Brainstem control of locomotion and muscle tone with special reference to the role of the mesopontine tegmentum and medullary reticulospinal systems

Effects of the mesopontine stimulation upon soleus EMGs, which are shown in Fig. 3B, well resembled to those shown in Fig. 1A. Stimulation of the dorsal CNF bilaterally increased the level of muscle tone (top traces in Fig. 3B), and that of the ventral CNF evoked locomotion (second traces). A mixture of locomotion and muscle tone suppression was evoked from the dorsal PPN (third traces). Stimuli applied to the PPN and to the ventral PPN resulted in muscular atonia (fourth and fifth traces) and hypotonia (bottom traces), respectively. The identical stimuli applied to the medial PRF, which corresponds to the nucleus reticularis pontis oralis (NRPo), also evoked muscular atonia (bottom traces in Fig. 1B). Totally effects from 49 sites on the coronal plane of the mesopontine tegmentum were examined in this animal (Fig. 1Da). Locomotory activities were evoked from the medial and ventral part of the CNF (n = 3, blue circles; locomotion-evoking sites). Stimuli of the dorsal and lateral parts of the CNF in addition to the locus coeruleus (LC) increased the level of muscle tone (n = 4, violet circles; hypertonus- evoking sites). In contrast, muscular atonia (n = 9, red circles; atonia-evoking sites) and hypotonia (n = 4, orange circles; hypotonus evoking sites) were induced by stimuli applied to the PPN and NRPo. A mixture of muscle tone suppression and locomotor rhythm was evoked by stimuli applied to the dorsal part of the PPN (n = 2, green circles).

Effects of the mesopontine stimuli were markedly altered by injections of atropine sulfate into the left and right NRPo (downward arrows in Fig. 1A). First, the CNF-induced locomotor activities were facilitated; the cycle time of the locomotion was reduced from 1.0 s (control) to 0.7 s after atropine administration (second traces in Fig. 1C). In addition, stimulation of the dorsal PPN elicited locomotion (third traces). Stimuli applied to the dorsal CNF (top traces) and to the PPN (fourth traces) also generated rhythmic activities. Second, suppressive effects from the PPN and NRPo were greatly attenuated; the latency to muscular atonia was increased (fifth and bottom traces). Stimulation of the ventral PPN even evoked movements (sixth traces). Consequently, as shown in Fig. 3Dc, pontine atropine injections increased the numbers of locomotion-evoking sites (n = 8) and hypertonus-evoking sites (n = 6). Instead, a number of atonia-evoking sites (n = 1) was reduced. Based on the above findings, following suggestions can be made. First, the cholinergic PPN-PRF projection is involved in the regulation of postural muscle tone and locomotor rhythm. Second, functional topographical organization may exist in the mesopontine tegmentum with respect to the regulation of postural muscle tone and locomotion. Third, the functional organization is altered depending on the activity of the cholinergic PPN-PRF projection.

