Chapter 10 The ventro-medial medullary projections to spinal motoneurons: ultrastructure, transmitters and functional aspects

G. Holstege. R. Bandler and C.B. Saper (Eds.) Progress in Bruin Research, Vol. 107

0 1996 Elsevier Science B.V. All rights reserved.

CHAPTER 10

The ventro-medial medullary projections to spinal motoneurons: ultrastructure, transmitters and functional aspects Jan C.Holstege Department of Anatomy. Erasmus University Medical School, P.O. Box I738, 3000 DR Rotterdam, The Netherlands

Introduction Like many projection systems in the central nervous system, the descending projections from the brain stem to the spinal cord have a history that runs parallel to the emergence of new tracing techniques. The degeneration technique showed that the reticular formation of pons and medulla gave rise to spinal projections which terminated primarily in the intermediate zone throughout the spinal cord (Kuypers et al., 1962; Nyberg-Hansen, 1965). Since these projections terminated mostly in the medial part of the intermediate zone, they were classified as the medial system of the descending brain stem pathways as defined by Kuypers (1981). Only few brain stem areas gave rise to projections that terminated in more lateral parts of the intermediate zone. They were classified as the lateral system of the descending brain stem pathways (Kuypers, 1981) and comprised the spinal projections from the red nucleus and the ventro-lateral pontine reticular formation. When the retrograde horseradish peroxidase (HRP) technique (Lavail and Lavail, 1972; Mesulam and Rosene, 1979) and the anterograde autoradiographic technique (Lasek et al., 1968; Cowan et al., 1972) became available, the descending brain stem projections were re-examined. The retrograde HRP technique showed that, apart from the areas already known from degeneration studies to project to the spinal cord, other spinal projecting

areas existed within the brain stem, such as the retroambiguus nucleus, the locus coeruleus and subcoeruleus, the lateral pontine tegmentum and several hypothalamic areas, including the paraventricular nucleus (Kuypers and Maisky, 1975; Crutcher et al., 1978; Kneisley et al., 1978; Leichnetz et al., 1978). In order to re-investigate the termination areas of the descending brain stem projections within the different laminae of the spinal cord, the anterograde autoradiographic tracing technique was used. This technique has the major advantage over the anterograde degeneration technique that it only labels fibers from neurons within the injection site, and not fibers of passage (Swanson, 1981). The anterograde autoradiographic studies (Basbaum et al., 1978; Holstege et al., 1979; Martin et al., 1979; Holstege, G. and Kuypers, 1982; Jones and Yang, 1985; Martin et al., 1985) confirmed most of the degeneration findings, but also identified several hitherto unidentified projections, including the massive projections from the caudal raphe nuclei and the adjacent reticular formation of the ventro-medial medulla and those from the area of the locus coeruleus and subcoeruleus. These projections terminated throughout the gray matter of the spinal cord, including the motoneuronal cell groups, and were confirmed more recently using phaseolus vulgaris leuco-agglutinin as an anterograde tracer (Jones and Light, 1990; Clark and Proudfit, 1991; Zagon and Bacon, 1991). Earlier studies (Carlsson

160

et al., 1964; Dahlstrom and Fuxe, 1965) using histofluorescence to identify monoamines, had already shown the presence of serotonin and noradrenalin both in the ventral and dorsal horn throughout the spinal cord and lesion studies (Dahlstrom and Fuxe, 1965; Nygren and Olson, 1977) had indicated that the monoaminergic terminals in the spinal cord originated from neurons in the brain stem, i.e. from the caudal raphe nuclei and the locus coeruleus and subcoeruleus. These anatomically and neurochemically identified “new” brain stem projections were difficult to fit into the medial or lateral system of descending brain stem pathways as defined by Kuypers (1981) and were therefore referred to as the third motor system (Kuypers, 1982). More recently, G. Holstege, who originally identified these projections in the cat (Holstege et al., 1979), coined the term emotional motor system (see Chapter 1) to include not only the descending brain stem projections but also their afferents from limbic areas of the brain. It was also pointed out that these limbic afferents may be subdivided in medial and lateral systems (Holstege, G., 1987; Holstege, G., 1991). In this concept of the motor system, the projections from the ventro-medial medulla, together with those of the locus coeruleus and subcoeruleus, are considered as the major output center of the lower brain stem for the connections associated with the medial part of the emotional motor system. In the remainder of this chapter the ventro-medial medullary projections to spinal motoneurons are considered in detail with respect to their ultrastructure, transmitters and function.

The ventro-medial medullary projections to spinal motoneurons Anatomical aspects of the ventro-medial medulla The boundaries of the area termed here the ventromedial medulla are not well defined. The delineation of this area is further complicated by species differences and the nomenclature. In general terms, the ventro-medial medulla comprises the area ranging from the caudal part of the inferior

olive to the caudal part of the trapezoid body. At the level of the inferior olive it also comprises the nucleus raphe obscurus, the ventral part of the nucleus raphe pallidus and the area immediately overlying the inferior olive and immediately lateral to it. More rostrally it comprises the nucleus raphe magnus and laterally adjacent areas up to the medial border of the facial nucleus. In the rat, according to the nomenclature of Andrezik and Beitz (1989, the ventro-medial medulla includes the ventral part of the paramedian reticular nucleus, the gigantocellular nucleus-ventral part and the ventro-medial part of the paragigantocellular nucleus at the level of the inferior olive. More rostrally, it comprises the gigantocellular nucleus-pars a and the rostra1 part of the paragigantocellular nucleus. In the cat, the magnocellular tegmental field of Berman (1968) corresponds to the gigantocellular nucleus-ventral part and pars a of the rat while the magnocellular nucleus of the cat corresponds mainly to the rat’s gigantocellular nucleus-pars a (Andrezik and Beitz, 1985; see also Siegel, 1994). It is important to note that the more dorsally located gigantocellular nucleus, which comprises many spinal projecting cells, including giant cells, is not considered part of the ventro-medial medulla.

