 90 min at room temperature in biotinylated donkey anti-goat IgG (1:2,000) followed by streptavidin-HRP (1:40,000, Jackson Immunores. Laboratories). Finally, the sections were immersed in 0.02% 3,3'-diaminobenzidine-4HCl (DAB, Sigma) containing 0.003% H2O2 and 0.6% nickel ammonium sulfate in 0.05 M Tris-HCl buffer (pH 7.6) for 10-15 min at room temperature. The same sections were then incubated for 4-6 days at 4C in rabbit antiserum to Methionin-Enkephalin (M-Enk, 1:5,000, UCB), 90 min at room temperature in swine anti-rabbit IgG (1:400) and then in rabbit peroxidase-antiperoxidase (1:400, PAP, DAKO). After rinses, the sections were reacted for 15-30 min at room temperature with 0.025% DAB solution containing 0.006% H2O2. The CTb reaction products obtained by the DAB-nickel histochemical procedure consisted of black punctate granules in the cell soma and dendrites, whereas the M-Enk immunohistochemical reaction product revealed using DAB appeared as a homogeneous light brown staining of the cell

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Figure 2. Series of drawings of 20 m frontal sections from rostral to caudal level of the rostral ventromedial medulla illustrating the location of singly M-Enk+ (), singly CTb+ (o) and CTb+/M-Enk+ double-labeled (*) cells in the Mc after tracer injection in the right FN (left column, cat Q108) and TMN (right column, cat P115). Each symbol represents one labeled cell body. Abbreviations according to the atlas of Berman (1968): 7 (facial nucleus), A (nucleus ambiguus), Gc (nucleus reticularis gigantocellularis), IO (inferior olivary complex), lvs (lateral vestibulospinal tract), Mc (nucleus reticularis magnocellularis), P (pyramidal tract), RM (nucleus raphe magnus), Rpa (nucleus raphe pallidus), rs (rubrospinal tract).

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Table 1 Total (N cells) and Average (Mean SD) Numbers of Singly CTb+, Singly M-Enk+ and Double-Labeled CTb+/M-Enk+ Cells in the Ipsilateral and Contralateral Mc, Following Tracer Injection in the FN or TMN. The counts were based on two representative CTb injection cases for each motor nucleus.
Singly CTb
Total Mc (Mc-Tot) Lateral Mc (Mc-Lat) Large Cells (in Mc-Lat)

Singly Enk
in Mc-Lat

Double CTb-Enk
in Mc-Lat

FN (Mc ipsi) TMN (Mc ipsi) FN (Mc contra)

N cells (16 sections) Mean SD N cells (14 sections) Mean SD N cells (16 sections) Mean SD

243 15.2 4.7 184 13.1 3.8 69 4.3 2.5 41 2.9 2.1

152 9.5 4.2 121 8.6 2.7 38 2.4 2.4 20 1.4 1.2

105 6.6 2.4 97 6.9 2.2 28 1.8 1.7 13 0.9 0.9

332 20.8 7.8 288 20.6 5.9 304 19 7.3 298 21.3 6.5

58 3.6 1.8 63 4.5 2.4 15 0.9 0.9 7 0.5 0.7

TMN (Mc contra) N cells (14 sections) Mean SD

Table 2 Proportions of the Different Populations of Labeled Cells Encountered within the Ipsilateral and Contralateral Mc, Following Tracer Injection in the Right FN or TMN (based on counts reported in Table 1)
Singly CTb Proportions FN (Mc ipsi) TMN (Mc ipsi) FN (Mc contra) TMN (Mc contra)
Mc-Lat/Mc-Tot

In the Mc-Lat
Large CTb/Singly CTb Double/Singly Enk

In the Mc-Tot
Double/Singly CTb

In the Mc-Lat
Double/Singly CTb Double/Large CTb

62.6% 65.8% 55.1% 48.8%

69.1% 80.2% 73.7% 65.0%

17.5% 21.9% 4.9% 2.3%

23.9% 34.2% 21.7% 17.1%

38.2% 52.1% 39.5% 35.0%

55.2% 64.9% 53.6% 53.8%

body (Fig. 1). The specificity of the antibody against M-Enk was assessed by the absorption test. Specific staining of the MEnk-like immunoreactive cell bodies was totally blocked when the primary antiserum was pre-incubated with an excess (100 g-1 mg/ml) of the synthetic peptide. Data analysis The distribution of the singly CTb (CTb+), singly M-Enk (M-Enk+) and double-labeled (CTb+/M-Enk+) cells within the Mc is illustrated in Figure 2 for one representative injection case restricted to the right FN (left column, cat Q108) and one restricted to the right TMN (right column, cat Q115). For this purpose, 4 sections at different rostro-caudal levels of the Mc were observed and drawn at low power magnification (x6.3) with a Leitz Orthoplan microscope equipped with an X/Y sensitive stage and a video camera connected to a computerized image data analysis system (Biocom, France). The labeled cells were plotted at higher power magnification (x16-25). Drawings were then assembled with Adobe Illustrator 7.0 software on a Macintosh computer. In order to precisely evaluate and directly compare the enkephalinergic projections to the FN and TMN, singly CTb+, singly M-Enk+ and double-labeled CTb+/M-Enk+ neurons were counted

bilaterally in the Mc. These counts (Table 1) and the proportions of the different contingents of labeled cells (Table 2) are provided for two representative cats for each motor nucleus injected (cats S106 and Q108 for the FN and cats P110 and Q115 for the TMN). The photomicrograph was taken with a Leitz microscope connected to a camera (Vario-orthoplan) and then scanned. To get an optimal reproduction of the staining, we modified the contrast and luminosity of the crude scan with Adobe Photoshop 4.0 on a Macintosh computer. The illustration plate was then printed with a color dye printer (Epson Stylus color).