Locomotor rhythm is generated by central pattern generator (CPG), which is composed of interneuronal networks in the spinal cord (Grillner 1981). The activity of the CPG is observed as rhythmic membrane oscillations in motoneurons during locomotion (Jordan et al. 2008; Shefchyk and Jordan 1985). To examine the supraspinal control of locomotor rhythm in motoneurons, the decerebrate cat was immobilized to remove the influence of sensory afferents. Findings are shown in Fig. 4. In a lateral gastrocnemius-soleus (LG-S) motoneuron, membrane potential was depolarized during the period of stimulation (top trace in Fig. 4B) applied to the dorsal CNF (filled circle in Fig. 4A). Stimulation of the ventral CNF (blue circle) first depolarized the membrane and then produced a sequence of rhythmic membrane oscillations with a cycle time of 1.1–1.4 s (second trace in Fig. 4B). Action potentials were generated on the depolarizing phase of the oscillations. Stimulation of the dorsal PPN (green circle in Fig. 4A) induced membrane hyperpolarization, which was associated with rhythmic firings of the motoneuron with intervals between 0.3 and 0.5 s (third trace in Fig. 4B). Even after termination of the firing, membrane oscillations remained (inset in Fig. 4Ba). The oscillations had a cycle time of appropriately 0.5 s. Stimulation of the ventral part of the PPN (red circle in Fig. 4A) stopped firing and hyperpolarized the membrane (bottom trace in Fig. 4B). These changes in the excitability of the motoneuron well reflect the changes in soleus EMGs shown in Fig. 1B. Following changes were observed after atropine injection into the medial PRF (downward arrow in Fig. 4A). First, stimulation of the dorsal and ventral CNF depolarized the membrane with faster time course (arrows in the first and second traces in Fig. 4C) compared to the control (open arrows in Fig. 4B). Second, locomotor activities were facilitated; the CNF-induced locomotion had oscillations with cycle times of 0.8–0.9 s (second trace in Fig. 4C). In addition, stimulation of the dorsal PPN continuously generated locomotor activity which was accompanied by rhythmic firing of the motoneuron (third traces in Fig. 4C). Third, the inhibitory effect evoked from the PPN was attenuated; stimulation of neither the dorsal nor ventral PPN blocked generation of action potentials (third and bottom traces in Fig. 4C).

Single pulse stimulation was applied to the mesopontine tegmentum (Fig. 5Ab), and PSPs evoked from each site were recorded (Fig. 5Aa). Mesopontine stimulation usually evoked a mixture of excitatory (EPSPs) and inhibitory postsynaptic potentials (IPSPs) with various latencies in hindlimb motoneurons. Stimulation of the dorsal CNF evoked EPSPs with a peak latency of around 25 ms (middle EPSP). Stimulation of the ventral CNF evoked a mixture of EPSP and IPSP within 20 ms (early-PSPs), which was followed by the middle EPSP. Stimulation of the mid and ventral part of the PPN evoked a sequence of EPSP and IPSP in the early latency, and they were followed by large IPSPs with a peak latency of 45 ms (late-IPSP). Stimulation of the dorsal PPN evoked a mixture of EPSPs and IPSPs whose time course was similar to those evoked from both the ventral CNF and PPN. The amplitude of the middle EPSP was the largest when the dorsal CNF was stimulated, and it was gradually reduced by stimuli applied to the ventral sites. Conversely, the late-IPSP was the most prominent when mid PPN was stimulated, and the amplitude was reduced by stimuli applied to either the dorsal or ventral site. Accordingly, neuronal mechanisms involved in the middle-EPSP and late-IPSP may contribute to the increase and decrease in the level of postural muscle tone, respectively. On the other hand, a mixture of the early-EPSP and IPSP was effectively evoked by stimuli applied to the ventral CNF and dorsal PPN (third and fourth traces). These sites well correspond to locomotion evoking area. The early-PSPs are superimposed in Fig. 5Ac with an expanded time scale; the minimum latency of the early-EPSPs was 6.8 ms (blue and green traces), and that of the IPSP (red trace) was approximately 1 ms later than the EPSPs. Therefore, the early-PSPs are possibly mediated by fast-conducting glutamatergic RSNs and spinal interneurons that are involved in the generation of locomotor rhythm (Jordan et al. 2008; Shefchyk and Jordan 1985).

Examinations were further made to elucidate whether cholinergic PPN neurons were involved in the generation of these PSPs. Triple pulses of stimuli applied to the PPN (red circle in Fig. 4Ab) evoked early-IPSPs which were followed by the middle-EPSP (asterisk in Fig. 5Ba) and late-IPSP (Fig. 4Ba). An injection of atropine into the PRF (filled arrow in Fig. 5Ab) greatly reduced the amplitude of the early- and late-IPSPs. On the other hand, the early-EPSP became evident (open arrow Fig. 4Bb). However, the middle-EPSP (asterisk in Fig. 5Bb) was unaffected. The difference of these PSPs (Fig. 5Bc) between before and after atropine injection reveals that the cholinergic PPN-PRF projection is involved in evoking both the early- and late-IPSPs. On the other hand, the early- and middle-EPSPs may be ascribed to the activation of non-cholinergic neurons in the mesopontine tegmentum.