Light microscopy of the ventro-medial medullary projections to spinal motoneurons The light microscopy of the descending brain stem projections to the spinal cord and their funicular trajectories have been extensively reviewed elsewhere (Kuypers, 1981; Martin, 1982; Kuypers, 1985; Holstege and Kuypers, 1987b; Holstege, G., 1991) and is not described in detail. With respect to the spinal projections, originating in the medial reticular formation of pons and medulla, it is important to note that the pontine projections terminate predominantly in the medial and central parts of the intermediate zone (lamina VII and VIII) throughout the spinal cord, with few if any direct projections to spinal motoneuronal cell groups. Similar projections originate in the dorsal part of the medullary medial reticular formation (Holstege, G. and Kuypers, 1982; Martin et al., 1985;

161

Matsuyama et al., 1988). However, the bulk of the spinal projections from the medulla is derived from the ventro-medial medulla. These projections terminate in all laminae of the spinal cord and are topographically organized in the sense that the more rostral parts of the ventro-medial medulla, i.e. the area of the nucleus raphe magnus, project preferentially to the dorsal horn (Basbaum et al., 1978; Holstege, G., 1988; Jones and Light, 1990), while the more caudally located nucleus raphe pallidus and adjacent reticular formation of the ventro-medial medulla project preferentially to the ventral horn including the motoneuronal cell groups (Holstege et al., 1979; Martin et al., 1979; Holstege, G. and Kuypers, 1982; Jones and Yang, 1985; Martin et al., 1985; Holstege and Kuypers, 1987b; Zagon and Bacon, 1991). Both areas were found to project to the intermedio-lateral cell columns of the thoraco-lumbar and sacral spinal cord (Loewy et al., 1981; Holstege, G. and Kuypers, 1982; Zagon and Bacon, 1991). This division is especially apparent in cat, but is less distinct in rat where, in the area of the caudal raphe magnus and the rostral pole of the inferior olive, ventral and dorsal horn projecting cells are intermingled (Jones and Light, 1990; Zagon and Bacon, 1991). The ventro-medial medullary projections do not show a clear preference for medial or lateral intermediate zone or for motoneurons innervating flexor or extensor muscles (cf. Holstege, J.C. and Kuypers, 1982). Retrograde double labeling studies (Martin et al., 1981; Huisman et al., 1984) have shown that a high percentage of the ventro-medial medullary neurons send collaterals to both cervical and lumbar levels, suggesting a diffuse organization of the descending projections from the ventro-medial medulla.

Electron microscopy The projections from the ventro-medial medulla to spinal motoneurons were investigated at the ultrastructural level using anterograde transport of [3H]leucine (Holstege and Kuypers, 1987a) or wheat-germ agglutinin (WGA)-HRP (Holstege, J.C., 1987). In the autoradiographic experiments,

midline injections were made that included the raphe pallidus, obscurus and caudal raphe magnus and the adjacent reticular formation. In additional experiments injections were made in the reticular formation of the ventro-medial medulla at the same rostro-caudal levels, but with limited involvement of the raphe nuclei. In both experiments, strong labeling was observed in the motoneuronal cell groups, throughout the spinal cord. Ultrastructural analysis of the autoradiograms from the L5 and L6 lumbar segments showed many structures carrying silvergrains. However, it is inherent to the EM autoradiographic technique that the presence of a silvergrain over a particular profile does not necessarily mean that the underlying structure is radioactively labeled, since silvergrains may be formed as a result of radiation from [3H]leucine in a nearby structure or from "background" radiation. Therefore, a statistical analysis has to be applied on the individual silvergrains and their underlying structures. The outcome of such an analysis (termed circle analysis) will determine which tissue compartments have a high probability of containing radioactivity and are therefore considered as labeled. In an alternative method, termed the cluster method, it is assumed that a structure is radioactively labeled if a cluster of silvergrains (e.g. 6 or more) is centered on that structure. It seems likely that with the cluster method, only strongly labeled structures are identified, which may originate preferentially from the center of the injection site. With the circle method more weakly labeled structures, possibly derived from the periphery of the injection site, are identified in addition to the strongly labeled structures. For clarity, only results obtained with the cluster method are presented here, since they were basically similar but somewhat more pronounced than the results obtained with the circle method. For details on the results obtained with both types of analysis and on the analysis of electron microscopic autoradiograms in general, see Holstege and Kuypers (1987a) and Holstege and Vrensen (1988). In the analysis of the EM autoradiograms special attention was paid to radioactively labeled

162 TABLE 1 Types of terminals distinguished in rat motoneuronal cell groups S-type Terminals containing spherical vesicles (40-50 nm in diameter) and an occasional large dense core vesicle (80-120 nm). They usually show asymmetrical synaptic junctions. F-type Terminals containing flattened synaptic vesicles (2535 x 50-60 nm in diameter) often in combination with spherical vesicles. They usually show symmetrical synaptic junctions. G-type Terminals, characteristically containing large granular vesicles (70-120 nm), sometimes elongated (up to 180 nm). In addition many clear vesicles, spherical or flattened (15 to 50 nm) and one or two large clear vesicles are present. The terminals usually show asymmetrical synaptic junctions. C-type Terminals containing spherical or flattened vesicles. The synaptic junctions lack pre- and postsynaptic membrane thickenings. The postsynaptic element is either a cell soma or a proximal dendrite. A postsynaptic subsurface cistern is present along the entire length of the synaptic apposition, with a Nissl body located beneath the cistern. P-type Terminals, usually small, containing slightly flattened vesicles. They are presynapric to large S-type terminals which sometimes are associated with postsynaptic dense bodies. Synaptic junctions of P-type terminals are not very pronounced.