RESULTS
Inputs to the TMN and FN from the Mc As previously described, pressure injection of 0.1 l of CTb typically resulted in very small deposits, with a spread of approximately 400 m from the needle point (Fort et al., 1989, 1990). Following CTb injection restricted to the FN (n = 5) or the TMN (n = 2), similar distributions of retrogradely-labeled within the brainstem reticular formation were observed between the different animals. The lateral medullary reticular formation, mainly the nucleus reticularis parvicellularis (Pc),

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contained the largest number of CTb+ neurons (not illustrated). A substantial number of CTb+ neurons were also seen in the rostral ventromedial medulla, bilaterally with a clear ipsilateral dominance (Table 1). In this region, they occupied a relatively small rostro-caudal and medio-lateral extension of the Mc, from the level of the caudal half of the FN to the rostral third of the inferior olivary complex (P7.5 to P9 levels according to the atlas of Berman, 1968) (Fig. 2). A precise analysis further showed that the number and pattern of distribution of CTb+ cells were similar after injections in the TMN or FN. Indeed, the mean number of ipsilateral CTb+ cells for a section was approximately 15 and 13 cells in the Mc (Mc-Tot) following tracer injections in the FN and TMN, respectively. Furthermore, approximately two-thirds of these CTb+ cells (Table 2, first column) were clustered in the lateral part of the Mc (Mc-Lat). Rostrally, they were just medial and ventromedial to the FN in the lateral vestibulo-spinal tract (lvs) (Fig. 2 A,A' and B,B'). Slightly more caudally, the cluster of CTb+ cells was dorsolateral to the inferior olivary complex and dorsal to the rubro-spinal tract (rs) (Fig. 2 B-C, B'-C'). At the most caudal level, they were ventral to the lateral part of the nucleus reticularis gigantocellularis (Gc) in an area medial to the nucleus ambiguus (A) (Fig. 2 D and D'). In all injection cases considered, the medial part of the Mc contained a moderate number of CTb+ cells while only a small number were seen in the raphe magnus and pallidus nuclei. Of particular interest were also the cytological similarities of the CTb+ cells within the Mc-Lat following tracer injections in the FN or TMN. Indeed, within this area, a very large majority of the CTb+ cells (around 70% and 80% for the FN and TMN, respectively; Tables 1 and 2, second column) were medium to large in size (30 x 20 m), round to ovoid in shape, and multipolar. M-Enkephalin-immunoreactive neurons projecting to the TMN and FN By means of colchicine treatment, two main groups of MEnk-like immunoreactive (M-Enk+) cell bodies were labeled within the rostral ventro-medial medulla: the former in the raphe magnus and pallidus nuclei and the latter in the lateral part of the Mc (Mc-Lat) (Fig. 2). Within the Mc-Lat, the MEnk+ cells were numerous (approximately 20 cells for a section, Table 1), situated ventro-medially to the FN and more caudally, dorsolaterally to the inferior olivary complex. As illustrated in Figure 2, after all injections in the TMN or FN, the double-labeled (CTb+/M-Enk+) cells formed at the most rostral level a cluster in the Mc-Lat, medio-ventral to the FN and ventrally to the lateral vestibulo-spinal tract (lvs). Slightly more caudally, the CTb+/M-Enk+ neurons were in the Mc-Lat and the adjacent nucleus paragigantocellularis lateralis (PGCL) in and around the lateral vestibulo-spinal tract. The caudal extension of the group was located in the Mc-Lat within the lvs, as well as more dorsally in the Gc and the Pc just lateral to it. At all levels, the medial Mc contained only occasional CTb+/M-Enk+ cells. In the Mc-Lat, the mean number of CTb+/M-Enk+ cells for a section was approximately 4 for the FN and TMN, respectively (Table 1). Of the singly M-Enk+ neurons in this area, around 20% for both the FN and TMN

were retrogradely-labeled (Table 2, third column), while the CTb+/M-Enk+ cells represented approximately 40% and 50% of the CTb+ cells encountered in this area (Table 2, fifth column). The CTb+/M-Enk+ cells were all medium to large in size (30 x 20 m) and round to ovoid in shape (Fig. 1). They accounted for a large majority of the medium- to large-sized retrogradely labeled cells within the Mc-Lat (around 55% and 65% for the FN and TMN, respectively; Table 2, sixth column).