Consequently findings in Figs. 3, 4 and 5 lead us following suggestions. (1) Cholinergic neurons in the PPN are involved in muscle tone suppression. (2) Non-cholinergic neurons in the dorsal CNF are involved in muscle tone augmentation. (3) Both the cholinergic and non-cholinergic neurons in the mesopontine tegmentum may be required to generate the locomotor rhythm.

The PPN is located in the ventrolateral portion of the caudal mesencephalic reticular formation (Olszewski and Baxter 1954), and is composed of a heterogeneous population of neurons containing gamma-amino-butyric acid (GABA), and glutamate in addition to acetylcholine (ACh) (Clements and Grant 1990; Ford et al. 1995; Lavoie and Parent 1994; Mena-Segovia et al. 2008, 2009; Saitoh et al. 2003; Takakusaki et al. 1996). Cells in the PPN are also characterized by other neuronal markers, including calcium-binding proteins and neuropeptides (Fortin and Parent 1999; Vincent 2000; Vincent et al. 1983). The cholinergic neurons serve to delineate PPN boundaries, identifying a pars compacta and a pars dissipata (Mesulam et al. 1989). The CNF lies dorsal to the PPN and ventral to the superior and inferior colliculi. However, it is often difficult to clearly distinguish the boundary of these nuclei.

Garcia-Rill et al. (1987, 2011) refined the anatomical boundary of the MLR in rats to a restricted region of the mesopontine tegmentum including the PPN, and they used markers of cholinergic neurons to convincingly demonstrate that locomotion was induced by the activation of cholinergic neurons within the PPN (Skinner et al. 1990a, b, c). Similarly, Mogenson et al. (Brudzynski and Mogenson 1985; Milner and Mogenson 1988; Brudzynski et al. 1993) ascribed the effects of electrical stimulation, drug injection and lesions to the actions of the PPN in evoking locomotion in rats. Since then, cholinergic PPN neurons have been widely considered a key element of the MLR. However, the same procedures in the cat and rat also produced a numbers of results showing that the effective sites were mainly located in and around the CNF including a vicinity in the PPN (Amemiya and Yamaguchi 1984; Brudzynski et al. 1986; Coles et al. 1989; Depoortere et al. 1990; Mori et al. 1989; Shik et al. 1966; Shik and Orlovsky 1976; Sterman and Fairchild 1966; Takakusaki et al. 2003a). In addition, the activity-dependent expression of c-Fos following treadmill locomotion rats was not detectable in the PPN but in the CNF (Jordan 1998). Similarly, 2-deoxyglucose labeling revealed an increased activity only in the CNF following MLR-evoked locomotion in cats (Shimamura et al. 1987). A recent study by Gut and Winn (2015) shows that complete lesions of PPN did not cause any gait deficits in the rat, throwing doubt on question as to the status of PPN as a motor control structure. Paradoxically, the PPN is labeled with c-Fos during rapid REM (Shiromani et al. 1992, 1995, 1996), indicating that the PPN rather contributes to the generation of REM sleep (Datta 2002; Ford et al. 1995; Jones 1991, 2005; Koyama and Sakai 2000; Lai et al. 1993; Mitani et al. 1988; Semba 1993).