terminals. From previous studies on the spinal motoneuron pool in different species (Bodian, 1966; Conradi, 1969; McLaughlin, 1972a; Bernstein and Bernstein, 1976) a general picture of the neuropil emerged, especially with respect to the different types of terminals. Basically there are 5 different terminal types (see Table 1 for definitions), which can be distinguished on the basis of their vesicle shape: either spherical (S-type), flattened (F-type) or granular (G-type) or their postsynaptic element, either another terminal (in case of the P-type) or a soma or dendrite with a subsynaptic cistern (in case of the C-type). It is important to realize that the overwhelming majority of the terminals in the spinal motoneuron pool are of the S- and F-type, whereas the G-, P- and C-types each represent no more than 1% of all the terminals. This indicates that most afferent connections

are represented by the F- and S-type terminals which, therefore, must originate from many different sources. After injections of [3H]leucinein the caudal raphe nuclei it was found that three types of terminals were labeled: F-type terminals, S-type terminals and G-type terminals (Figs. 1 and 2). After injection in the adjacent reticular formation of the ventro-medial medulla, the same terminal types were labeled but with a different frequency (Figs. 1 and 2). Thus, after raphe injections a larger number of G-type terminals was labeled (Fig. lA), while after ventro-medial reticular formation injections the F-type terminals were more abundant (Fig. 1B). The S-type terminals that were labeled, originated mainly from the reticular formation of the ventro-medial medulla. In addition a limited number of C-type terminals was labeled (Fig. 2). Although there were relatively few (3% of the total number of labeled terminals) their characteristics and labeling were very clear. This showed that Ctype terminals are not derived exclusively from nearby interneurons or short propriospinal connections as suggested previously (McLaughlin, 1972b; Matsushita and Ikeda, 1973). There is some evidence that C-type terminals contain acetyl-choline (Nagy et al., 1993). If this is indeed the case, the labeling of C-type terminals from the lower brain stem would mean the existence of a (minor) cholinergic brain stem projection to spinal motoneurons. With respect to the frequency that a labeled terminal profile showed a synaptic specialization in a single thin section, it was found that the S- and F-type terminals established a synapse in about 50% of the cases (Tables 2 and 3). They synapsed mostly with proximal dendrites (defined as dendrites containing ribosomes), while much fewer contacted distal dendrites (without ribosomes) or cell somata. In this respect it is important to note that only about 4% of the total surface area of a spinal motoneuron is accounted for by the cell soma (Zwaagstra and Kernell, 1981; Ulfhake, 1984), indicating that the large majority of the terminals on a motoneuron are on its dendrites. The G-type terminals behaved very differently as

163

Fig. 1. Electron micrographs of autoradiographically labeled terminals in the L5 or L6 lumbar motoneuronal cell groups after injections of [3H]leucine in the caudal raphe nuclei (A), the reticular formation of the ventro-medial medulla (B) and the reticular formation of the ventro-medial medulla combined with retrograde transport of HRP from the hindlimb muscles (C). G, G-type terminal containing several dense cored vesicles; F, Ptype terminal. Small arrows indicate synaptic junction, large arrows indicate HRP reaction product. Bar = 0.5 pm, For details, see Holstege and Kuypers (1987a).

164

3 5

Reticular 60 50

I-

8

40

-1

W

2 30 -1

8 S-type

% 70 v)

2

60

$

50

z

1

F-type G-type C-type

Raphe

tablish a synaptic junction at all. The latter possibility, i.e. the existence of non-synaptic G-type terminals, has been investigated in detail in cat motoneuronal cell groups using serial section analysis (Ulfhake et al., 1987). This study showed that non-synaptic G-type terminals, which contained serotonin, substance P or thyrotropin releasing hormone (TRH), do exist. This makes it likely that several of the G-type terminals release their transmitter(s) non-synaptically (see also Holstege and Bongers (1991b) and Bach-y-Rita (1993) for a discussion on non-synaptic release of transmitters). In order to ascertain that the terminals in the motoneuronal cell groups, which originated from neurons in the lower brain stem, actually terminated on motoneuronal dendrites and not on dendrites of local interneurons radiating into the motoneuronal cell groups, an additional experiment (Holstege and Kuypers, 1987a) was executed in which the anterograde labeling of terminals with r3H]leucine from the lower brain stem was com-

I-

TABLE 2

40

1 30

Synaptic frequency and postsynaptic structures of clusterlabeled terminals after ventro-medial reticular formation injections

v)

3

6

10

Type of terminal

(%I

8

S-type F-type G-type C-type Fig. 2. Frequency of the different types of terminals, labeled with a cluster of silvergrains after injections of [3H]leucine in the reticular formation of the ventro-medial medulla or the caudal raphe nuclei in the rat. For details, see Holstege and Kuypers (1987a).

compared to the S- and F-type terminals, since on average only 12% of the G-type terminals showed a synaptic contact (Tables 2 and 3). This indicated either that the synaptic contacts of G-type terminals were smaller and consequently less frequently encountered than those of the S- and F-type terminals, or that several G-type terminals did not es-

% Synapse

Postsynaptic element (%)

S-type: 16 i 5 (n = 139)

41 i 5

CS: 5 i 6 pD: 8 0 i 9 dD: 1 5 i 8

F-tpe: 6 0 i 6

51 i 2

CS: 8 i 4 pD: 7 6 i 5 dD: 1 6 i 4

G-type: 20 i 4 ( n = 197)

9i4

(n = 548)

C-type: 3 i 2 (n = 26)

100

cs:

0 pD: 8 2 i 14 dD: M i 1 4 CS: 62 i 10 pD: 3 8 i 10 dD: 0

CS, cell soma; pD, proximal dendrite; dD, distal dendrite. For details, see Holstege and Kuypers (1987a).

165 TABLE 3 Synaptic frequency and postsynaptic structures of clusterlabeled terminals after caudal raphe injections Type of terminal

(%I

S-type: 5 * 4 (n = 17)

% Synapse

Postsynaptic element (%)

34

cs:

0

*

pD: 100 9 dD: 0

F-type: 33 (n = 149)

*7

55*11

cs:

G-type: 59 ( n = 231)

*8

14*6

cs: 4 * 5 pD: 6 6 i 16 dD: 3 0 i 1 6

4*4 pD: 71*14 dD: 25* 14

CS, cell soma; pD, proximal dendrite; dD, distal dendrite. For details, see Holstege and Kuypers (1987a).

bined with the retrograde labeling of motoneurons from the hindlimb. Analysis of this material showed that in approximately 50% of the cases an autoradiographically labeled terminal established a synaptic contact, the post-synaptic element was labeled with HRP. Since free HRP, in contrast to WGA-HRF', is not transported transneuronally (ZAborsky and Heimer, 1989), all HRP-labeled elements were considered part of a motoneuron (innervating a hindlimb muscle) (Fig. 1C). Since neither all motoneurons nor all parts of a motoneuron were labeled, this result must be an underestimate of the actual number of cases a motoneuron was contacted. Thus it may be concluded that within the lumbar motoneuronal cell groups the large majority of the postsynaptic structures contacted by the descending projections from the ventro-medial reticular formation belong to motoneurons.

