The Mesencephalic Locomotor Region: Beyond Locomotor Control

Abstract

The mesencephalic locomotor region (MLR) was discovered several decades ago in the cat. It was functionally defined based on the ability of low threshold electrical stimuli within a region comprising the cuneiform and pedunculopontine nucleus to evoke locomotion. Since then, similar regions have been found in diverse vertebrate species, including the lamprey, skate, rodent, pig, monkey, and human. The MLR, while often viewed under the lens of locomotion, is involved in diverse processes involving the autonomic nervous system, respiratory system, and the state-dependent activation of motor systems. This review will discuss the pedunculopontine nucleus and cuneiform nucleus that comprises the MLR and examine their respective connectomes from both an anatomical and functional angle. From a functional perspective, the MLR primes the cardiovascular and respiratory systems before the locomotor activity occurs. Inputs from a variety of higher structures, and direct outputs to the monoaminergic nuclei, allow the MLR to be able to respond appropriately to state-dependent locomotion. These state-dependent effects are roughly divided into escape and exploratory behavior, and the MLR also can reinforce the selection of these locomotor behaviors through projections to adjacent structures such as the periaqueductal gray or to limbic and cortical regions. Findings from the rat, mouse, pig, and cat will be discussed to highlight similarities and differences among diverse species.

Full text

REVIEW
published: 09 May 2022
doi: 10.3389/fncir.2022.884785
Edited by:
Marie-Claude Perreault,
Emory University, United States
Reviewed by:
Clémentine Bosch-Bouju,
Institut Polytechnique de Bordeaux,
France
Marc Kaufman,
The Pennsylvania State University,
United States
*Correspondence:
Brian R. Noga
bnoga@miami.edu
Patrick J. Whelan
whelan@ucalgary.ca
Received: 27 February 2022
Accepted: 14 April 2022
Published: 09 May 2022
Citation:
Noga BR and Whelan PJ (2022) The
Mesencephalic Locomotor Region:
Beyond Locomotor Control.
Front. Neural Circuits 16:884785.
doi: 10.3389/fncir.2022.884785
The Mesencephalic Locomotor
Region: Beyond Locomotor Control
Brian R. Noga1* and Patrick J. Whelan2,3*
1The Miami Project to Cure Paralysis, Department of Neurological Surgery, Miller School of Medicine, University of Miami,
Miami, FL, United States, 2Hotchkiss Brain Institute, University of Calgary, Calgary, AB, Canada, 3Department of
Comparative Biology and Experimental Medicine, University of Calgary, Calgary, AB, Canada
The mesencephalic locomotor region (MLR) was discovered several decades ago in the
cat. It was functionally defined based on the ability of low threshold electrical stimuli within
a region comprising the cuneiform and pedunculopontine nucleus to evoke locomotion.
Since then, similar regions have been found in diverse vertebrate species, including the
lamprey, skate, rodent, pig, monkey, and human. The MLR, while often viewed under
the lens of locomotion, is involved in diverse processes involving the autonomic nervous
system, respiratory system, and the state-dependent activation of motor systems. This
review will discuss the pedunculopontine nucleus and cuneiform nucleus that comprises
the MLR and examine their respective connectomes from both an anatomical and
functional angle. From a functional perspective, the MLR primes the cardiovascular and
respiratory systems before the locomotor activity occurs. Inputs from a variety of higher
structures, and direct outputs to the monoaminergic nuclei, allow the MLR to be able
to respond appropriately to state-dependent locomotion. These state-dependent effects
are roughly divided into escape and exploratory behavior, and the MLR also can reinforce
the selection of these locomotor behaviors through projections to adjacent structures
such as the periaqueductal gray or to limbic and cortical regions. Findings from the rat,
mouse, pig, and cat will be discussed to highlight similarities and differences among
diverse species.
Keywords: locomotion, motor control, brainstem, spinal cord, dopamine, aminergic
INTRODUCTION
Building on work by Graham-Brown, a renaissance in the study of locomotion started in the
1960s, driven by work by Anders Lundberg, Mark Shik, Grigori Orlovsky, and Sten Grillner
(Stuart and Hultborn, 2008; Sharples and Whelan, 2020). It was generally recognized that
the brainstem could elicit locomotor activity, coordinating in some way with spinal cord
centers. This led to the publishing of work by Shik and Orlovskii in 1966 of an area of the
brain bounded by the cuneiform (CnF) nucleus named the mesencephalic locomotor region
or MLR, having a linear dimension of 1 mm, and when stimulated, produced locomotor
Abbreviations: CnF, cuneiform nucleus; DBS, deep brain stimulation; dlPAG, dorsolateral PAG; dmPAG, dorsomedial
PAG; FOG, freezing-of-gait; KF, Kölliker-Fuse nucleus; l-DOPA, l-3,4-dihydroxyphenylalanine; LH, lateral hypothalamus;
lPAG, lateral PAG; LC, locus ceruleus; LPGi, lateral paragigantocellular nucleus; LTD, laterodorsal tegmental nucleus;
LRN, lateral reticular nucleus; MRF, medial reticular formation; MLR, mesencephalic locomotor region; NTS, nucleus
tractus solitarii; PAG, periaqueductal gray; PD, Parkinson’s disease; PPN, pedunculopontine nucleus; RfN, raphe nucleus;
RS, reticulospinal; RVLM, rostral ventrolateral medulla; SC, superior colliculus; SLR, subthalamic locomotor region; SNc,
substantia nigra pars compacta; SNr, substantia nigra pars reticulata; vlPAG, ventrolateral PAG; vGlut2, vesicular-glutamate
transporter 2; VRG, ventral respiratory group; VTA, ventral tegmental area; V1, primary visual cortex.