Our findings using decerebrate cat preparation revealed that optimal sites for evoking locomotion, i.e., MLR, are mostly located in the ventral part of the CNF and the vicinity in the dorsal part of the PPN (Fig. 2; Takakusaki et al. 2003a). Moreover, neurons in the dorsal part of the CNF and the ventral of the PPN may contribute to the increase and decrease in the level of postural muscle tone, respectively. Therefore, the MLR is, functionally, surrounded by the areas involved in the augmentation and suppression of postural muscle tone. The atonia induction zone well corresponds to PPN pars compacta where abundant cholinergic neurons are located (Fig. 2). Importantly, such a functional organization of the mesopontine tegmentum may be altered depending on excitability of the cholinergic neurons in the PPN and cholinoceptive neurons in the medial PRF. If the excitability of these neurons is higher, the excitation of the mesencephalic neurons may reduce muscle tone, whereas it may increase the level of muscle tone and/or elicit locomotion when excitability of these neurons is lower.

It is of worth to note that the effects by stimulating the MLR can be separated by changing parameters of electrical stimuli. Based on the findings in Fig. 1, neurons that are involved in the locomotor rhythm may have a threshold higher than those which contribute to the augmentation of muscle tone (Fig. 1Ac). Additionally, they may optimally respond to the stimulation with a frequency of around 50 Hz (between 20 and 100 Hz, Fig. 1Ad). In other words, neurons responsible for muscle tone suppression may respond to the stimuli with wide frequency ranges. Accordingly, the MLR may be composed of functionally different groups of neurons; neurons which contribute to the control of muscle tone and those to the generation of locomotor rhythm are intermingled.

A large body of findings in quadruped animals indicates that cholinergic neurons in the PPN may not be principal neural elements of the MLR. Nonetheless, our studies suggest that cholinergic PPN neurons may play a crucial role in the control of posture and locomotion. Specifically, cholinergic neurons are critically involved in the suppression of muscle tone in addition to the modulation of the locomotor rhythm (Figs. 3, 4, 5, Takakusaki et al. 2003a, 2004a, 2011). We propose that the muscle tone inhibitory system arising from cholinergic neurons in the PPN via the cholinoceptive PRF neurons and the medullary RSNs regulates the excitability of spinal cord interneurons that comprise CPG as well as that of motoneurons. Therefore, the locomotor rhythm and postural muscle tone can be simultaneously modulated (see section “The inhibitory system”, Fig. 15).

A considerable population of cholinergic PPN neurons contains glutamate (Lavoie and Parent 1994). Then, what is the possible role of the glutamatergic neurons? Jordan et al. (2008) suggest that non-cholinergic neurons in the MLR area elicit locomotion via fast-conducting RSNs in the medial MRF. Findings in this article also suggest that non-cholinergic neurons contribute to both the regulation of locomotor rhythm and the level of postural muscle tone. This suggestion is based on following observations; pontine atropine injections facilitated locomotory activities evoked from the ventral CNF and enhanced excitatory effects from the dorsal CNF despite of attenuation of the PPN-induced inhibitory effects (Figs. 3, 4). Similarly, EPSP components with the early and middle latencies were not attenuated by the pontine atropine injection (Fig. 5). The former and the latter may contribute to the generation of locomotor rhythm and muscle tone augmentation, respectively (see section “Changes in postsynaptic potentials evoked by mesencephalic stimulation”).

The PPN also has descending projections to the medioventral part of the MRF (Nakamura et al. 1989) and spinal cord (Skinner et al. 1990a, b, c; Spann and Grofova 1989). There is a suggestion that both cholinergic and non-cholinergic (glutamate and substance P) projections to the medioventral MRF are likely involved in the initiation of locomotion (Kinjo et al. 1990; Skiner et al. 1990a, b, c). It should be noted that substance P containing neurons in the mesopontine tegmentum are severely damaged in patients of PD (Gai et al. 1991). PPN neurons with projections to the spinal cord are possibly non-cholinergic in nature (Skinner et al. 1990a, b, c). Role of the spinal projecting neurons has not been examined. A recent study using in rats by Sherman et al. (2015) showed that glutamatergic RSNs just medial to the PPN contributed to the locomotor behaviors.