Transmitters in the ventro-medial medullary projections to spinal motoneurons Uchizono (1966) first suggested that terminals with flattened vesicles (F-type) were inhibitory, while terminals with spherical vesicles (S-type)

were excitatory. The presence of spherical and especially flattened vesicles is dependent not on aldehyde fixation (Fukami, 1969) but on the osmolarity of the fixation and rinsing fluids (Bodian, 1970; Valdivia, 1971). Thus the differences in vesicle shape are in fact an artefact which, however, is remarkably reproducible. Indeed, in the motoneuronal cell groups F-type terminals are preferentially labeled for the inhibitory transmitters y-amino butyric acid (GABA) and glycine (Ljungdahl and Hokfelt, 1973; McLaughlin et al., 1975; Holstege and Calkoen, 1990; Destombes et al., 1992), while S-type terminals usually were not (in line with the idea that they contain an excitatory transmitter). G-type terminals were found to be immunoreactive for serotonin and, in several cases, for substance P and TRH (Ulfhake et al., 1987). GABA immunoreactivity in G-type terminals in the motoneuronal cell groups has been described (see also Fig. 3C), indicating coexistence of GABA and serotonin (Holstege and Calkoen, 1990). P-type terminals show the strongest immunoreactivity for GABA (McLaughlin et al., 1975; Holstege and Calkoen, 1990), which is in accordance with their presumed function: presynaptic inhibition of dorsal root (Ia) afferents (Conradi et al., 1983). It seems likely that S-type terminals contain an excitatory transmitter, probably glutamate, but some S-type terminals (Houser et al., 1983; Lagerback et al., 1978) as well as some C-type terminals (Nagy et al., 1993) may contain acetylcholine. These data support the idea that the classification of terminals in different types on the basis of their morphology, is meaningful with respect to their transmitter content or origin. The ultrastructural studies on the descending projections from the ventro-medial medulla to spinal motoneurons showed that three types of terminals were involved: F-, S- and G-type terminals (Fig. 2). The transmitters associated with each of these terminal types are discussed below. They are serotonin and various coexisting peptides, associated with the G-type terminals; GABA and glycine, associated with the F-type terminals; and acetylcholine and glutamate, associated with the Stype terminals.

166

Fig. 3. (A) Light micrograph showing WGA-HRP injection site in the rat lower brain stem at the level of the rostra1 inferior olive. Some WGA-HRP was present in the pyramidal tract area, however there was no anterograde labeling of pyramidal tract fibers in the dorsal funiculus of the spinal cord, indicating that there was no significant uptake of WGA-HRP by the pyramidal fibers; bar = 1 mm. (B,C) Electron micrographs from the rat L5 lumbar motoneuronal cell groups showing terminal profiles labeled with WGA-HRP reaction product (large arrows), after injection as in (A), with many gold particles indicating the presence of GABA. Small arrows indicate a synaptic junction. F, F-type terminal; G, G-type terminal, containing many dense cored vesicles. Bar = 0.5 pm.

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Serotonin and associated peptides

Immunocytochemical studies have shown that Gtype terminals in the ventral horn, including the motoneuronal cell groups, contained serotonin, often with colocalized substance P and TRH (Pelletier et al., 1981; Vacca et al., 1982; Wessendorf and Elde, 1985; Ulfhake et al., 1987). Since there are no (Steinbusch, 1981) or very few (La Motte et al., 1982) serotonergic cells in the mammalian spinal cord, the G-type terminals are thought to originate primarily from serotonergic neurons in the caudal raphe nuclei of the lower brain stem, which project to the spinal ventral horn, as first observed by Dahlstrom and Fuxe (1965) and confirmed in many subsequent studies mostly using a combination of retrograde tracing with serotonin immunocytochemistry (Johansson et al., 1981; Skagerberg and Bjorklund, 1985; Bowker and Abbott, 1990). Many of the serotonergic neurons in the medulla were found to contain additional transmitters, especially peptides like substance P (Hokfelt et al., 1978; Bowker et al., 1983; Arvidsson et al., 1990), TRH (Hokfelt et al., 1975; Johansson et al., 1981; Arvidsson et al., 1990), enkephalin (Hunt and Lovick, 1982) and galanin (Melander et al., 1986). In accordance with these observations, the serotonergic fibers in the ventral horn contain colocalized peptides, mostly substance P, TRH (Johansson et al., 1981; Gilbert et al., 1982; Arvidsson et al., 1990) and galanin (Arvidsson et al., 1991) and only rarely enkephalin (Tashiro et al., 1988). Some of the spinal projecting neurons in the ventromedial medulla contain peptides, like somatostatin, cholecystokinin or enkaphalin, but not serotonin (Bowker and Abbott, 1990). There is also evidence for coexistence of serotonin with GABA in neurons of the ventro-medial medulla (Millhorn et al., 19881, which project to the spinal cord (Millhorn et al., 1987; Reichling and Basbaum, 1990), including the motoneuronal cell groups (see later). More recently, coexistence of serotonin with glutamate was reported in neurons of the ventromedial medulla (Kaneko et al., 1990; Nicholas et al., 1992) (see later). Taken together these findings indicate that the serotonergic projection to spinal

motoneurons is very heterogeneous with respect to its many colocalized transmitters. The effects of serotonin on motoneurons are mainly modulatory such that it facilitates the effect of other excitatory inputs, especially glutamate (White and Neuman, 1980; White, 1985) and these effects are enhanced by substance P and TRH (for details see Chapter 11). The phenomenon of plateau potentials in motoneurons (Kiehn, 1991), which allows short inputs to trigger a maintained motor output, may in part underlie the serotonininduced facilitation. GABA and glycine