DISCUSSION
In this study, we demonstrated that the TMN and FN receive a major enkephalinergic input from neurons in the lateral part of the Mc (Mc-Lat) located in the rostral ventro-medial medulla. Our detailed comparison of the results further suggest that the Methionin-enkephalin (M-Enk) positive neurons projecting to these two motor nuclei could belong to the same population of cells and might therefore contribute to the simultaneous hyperpolarization of the FN and TMN motoneurons during PS episodes. In the following part of the discussion, we report experimental data, mainly in cats supporting this hypothesis. It is well known in cats that serotonin is frequently colocalized with M-Enk in neurons of the rostral ventro-medial medulla (Hunt and Lovick, 1982; Lger et al., 1986). However, we previously observed no or only occasional serotoninimmunoreactive neurons retrogradely labeled in the Mc-Lat following CTb injections in the FN or TMN (Fort et al., 1989, 1990). These data indicate that the M-Enk neurons in the McLat are not serotonergic "PS-off cells" (reviewed in Jacobs and Fornal, 1993). Besides, electrophysiological studies in freely-moving cats reported a few cells in the Mc with a tonic increase of their firing rate selectively during PS episodes (namely "PS-on" cells) projecting to the TMN as shown by antidromic activation (Sakai et al., 1979, 1981; Nakamura, 1986). These cells were located in the same region as the M-Enk neurons we found projecting to the TMN and FN, namely in the Mc-Lat. Moreover, this region receives a projection from the dorsomedial pontine area responsible for the muscle atonia during PS (Sakai et al., 1981). Furthermore, it has recently been reported that the rostral ventro-medial medulla contained numerous C-fos-positive neurons following PS hypersomnia induced by carbachol micro-injections in the dorsal pontine tegmentum (Yamuy et al., 1993). Combining C-fos immunohistochemistry and retrograde-tracing with CTb, the same authors further found in the Mc-Lat a population of doublelabeled neurons specifically activated during PS and projecting to the TMN (Morales et al., 1996). Therefore, it is tempting to hypothesize that the M-Enk neurons in the lateral Mc with inputs to the FN and TMN correspond to the inhibitory "PSon" premotoneurons in the cat. However, it has been demonstrated that the major component of the suppression of the masseteric and spinal motor activity during PS is ascribable to a strychnine-sensitive postsynaptic inhibition during PS (Soja et al., 1987a, 1987b; Chase et al., 1989; Soja et al., 1991). These results indicate that

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the amino acid glycine is the principal mediator of the hyperpolarization of motoneurons during PS. The localization of the glycinergic premotoneurons responsible is still a matter of debate. It was originally hypothesized that, during PS, excitatory neurons in the nucleus reticularis magnocellularis tonically stimulate glycinergic premotoneurons localized in the parvocellular reticular nucleus (Pc) for the cranial motoneurons or the intermediate zone of the spinal cord for the spinal motoneurons (Magoun and Rhines, 1946; Pompeiano, 1967; Sakai et al., 1979, 1981). Later, a number of studies suggested that the glycinergic neurons responsible for the hyperpolarization of cranial and spinal motoneurons could be directly located in the Mc (Fort et al., 1989, 1990; Holstege and Bongers, 1991; Fort et al., 1993; Yamuy et al., 1993; reviewed in Holstege, 1996). If this hypothesis is correct, Methioninenkephalin could be co-contained in these glycinergic neurons. However, in recent studies in the rat, it has been shown that the Pc provides a strong glycinergic projection to the FN or the TMN and the Mc only a weak one (Li et al., 1996; Rampon et al., 1996; Li et al., 1997). These contradictory results might be explained by species differences. Another possibility is that the glycinergic neurons responsible for the hyperpolarization of cranial motoneurons during PS are localized in the Pc. If this is the case, the M-Enk neurons localized in the Mc-Lat and projecting to the cranial motoneurons would not be glycinergic. They would facilitate the hyperpolarization induced by glycinergic neurons from the Pc. Finally, numerous neuroanatomical studies in cats demonstrated that, in addition to the TMN and FN, the Mc-Lat sends efferent projections to spinal motoneurons (Sakai et al., 1981; Alstermark et al., 1987; Ohta et al., 1990). It has further been shown combining retrograde tracing with M-Enk immunohistochemistry that this projection is also in part enkephalinergic in nature (Fung et al., 1994). Altogether, these data suggest that neurons within the lateral part of the Mc, containing M-Enk, could participate in the hyperpolarization of the cranial and also of spinal motoneurons during PS in the cat. Conclusion and new hypothesis Our experimental data suggest that in the cat the enkephalinergic neurons in the lateral part of the Mc could participate in the hyperpolarization of the cranial motoneurons during PS. These neurons could also participate in the hyperpolarization of spinal motoneurons during this state of sleep. Further anatomical and electrophysiological investigations focused on this enkephalinergic cell group are necessary in cats to test this hypothesis.
ACKNOWLEDGMENTS

The investigations in this report were supported by INSERM, CNRS and DRET (grants 90/1615 and 91/130). We would like to express our gratitude to Denise Salvert and Colette Buda for their skillful technical assistance.

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