When carbachol (a long-acting cholinergic agonist resistant to choline-esterases) was injected into the right NRPo, postural muscle tone was bilaterally reduced (Fig. 10Ba). Muscle tone was restored by an injection of atropine sulfate into the same site and pinna stimulation (i.e., pinching the scapha indicated by filled triangles; Fig. 10Bb). The carbachol-induced atonia usually lasted for more than 1 h if atropine was not administered. In a different animal, serotonin was injected into the inhibitory site in the NRPo. With this, bilateral contractions of the soleus muscles were increased (Fig. 10Ca). However, the effects were completely suppressed following a subsequent injection of carbachol into the same site (Fig. 10Cb). The carbachol-induced atonia was associated with reduced excitability of the soleus motoneuron (Fig. 10Da). Spontaneous firing of the motoneuron ceased, and the membrane potential was hyperpolarized. The reduced excitability of motoneurons is attributed to both postsynaptic inhibition (Chase and Morales 1990; Takakusaki et al. 1993a) and withdrawal of excitatory inputs impinging on the motoneurons (dis-facilitation; Takakusaki et al. 1993a). Subsequent serotonin injection started to depolarize the membrane and generated action potentials (Fig. 10Db). Several minutes after the injection, firing of the motoneuron was re-established. These changes in the motoneuron were associated with contractions of the soleus muscles. Consequently, the excitability of cholinoceptive and/or serotonin receptive neurons in the medial PRF, specifically the NRPo, is critically involved in the control of postural muscle tone (Takakusaki et al. 1993a, 1994).

Changes in the firing rates of the medullary RSNs were examined during chemically induced alteration of muscle tone. RSNs in Fig. 11A, B were recorded from the NRGc. They received excitatory input from the NRPo (Fig. 11Aa, Ba) and projected to the lumbar segments (Fig. 11Ab, Bb). Following carbachol injection, the firing frequency of both RSNs was increased in accordance with reduction in muscle tone (Fig. 11Ac, Bc). The RSN in Fig. 11A had a firing frequency of more than 20 Hz and muscular atonia was still evident 40 min after carbachol injection. The firing frequency was reduced by atropine injection; however, the effect was only transient and the firing rebounded after the atropine injection. Approximately 20 min after the first injection, the second higher dose atropine injection reduced the firing frequency of the RSN and partially restored muscle tone. The firing frequency of the RSN in Fig. 11B was greater than 40 Hz 20 min after carbachol injection. In this cat, a subsequent serotonin injection steadily reduced the RSN’s firing frequency. At about 10 min, the firing frequency was less than 10 Hz and the tonic muscle contractions resumed. The RSN in Fig. 11C was recorded from the NRMc. Spontaneous firing of this RSN was suppressed by stimulating the NRPo (Fig. 11Ca), indicating the inhibitory input from the NRPo. Carbachol injection reduced the firing of this RSN as well as muscle tone (Fig. 11Cc). However a subsequent serotonin injection restored the firing frequency of the RSN and the level of muscle tone.

During the states of hypertonus (before carbachol injection) and carbachol-atonia, the firing frequency, conduction velocity (CV) and the location of medullary RSNs were investigated (Fig. 12).

Here we define RSNs which had firing frequency greater than 10 Hz during hypertonus state and atonia state as hypertonus-related RSNs (n = 76 of 162 RSNs) and atonia-related RSNs (n = 75 of 130 RSNs), respectively. Hypertonus-related RSNs were located in the ventromedial part of the MRF (blue circles on the left in Fig. 12Aa), whereas atonia-related RSNs were located in the dorsomedial part of the MRF (red circles on the left in Fig. 12Ba). The hypertonus-related RSNs had a CV (80.2 ± 11.6 m/s, mean ± standard deviation; SD, Fig. 12Ab), which was slower than that of the atonia related RSNs (90.3 ± 12.7 m/s, Fig. 12Bb). On the other hand, 86 RSNs were inactive in hypertonus state. These RSNs were mostly distributed in the dorsomedial part of the MRF (denoted by dots on the right in Fig. 12Aa), and had a CV of 92.0 ± 12.2 m/s (Fig. 12Ab). Moreover, inactive RSNs (n = 55) during atonia state were located in the ventromedial part of the MRF (Fig. 12Ba). They had a CV of 76.8 ± 16.2 m/s.