The main inhibitory transmitters in the spinal cord and lower brain stem are GABA and glycine. Within the motoneuronal cell groups both transmitters have been identified mostly in F-type terminals (Ljungdahl and Hokfelt, 1973; McLaughlin et al., 1975; Van den Pol and Gorcs, 1988; Holstege and Calkoen, 1990; Destombes et al., 1992), which is in line with the idea that terminals with flattened vesicles (F-type terminals) contain an inhibitory transmitter. In addition it was shown recently in cat (Ornung et al., 1994; Taal and Holstege, 1994) that GABA and glycine were frequently colocalized: nearly two out of every three GABAergic terminals also contained glycine and vice versa (Taal and Holstege, 1994). There is evidence for a GABAergic projection to the spinal cord originating in the ventro-medial medulla (Millhorn et al., 1987). In order to determine whether these GABAergic projections terminated in the motoneuronal cell groups and whether also glycinergic projections existed, the combination of WGA-HRP anterograde tracing with GABA and glycine postembedding immunocytochemistry was employed (Holstege, J.C., 1991; Holstege and Bongers, 1991a). Injections of WGA-HRP were made in the ventro-medial medulla at the level of the rostra1 inferior olive and the caudal facial nucleus, with limited involvement of the nucleus raphe magnus (Fig. 3A). This area contained the largest number of GABAergic neurons, projecting to the spinal cord (Millhorn et al.,

168

Fig. 4.Electron micrograph from the rat L5 lumbar motoneuronal cell groups showing a terminal profile labeled with WGA-HRP reaction product (large arrows), after a WGA-HRP injection similar to the one shown in Fig. 3A. Gold particles indicate the presence of glycine. Small arrows indicate a synaptic junction. F, F-type terminal; bar = 0.5 pm.

1987). After 3-4 days survival, vibratome sections from the lumbar spinal cord were reacted for WGA-HRP and processed for electron microscopy. Ultrathin sections containing the HRP reaction product were treated using the postembedding immunogold technique with GABA antibodies. A quantitative analysis of the material showed (Fig. 5) that approximately 40% of the terminals, anterogradely labeled with WGA-HRP from the ventro-medial medulla, were immunoreactive for GABA (Fig. 3B and C). In a separate experiment (Holstege and Bongers, 1991a), using the same technique with a glycine antibody (Fig. 4), it was found (Fig. 5 ) that 15% of the WGA-HRP labeled terminals were immunoreactive for glycine. The existence of a glycinergic projection to the spinal cord is further supported by the finding that glycinergic cells are present in the ventro-medial medulla (Fort et al., 1993) and that neurons in the same area are retrogradely labeled with [3H]glycine from the spinal cord (Holstege et al.,

1991). Most, but not all, of the terminals belonging to the descending GABAergic and glycinergic projections were of the F-type (Figs. 3B and 4).A few were of the S-type and some of the GABAergic but none of the glycinergic terminals were of the G-type (Fig. 3C), suggesting coexistence of GABA and serotonin in the descending projections from the lower brain stem to spinal motoneurons. This conclusion, which is partly based on morphological criteria (GABA in G-types signifies coexistence of GABA and serotonin), is supported by the finding that some of the neurons, which contained glutamic acid decarboxylase (the GABA synthesizing enzyme) and projected to the spinal cord, also contained serotonin (Millhorn et al., 1987; Millhorn et al., 1988). Taken together these data indicate (Fig. 5 ) that a substantial part of the descending monosynaptic projections to motoneurons contains GABA (partly coexisting with serotonin) and glycine. Many physiologic studies have shown that

169

0 40

-

30

-

20

-

10

-

= F-type = S-type

= G-type = C-type

I

GABA

Glycine

?

Fig. 5 . Histogram compiled from the results of two different studies (Holstege, J.C., 1991; Holstege and Bongers, 1991a). Bars indicate the percentages of GABAergic terminals, glycinergic terminals and terminals with an unidentified transmitter, derived from the ventro-medial medullary reticular formation. Within these groups, the proportion of the different types of terminals is indicated. Terminals in the group with unidentified transmitter may contain serotonin (in the G-type terminals) and acetylcholine (in the C-type terminals), while the remaining S- and F-type terminals may contain glutamate.

GABA and glycine exert an inhibitory effect on motoneurons. Direct inhibition of motoneurons from the ventro-medial medulla can be elicited, including monosynaptic inhibition (Magoun and Rhines, 1946; Llinas and Terzuolo, 1964). However, most of these studies indicate that supraspinal inhibition, especially of limb motoneurons, is produced through excitation of inhibitory interneurons (see Wilson and Peterson (1981) for review). Thus, the direct inhibitory projection to motoneurons may either be additional to the inhibition through interneurons or subserve a very different

function, like controlling the general level of excitability of the spinal motoneurons (see later).

The excitatory component

In the previous section it was argued that in the motoneuronal cell groups, the S-type terminals may contain an excitatory transmitter, since they contain only spherical vesicles and were generally not labeled for GABA or glycine. Thus, the consistent presence of S-type terminals in the descending projections from the ventro-medial medulla is indicative of an excitatory transmitter in part of this connection. The most likely excitatory transmitter candidates for the S-type terminals are acetylcholine and glutamate. Previous studies have suggested the presence of a cholinergic projection from the lower brain stem to the spinal cord (Bowker et al., 1983; Jones et al., 1986), which was subsequently denied (Sherriff et al., 1991). The finding that in the motoneuronal cell groups, 3% of the terminals originating from the reticular formation of the ventro-medial medulla were of the C-type (Holstege and Kuypers, 1987a), suggests that a supraspinal cholinergic projection does exist, since C-type terminals in all likelihood contain acetyl-choline as a transmitter (Nagy et al., 1993). Since this descending cholinergic projection is very limited, it seems most likely that glutamate is the most important excitatory transmitter in the descending projections from the ventromedial medulla. Glutamate usually has a clear-cut excitatory effect on spinal motoneurons, through the activation of different types of ionotropic glutamate receptors, details of which have been reviewed elsewhere (Watkins, 1984; Headley and Grillner, 1990). Whether other excitatory amino acids, especially aspartate, should also be considered as important transmitters in the brain is uncertain (Orrego and Villanueva, 1993); see also Shupliakov et al. (1993). Therefore, these possibilities are not considered here. Recently it was reported that glutamate is present in many (Kaneko et al., 1990; Minson et al., 1991) or all (Nicholas et al., 1992) serotonergic cells of the lower brain stem, including those pro-

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jecting to the spinal cord. These data were obtained either with an antibody directed against glutamate (Nicholas et al., 1992) or with an antibody against phosphate-activated glutaminase, which is considered a key enzyme in the production of glutamate as a transmitter (Kaneko et al., 1990; Minson et al., 1991), but which is also present in GABAergic neurons. Coexistence of serotonin and glutamate was also observed in terminals within the motoneuronal cell groups, often together with substance P (Nicholas et al., 1992). However, it should be realized that the immunocytochemical identification of glutamate in neurons and terminals does not necessarily imply that glutamate has a transmitter function in those cells, since glutamate is present in the metabolic pool of all neurons. Therefore additional evidence is necessary to ascertain the presence of glutamate in the descending projections from the ventromedial medulla. If glutamate is indeed present as a transmitter the serotonergic projection to the motoneuronal cell groups as well as in some non-serotonergic fibers, this would imply that glutamate is localized in G-type terminals and in most of the S-type terminals originating in the ventro-medial medulla. In the G-type terminals glutamate may even be regarded as the “main” transmitter with serotonin acting as a modulator. If this is true, it would put the serotonergic raphe projection to the spinal cord in a different perspective. The low synaptic incidence of G-type terminals (Tables 2 and 3) and the finding that some of the G-type terminals do not exhibit synaptic junctions (Ulfhake et al., 1987), strongly suggest non-synaptic release of serotonin with coexisting peptides and possibly also glutamate. The effect, produced by a transmitter subsequent to its release, depends not only on characteristics of the receptors involved, but also on the rate of degradation or the efficiency of (re-)uptake of that transmitter from the extracellular space by local neuronal and glial elements. Inside the synaptic cleft, these mechanisms may be much more efficient than outside, leading to a much wider spread of transmitter after non-synaptic release as compared to synaptic re-

lease. Thus, a whole area (like the motoneuronal cell groups) may be “flooded” with transmitter after its non-synaptic release. In the spinal cord non-synaptic release may be mimicked by intrathecal administration of transmitter or application to the bath in case of in-vitro brain stem spinal cord preparations. Also in these cases the spinal cord is flooded with transmitter, producing very specific rhythmic effects (see later). It may be concluded that the transmitters in the descending projections from the ventro-medial medulla to spinal motoneurons can be subdivided in three groups (Fig. 6): the inhibitory transmitters GABA and glycine (represented mainly by the Ftype terminals), the excitatory transmitter glutamate (represented mainly by the S-type terminals) and the modulatory transmitter serotonin colocalized with several peptides (represented by the Gtype terminals). In addition, there may be extensive colocalization of serotonin with glutamate and, to a limited extent, with GABA.

Glutamate?

i TRH/Subst Serotonin i GABA P i Glycine I

; Glutamate? : VENTRO-MEDIAL MEDULLA

I

Spinal motor system

I

Fig. 6 . Schematic drawing showing the transmitters present in the different neurons of the ventro-medial medulla, their main afferent projections, their descending projections to the spinal motor system and the terminal types involved in these projections.

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Behavioral context of the ventro-medial medulla The multitude of transmitters in the spinal projections from the ventro-medial medulla suggests a very complicated and diverse control over sensory and motor processing in the spinal cord. This makes it difficult to assess the function of the ventro-medial medulla. However, there are several aspects of behavior in which the ventro-medial medulla was found to play an important role. With respect to motor behavior, there are two phenomena which have been extensively investigated: locomotion as elicited in the mesencephalic locomotor region and muscle atonia during rapid eye movement (REM) sleep. The ventro-medial medulla plays a key role in both these opposing aspects of motor behavior. Therefore the function of the ventro-medial medulla is considered in detail with respect to these two types of motor behavior. In addition the functional role of the ventro-medial medulla in relation with the peri-aqueductal gray (PAG) is discussed. Locomotion Electrical (Shik et al., 1966; Skinner and GarciaRill, 1984) and chemical (Garcia-Rill et al., 1990) stimulation in the mesencephelic locomotor region (MLR), an area in the posterior midbrain tegmentum at the level of the inferior colliculus, elicits locomotion on a treadmill in decerebrate cats and rats. A characteristic feature of the MLR is that increased stimulation in that area will speed up locomotion from stepping via trot to gallop. The locomotion effects of MLR stimulation are mediated by neurons in the ventro-medial medulla since anterograde tracing studies have shown direct connections between the MLR and the ventro-medial medulla (Garcia Rill et al., 1983; Steeves and Jordan, 1984) which were confirmed electrophysiologically (Garcia-Rill and Skinner, 1987b), while direct projections to the spinal cord were virtually absent (Steeves and Jordan, 1984; Garcia-Rill and Skinner, 1987b). Furthermore, electrical and chemical stimulation within the ventro-medial me-

dulla (Garcia-Rill and Skinner, 1987a; Atsuta et al., 1990; Kinjo et al., 1990) have also elicited locomotor behavior, and cooling of ventro-medial medulla prevented locomotion through MLR stimulation (Shefchyk et al., 1984; Noga et al., 1991). Locomotion can also be evoked from an area located laterally in pons and medulla (Mori, 1987), the ponto-medullary locomotor strip, but these effects also appeared to be mediated by cells in the ventro-medial medulla (Noga et al., 1991). Taken together, the various findings indicate that the ventro-medial medulla acts as an essential relay for the effects of the MLR on the spinal cord. Rhythmic movements resembling locomotion can also be generated in the spinal cord when it is isolated from the rest of the brain. The neuronal networks, responsible for these rhythmic movement have been termed central pattern generators (Grillner, 1985). Fictive locomotion, i.e. locomotor-like activity in motoneurons and their axons in pharmacologically paralyzed animals, has been used to assess the effects of various substances on the central pattern generator. Following the pioneering work in invertebrates (Grillner and Dubuc, 1988), experiments with the in vitro preparation of the isolated spinal cord or the brain stem-spinal cord preparation of neonatal rats have shown similar results: glutamate receptor agonists, when bath-applied to the isolated spinal cord, evoked fictive locomotion (Kudo and Yamada, 1987; Smith et al., 1988; Cazalets et al., 1992). Bath application of other substances like noradrenalin (Cazalets et al., 1990), serotonin (Cazalets et al., 1992), dopamine or acetyl-choline (Smith et al., 1988) also could elicit fictive locomotion, but these effects may ultimately be dependent on the activation of NMDA receptors (Smith et al., 1988). This is in line with the finding (Steeves et al., 1980) that depletion of serotonin and/or noradrenalin in the spinal cord did not abolish MLRinduced locomotion in decerebrate cats. Combined administration of serotonin and NMDA induced locomotor-like rhythmic activity, which showed a much more stable and reproducible motor pattern than with either NMDA or serotonin alone (Sqalli-