The firing properties of medullary RSNs have been well characterized during locomotion in acute decerebrate cats (Iwakiri et al. 1995; Orlovsky 1972; Perreault et al. 1993) as well as in chronic alert cats (Drew et al. 1986; Matsuyama and Drew 2000a, b; Schepens et al. 2008). However, the role of medullary RSNs in the integrative control of postural muscle tone and locomotion is unclear. To understand this issue, the firing properties of presumed muscle tone-related medullary RSNs were examined during locomotion. Representative results are shown in Fig. 13, where two RSNs were recorded in high decerebrate (subthalamic) cats. The RSN in Fig. 13A was recorded from the ventromedial MRF (NRMc; Fig. 13Aa). During standing this neuron exhibited spontaneous firing (Fig. 13Ab). This spontaneous firing was inhibited by stimulating the NRPo and the PPN (Fig. 13Ad, Ae), indicating that this NRMc-RSN relates to hypertonus. During locomotion, this RSN exhibited rhythmic firing in relation to the step cycles (Fig. 13Af). However, stimulation of the PPN suppressed the firing of the RSN as well as locomotion (Fig. 13Af). On the other hand, the RSN in Fig. 13B was recorded from the dorsomedial MRF (NRGc; Fig. 13Ba). Because this cell did not exhibit spontaneous firing during standing (Fig. 13Bb), but was orthodromically excited by NRPo and PPN stimulation (Fig. 13Bd, Be), the NRGc-RSN was considered to be an atonia-related RSN. Although the RSN was silent (Fig. 13Bb), it began to discharge with a frequency between 10 and 20 Hz during locomotion (Fig. 13Bf). An activation of spinoreticular tract neurons (Antonino-Green et al. 2002; Huber et al. 1999) during locomotion may induce such a firing of this RSN. The firing frequency was further increased by PPN stimulation, which suppressed locomotion.

Iwakiri et al. (1995) classified medullary RSNs into four groups based on convergent inputs from the muscle tone inhibitory region in the PRF (NRPo) and the MLR. Of 250 RSNs, 126 neurons were excited by stimulating either the MLR (n = 25) or the NRPo (n = 67) or both (n = 34), indicating that half of the medullary RSNs contributed to the control of muscle tone and locomotion. Group 1 RSNs (n = 13) was excited following stimulation only from the MLR (13/59 = 22.0 %, Fig. 14Aa). Group 2 RSNs (n = 12) was excited following stimulation to the MLR and inhibited following stimulation to the NRPo (Fig. 14Ab). Group 3 RSNs (n = 34) was excited following stimulation to both the MLR and the NRPo (Fig. 14Ac). It should be noted that the majority (46/59 = 80.0 %) of MLR-activated RSNs received either inhibitory (Group 2 RSNs; 12/59 = 20.3 %) or excitatory (Group 3 RSNs; 34/59 = 57.7 %) inputs from the NRPo. Group 4 RSNs (n = 67) were excited following stimulation to the NRPo (Fig. 14Ad). We found that the conduction velocity was faster in RSNs that belonged to the Group 3 (94.3 ± 15.5 m/s) and Group 4 (95.2 ± 15.7 m/s) than for those in the Group 1 (85.6 ± 12.6 m/s) and Group 2 (85.4 ± 16.3 m/s; Fig. 12B).