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Houssaini et al., 1993). In addition it was found that the period of the rhythmic movement could be set by combining the neurotransmitters in different concentrations. The effects of glutamate receptor agonists were also tested on the spinal cord of adult decerebrate cats (Douglas et al., 1993). In this preparation, fictive locomotion induced by electrical stimulation of the MLR was blocked by intrathecal administration of NMDA and nonNMDA receptor antagonists, while NMDA receptor agonists induced locomotion in resting animals. Most studies have concentrated on the excitatory (glutamate) and modulatory (monoaminergic) transmitters involved in fictive locomotion and much less on the effects of inhibitory transmitters, like GABA and glycine. In lower vertebrates it seems likely that glycine is involved in mediating reciprocal inhibition between the left and right side (Cohen and Harris-Warrick, 1984), while GABA, and GABAB receptor antagonists had little effect on individual segments and appeared to be involved mainly in intersegmental coordination (Grillner et al., 1991). Recently, in an isolated rat neonatal spinal cord brain stem preparation, NMDA induced locomotor activity was found to be slowed down or stopped altogether with simultaneously applied GABA in a dose dependent manner (Cazalets et al., 1994). In the same study it was found that GABAAand GABABreceptor antagonists had differential effects on the locomotion rhythm, depending on the concentrations of the GABA antagonists and the glutamate receptor agonists. Interestingly, NMDA bath-applied to the brain stem alone could not evoke locomotion without the presence of GABA receptor antagonists around the isolated lumbar cord, possibly indicating descending GABAergic activity. It was concluded from these findings that the balance between excitatory (glutamate) and inhibitory (GABA) influences determined the final motor output. This conclusion fits very well with the presence of GABA and (presumably) glutamate in the descending projections from the ventro-medial medulla to the spinal cord.

Muscle atonia during REM sleep

During the REM sleep phase there is a complete suppression of muscle tone (atonia), which is the result of an active inhibition of motoneurons, under direct control of the brain stem, rather than of a withdrawal of excitatory input (Chase and Morales, 1990). Cholinergic stimulation with carbachol in the dorsal pontine tegmentum has been shown to produce muscle atonia and lesions in this area will lead to REiM sleep without atonia (Jones, 1991). The dorsal pontine tegmentum projects to an area in the ventral medulla that is essential for triggering muscle atonia. Chemical lesions (which only compromise neurons and not fibers of passage), placed in the cat ventro-medial medulla and more dorsal areas (Schenkel and Siegel, 1989) led to REM sleep without atonia and chemical stimulation in the ventro-medial medulla triggered muscle atonia (Lai and Siegel, 1988). The latter study showed that two cell groups exists that give rise to motoneuron inhibition: a rostra1 area, corresponding the caudal magnocellular nucleus, which is sensitive to glutamate and a more caudal region located directly over the inferior olive and in the midline, corresponding to the nucleus paramedianus, which is sensitive to acetylcholine. These areas are located ventrally in the medulla and much less in the gigantocellular nucleus, which is located more dorsally. This localization in the ventro-medial medulla fits very well with a recent study in cat on C-fos activity related to REM sleep (Yamuy et al., 1993), which showed that in the ventral medulla many cells expressed C-fos activity especially in the raphe magnus area and cells located directly over the inferior olive. Intracellular recordings from lumbar motoneurons in intact unanesthetized cats during the different phases of sleep and wakefulness (Morales et al., 1987) have shown that the active inhibition of motoneurons during REM sleep is achieved by large amplitude inhibitory postsynaptic potentials which are only present during REiM sleep. In addition small inhibitory potentials are present, but

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these are also present during other sleep states and waking (Morales et al., 1987). The large inhibitory potentials are generated by glycine, since they were antagonized by strychnine, both after medullary stimulation (Soja et al., 1987) and during spontaneous occurrence in REM sleep (L6pezRodriguez et al., 1990; Soja et al., 1991), while with GABA antagonists no effects were seen (Soja et al., 1987), implying that GABA is not involved in the inhibition of motoneurons during REM sleep. In addition to the overwhelming inhibition of motoneurons, there is also an excitatory input to motoneurons during REM sleep, which occasionally breaks through the inhibition, leading to muscle twitches, including the characteristic rapid eye movements. Apparently the transmitter in these excitatory inputs to motoneurons is glutamate, acting primarily through non-NMDA receptors (Chase and Morales, 1990). Thus inhibition and excitation are both present during REM sleep, hence the name paradoxical sleep that is also used for this sleep state. The above data fit very well with a study in narcoleptic dogs (Siege1 et al., 1991). Dogs with this disorder (which has a homologue in humans) show episodes of cataplexy, an abrupt loss of muscle tone during waking. This phenomenon, often triggered by a strong emotion, is thought to result from a sudden activation during waking of cells normally responsible for muscle atonia during REM sleep. Recordings from the ventro-medial medulla in these dogs during REM sleep and during cataplexic attacks showed that the cells that were active during REM sleep could be subdivided in two groups: those that were also active during cataplexy and those that were not. It seems likely that the cells that were active during both episodes are responsible for normal muscle atonia during REM sleep and abnormal atonia during waking, while the cells active during REM sleep but not during cataplexy were related to the excitatory input to motoneurons that normally occurs during REM sleep. Also in this case, the cells presumed to be responsible for the muscle atonia during REM sleep and cataplexy were located in the ventro-medial medulla rather

than in the gigantocellular nucleus. These data suggest the existence of descending pathway from the ventro-medial medulla to spinal motoneurons, which is active during REM sleep. The direct glycinergic projection from the ventro-medial medulla to spinal motoneurons would fit in this scheme. Based on the results of their physiological experiments it has been proposed by Takakusaki et al. (1989, 1994), that reticulospinal neurons in the gigantocellular nucleus exert a general inhibition of spinal motoneurons through a disynaptic pathway. In this situation the inhibition of motoneurons is effectuated by local inhibitory interneurons. However, the location of the cells in the gigantocellular nucleus does not correspond with data of other authors (see above) and the presumed inhibitory interneurons have, so far, not been identified. Local interneurons like the Renshaw cell and, by inference, the Ia or Ib inhibitory interneurons (Morales et al., 1988) are not the source of this inhibition. In fact, it was found that Renshaw cells were actively inhibited during REM sleep (Morales et al., 1988), as were the neurons giving rise to ascending projections from the spinal cord to brain stem and thalamus (Soja et al., 1993). This suggests that there may be a generalized inhibition in the spinal cord, probably including the inhibitory interneurons. Such a generalized inhibition may be achieved most effectively by a direct supraspinal inhibition, i.e. the glycinergic projection from the ventro-medial medulla.

PAG and limbic afferents Apart from the afferents from the MLR and surrounding areas, involved in locomotion, and those from the dorsal pons, involved in muscle atonia during REM sleep, there is a substantial input to the ventro-medial medulla from the limbic system, including the caudal part of the medial hypothalamus, the bed nucleus of the stria terminalis and the central nucleus of the amygdala (see Holstege, G. (1991) for review). There are also strong projections from the peri-aquaductal gray (PAG), which are especially interesting, since they mediate very

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different types of behavior (see Bandler and Shipley (1994) for review). When chemically stimulated in a freely moving cat, the caudal part of the lateral PAC evoked a flight reaction, including rapid locomotion and jumping. In contrast, stimulation of the adjacent ventro-lateral area of the PAC evoked suppression of movement, i.e. immobolization and relaxation, like after defeat or when wounded. Both areas of the PAC strongly project to both serotonergic and non-serotonergic spinal projecting neurons in the ventro-medial medulla (Lakos and Basbaum, 1988). Since the terminals of these projections contained rounded vesicles (Lakos and Basbaum, 1988), and did not contain GABA (Reichling and Basbaum, 1990), it seems likely that they are excitatory in nature. Thus two adjacent areas in the PAC with opposite effects on behavior both project to the ventromedial medulla probably using an excitatory transmitter.

Synthesis The ventro-medial medullary projections to the spinal cord are very heterogeneous in many respects: they terminate in virtually all areas of the gray matter, they contain a vast number of transmitters, they receive afferents from many different areas and are involved in many different types of behavior. In fact one of the characteristics of the ventro-medial medulla is that opposing elements seem to come together in this area, e.g. glutamate and glycine, locomotion and muscle atonia, flight and quiescent behavior. Although somewhat confusing, these phenomena may all be considered part of a continuum ranging from strong inhibition to strong activation of motor behavior. Recordings from serotonergic (and possibly glutamatergic) cells in the ventro-medial medulla (especially the raphe magnus) of behaving cats showed that the activity of these cells is strongly correlated with the state of arousal of the animal, such that the discharge rate of these cells is highest during active waking and lowest or absent during REM sleep (Jacobs and Azmitia, 1992). Electrical stimulation in the midline of the ventral tegmental

field of the caudal pons in freely moving animals, probably activating the rostra1 ventro-medial medulla, resulted in non-goal-directed locomotion, similar to that evoked by MLR stimulation in a decerebrate cat, while stimulation of the MLR led to fast walking and running (Mori et al., 1989). Thus stimulation of the midline structures, where the serotonergic and presumed glutamatergic cells are especially concentrated, results in activation of the motor system, while these cells become increasingly less active when proceeding from active waking to quiet waking and drowsiness and they even become almost silent during REM sleep. It may be hypothesized that the ventro-medial medulla can effectuate the activation of the spinal motor system by the excitatory component of their projections to the spinal ventral horn. These projections may release glutamate together with serotonin from G- and S-type terminals. If the release from G-type terminals occurs non-synaptically it would produce a rise in the concentration of glutamate and serotonin in the extracellular space. This would not give rise to a bizarre and unpredictable effect on the spinal motor system, since various studies (see earlier) have shown that a rhythmical behavior will be induced, strongly resembling locomotion. This means that non goaldirected locomotion can be accomplished merely by inducing a rise in extracellular glutamate and serotonin concentration. At the same time the motor pattern can be adjusted or modified by more precisely organized inputs, mainly to interneurons, originating in the vestibular nuclei, the dorsal medullary and the pontine tegmentum, the red nucleus, the cortex and other areas involved in steering movements as part of a goal-oriented motor behavior. General excitation of the spinal motor system, leading to locomotion and allowing goal-oriented movements to occur, may be halted or suppressed by a general GABAergic input to the spinal motor system. This was shown in in-vitro experiments with fictive locomotion (see earlier) but is also found in the serotonin or glutamate induced bistable behavior of motoneurons, i.e. the phenomenon that a cell can be switched to a higher level of

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excitability (Kiehn, 1991). This intrinsic property of motoneurons has a strong effect on the efficacy on the different synaptic inputs onto motoneurons. Bistable behavior can be turned off by small inhibitory potentials like those produced by GABA. Thus the transmitters that turn on or turn off the bistable behavior of motoneurons are both present in the descending projections from the ventromedial medulla. A powerful and unique type of inhibition of motoneurons is present during REM sleep. This type of inhibition, which produces complete muscle atonia, is mediated not by GABA but by glycine. The ventro-medial medulla is strongly implicated in muscle atonia and glycine is present in some of its descending projections. In this respect it is interesting to note that both activation (locomotion) and inhibition (muscle atonia) can be produced from the same area in the midbrain and rostral pontine reticular formation (Lai and Siegel, 1990), indicating that in those areas, as in the ventro-medial medulla, excitation and inhibition of the motor system are closely linked. Taken together, it may be concluded that the ventro-medial medulla is an essential relay for the production of very different types of behavior, in a continuum ranging from muscle atonia, via quiescent immobilization to locomotion and running. It seems likely that the balance between the GABA and glycine output on the one hand and the glutamate and serotonin output on the other hand, will determine the level of activation of the spinal motor system. In order to obtain the appropriate balance between the inhibitory and excitatory output of the ventro-medial medulla, the various afferent projections must differentially activate these output systems. How this is achieved is still unknown and will be an important goal for future research.

Acknowledgements The author would like to thank Dr. J. Voogd for reading the manuscript and Mr. E. Dalm for his help with the photography. Supported by Grant 900-550-072 from the Netherlands Research Organization (NWO).

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