We compared the locations of RSNs in relation to the control of postural muscle tone (Fig. 12) and locomotion (Fig. 14). In the Groups 1 and 2, the majority of the RSNs (18/25; 75 %) were distributed in the ventral part of the MRF corresponding to the NRMc (Fig. 14Aa, Ab). This distribution was similar to that reported by Garcia-Rill and Skinner (1987) who demonstrated that stimulation of the MLR mostly activated RSNs located in the ventromedial medulla. Moreover, the distribution is in agreement with the location of that of hypertonus-related RSNs (Fig. 12A) suggesting that these MLR-activated RSNs also contribute to increasing the level of muscle tone. The majority of RSNs in the Groups 3 and 4 (Fig. 14Ac, Ad) was located in the dorsomedial MRF corresponding to the NRGc, and their distribution was similar to that of the atonia-related RSNs (Fig. 12B). It is possible that the Group 3 RSNs (n = 34) was involved in muscle tone suppression and locomotor control. Surprisingly, only 13 RSNs (22.0 %) were excited following stimulation from the MLR without the influences of the NRPo (Group 1). In other words, considerable portion of the medullary RSNs may integrate supra-medullary signals related to the control of postural muscle tone and locomotion. Descending signals in these RSNs thus modulate the activity of the spinal locomotor network such that the level of muscle tone and locomotor patterns are simultaneously regulated.

A series of studies in our laboratory suggested that the muscle tone inhibitory system arises from cholinergic neurons in the PPN (Takakusaki et al. 1993a, 1994, 2001, 2003a, 2004a, c, 2005, 2011). Although the PPN is composed of a heterogeneous neuronal population, cholinergic neurons may activate cholinoceptive neurons in the medial PRF (NRPo), which in turn excite medullary RSNs located in the dorsomedial MRF (atonia-related RSNs; Fig. 12B). This inhibitory system inhibits α- and γ-motoneurons that innervate extensor and flexor muscles, in parallel with interneurons mediating reflex pathways via a group of spinal inhibitory interneurons in the Rexed lamina VII (Takakusaki et al. 2003b). Presynaptic inhibition of primary sensory afferents was also induced by this system (Chan and Barnes 1974; Takakusaki 2015). Accordingly, an activation of this system may inhibit whole neuronal constituents of spinal reflex loops including the CPG, resulting in the suppression of locomotion as well.

However, critical questions remain unanswered. First, the medullary-induced inhibitory effects are presumably mediated by neurons with a CV of 20–40 m/s (Habaguchi et al. 2002; Kohyama et al. 1998), which is much slower than that of atonia-related RSNs (Fig. 12Bb). The second question involves the lamina VII interneurons. Postsynaptic inhibition uses glycine and presynaptic inhibition uses GABA; therefore there is a need to determine whether the lamina VII interneurons contain both transmitters and contribute to both inhibitory processes. It should be noted that this muscle tone inhibitory system likely induces muscular atonia during REM sleep (Chase and Morales 1990). Similarly, cholinergic projection from the PPN to the paramedian MRF in the caudal medulla is proposed to induce atonia during REM sleep (Shiromani et al. 1990).

Muscle tone augmentation was induced by applying stimulation to the monoaminergic nuclei in the rostral pons such as the LC and RD, the RM in the caudal pons, and the raphe pallidus (RPa) in the medulla (Figs. 3, 6). These findings support a previous suggestion that descending monoaminergic pathways such as the coerulospinal and raphespinal tracts may comprise the muscle tone excitatory system (Fung and Barnes 1981; Holstege and Kuypers 1987; Sakai et al. 2000). Moreover, the reticulospinal tract descending from the ventromedial MRF may also belong to the muscle tone excitatory system (Figs. 6, 7, 8, 12). Because a population of reticulospinal fibers adjoining the raphe nuclei contains serotonin (Holstege and Kuypers 1987), the hypertonus-related medullary RSNs might use serotonin to increase the level of muscle tone. Consequently, serotonergic projection systems may play a vital role in modulating the level of muscle tone by direct projections to the spinal cord and indirect projections via the medullary reticulospinal tract. It is also possible serotonergic projections to the PRF and PPN may reduce the excitability of the inhibitory system (see also section “Properties of medullary RSNs in relation to locomotor control”, Fig. 15).

During muscle tone augmentation induced by pontine serotonin injection (Fig. 10), postsynaptic excitation and presynaptic facilitatory effects are induced in motoneurons (Takakusaki et al. 1993b). Such excitatory synaptic mechanisms may be induced by the activation of the ventromedial medullary RSNs (Fig. 12A). Alternatively these facilitatory effects are possibly attributed to the removal of inhibitory effects that are mediated by the atonia-related medullary RSNs (Fig. 12B).

Postural muscle tone is regulated by a counterbalance between the excitatory and inhibitory systems (Fig. 15). Such interactions may occur at the levels of the brainstem and spinal cord as follows. (1) Reciprocal connection exists between the cholinergic and monoaminergic nuclei. The mesopontine cholinergic neurons receive direct serotonergic projection from the DR and noradrenergic projection from the LC (Honda and Semba 1994). These monoaminergic projections may inhibit cholinergic neurons (Kobayashi et al. 2003; Leonard and Llinás 1994; Pal and Mallick 2006). Conversely, cholinergic input to DR inhibits serotonergic neurons by the mediation of GABA neurons (Yang and Brown 2014). (2) The medial PRF (NRPo) receives monoaminergic projections from the DR/LC (Semba 1993) and cholinergic projections from the PPN/LDT (Mitani et al. 1988; Lai et al. 1993). Therefore, the activity of each muscle tone control system is determined by the excitability of NRPo neurons in response to ACh and serotonin. (3) MRF neurons belonging to the inhibitory system may suppress the activity of LC neurons (Mileykovskiy et al. 2000). (4) Reciprocal changes in the muscle tone control systems are reflected by the release of neurotransmitters in the spinal cord. For example, Lai et al. (2010) demonstrated a combination of increased release of glycine and GABA and decreased release of serotonin and noradrenalin during atonia following stimulation of the medial MRF.

Previous studies suggested that signals from the MLR activate medullary RSNs which comprise the “locomotor system” that relays locomotor commands to the spinal locomotor network so that it generates an oscillatory pattern of locomotion (Grillner 1981; Armstrong 1986; Garcia-Rill and Skinner 1987; Rossignol 1996). Presumably, the RSNs in the locomotor system utilize glutamate to elicit the locomotor rhythm (Fenaux et al. 1991; Douglas et al. 1993; Hagevik and McClellan 1994; Jordan et al. 2008; Rossignol and Dubuc 1994). As shown in Figs. 12, 13, and 14, RSNs constituting the locomotor system may include a considerable population of the medullary RSNs which relate to the control of muscle tone, indicating that the level of muscle tone and the locomotor rhythm are simultaneously regulated by these RSNs (Fig. 15). MLR-induced locomotion is often preceded by the augmentation of postural muscle tone (Fig. 1), which is reflected by membrane depolarization of extensor motoneuron (Fig. 4). The muscle tone augmentation can also be ascribed to the recruitment of the monoaminergic muscle tone excitatory system, such as the coerulospinal and raphespinal tracts (Mori et al. 1992). The raphespinal tract is particularly important in the control of posture and locomotion (Jacob and Fornal 1993; Sławińska et al. 2012, 2014). Both the firing rates of serotonin neurons in the caudal RN (Veasey et al. 1995) and the serotonin levels in the spinal cord increase during locomotion (Gerin and Privat 1998). Xiang et al. (2013) used pseudorabies virus, which is a marker for synaptic connectivity in CNS by propagating retrogradely through chains of functionally connected neurons, and revealed that neurons in the area corresponding to the MLR were retrogradely infected via the medullary RSNs in mice. However, no serotonergic and catecholaminergic neurons were infected, indicating without the participation of the monoaminergic pathways in the MLR-induced locomotion.