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Noga and Whelan The Diversity of the MLR
activity (Shik et al., 1966, 1969). Anatomically the other nucleus
that constitutes the MLR is the pedunculopontine nucleus
(PPN). It appears that many principles are conserved across
vertebrate species from lamprey to humans (Eidelberg et al.,
1981; McClellan and Grillner, 1984; Garcia-Rill et al., 1985;
Masdeu et al., 1994; Dubuc et al., 2008; Caggiano et al., 2018;
Josset et al., 2018). While we use the term MLR in the review,
it will be argued that it would be better to refer to the PPN and
CnF separately, given the diversity of functions of the region.
MLR CONNECTIVITY
The MLR forms a central node in the initiation of locomotion
by higher brain centers (Figure 1A). It receives inputs from
the ipsilateral subthalamic locomotor region (SLR; Orlovskii,
1969; Mel’nikova, 1977; Sinnamon and Stopford, 1987), the
substantia nigra pars reticulata (SNr; Beresovskii and Bayev,
1988; Roseberry et al., 2016) and central amygdala (Roseberry
et al., 2016). It is reciprocally connected with the contralateral
MLR (Steeves and Jordan, 1984; Beresovskii and Bayev, 1988),
possibly facilitating or coordinating descending signal output
on both sides, and receives input from several sensory systems
(e.g., auditory, visual) via the superior colliculus and lateral
lemniscus, amongst others (Mitchell et al., 1988; Furigo et al.,
2010; Roseberry et al., 2016). Activation of the MLR is also
achieved by disinhibition of inhibitory SNr projections affecting
both postural muscle tone and locomotion (Garcia-Rill et al.,
1985; Takakusaki et al., 2003, 2016; Roseberry et al., 2016).
Recent work suggests a greater normalized projection to the
PPN compared to the CnF (McElvain et al., 2021). There is
also a strong reciprocal interconnection with the periaqueductal
gray (PAG; Mantyh, 1983; Beresovskii and Bayev, 1988; Sandner
et al., 1992; Ferreira-Netto et al., 2005; Caggiano et al., 2018)
which may be necessary for the mediation of rapid defensive
decision making or the control of locomotion during the
pursuit, initiated by activation of the amygdala (Han et al.,
2017). It is important to consider the components of the MLR
separately, as their functions are different. The differential
effects of PPN stimulation on locomotion correspond to the
diversity of anatomical projections to motor structures such
as the cerebellum, spinal cord, basal ganglia, and brainstem.
On the other hand, the more defined response of the CnF
is consistent with projection patterns to downstream medial
reticular formation (MRF) structures (Steeves and Jordan, 1984;
Sotnichenko, 1985; Garcia-Rill and Skinner, 1987; Dautan et al.,
2021). Similarly, projection patterns to the PPN are larger
(basal ganglia and brainstem) than the CnF and more diverse
(Caggiano et al., 2018; Dautan et al., 2021), and it has been
suggested that the PPN is involved more in the modalities of
movement rather than the execution of movement (Dautan
et al., 2021). The output of the CnF is connected with PAG
and other defensive areas of the brain suggesting an integrated
escape functionality (Edwards and de Olmos, 1976; Steeves
and Jordan, 1984; Dampney et al., 2013; Caggiano et al., 2018;
Opris et al., 2019). The field is at an exciting juncture where
electrophysiological data from pioneers such as Jankowska and
colleagues examining supraspinal projections (Jankowska et al.,
1993; Krutki et al., 2003) can now be married with circuit-specific
modulation (Ferreira-Pinto et al., 2021) to establish sufficiency
and necessity.
MLR FUNCTION—A TALE OF TWO NUCLEI
Considering both the CnF and PPN, the PPN has the most
functional diversity. The PPN promotes arousal (Moruzzi
and Magoun, 1949; Lee et al., 2014), likely through the
ascending reticular activating system. Indeed, Parkinsonian
patients implanted with deep brain stimulation (DBS) electrodes
report side effects of increases in general arousal (Stefani
et al., 2007). While chemogenetic stimulation of cholinergic
neurons does not alter waking time, activation of PPN vesicular-
glutamate transporter 2 (vGlut2) neurons has a robust effect
on awake time (Kroeger et al., 2017). Moreover, in terms of
locomotion, stimulation of the glutamatergic PPN cells induces
locomotor activity, and these cells receive input from the
amygdala and the basal ganglia (Roseberry et al., 2016, 2019).
The results from stimulation of glutamatergic PPN cells on
locomotion are mixed, with some suggesting they are involved
in slow exploratory-like locomotor activity (Caggiano et al.,
2018). Support for this notion is derived from increased time
head-dipping in hole-board tasks, PPN unit activity during
slower speeds, and optogenetic activity inducing slow locomotor
activity (Caggiano et al., 2018). However, other reports suggest
the opposite—that PPN stimulation produces locomotor arrest
(Josset et al., 2018; Dautan et al., 2021). Another group reported
arrest and locomotor behaviors in rats following optogenetic
stimulation of the PPN but with most animals producing
an abrupt increase in locomotor activity (Carvalho et al.,
2020). Interestingly, photostimulation consistently produced
either arrest or locomotion, suggesting that the subregion of
the PPN stimulated was important. A recent study in mice
shows that PPN vGlut2+photostimulation reliably inhibits the
distance traveled by mice (Dautan et al., 2021). The lack of
consensus may be due to the known heterogeneity of the PPN,
differences in the viral spread, and the target region of the
PPN (e.g., dorsoventral positioning; Chang et al., 2020). Unit
electrophysiological recordings from PPN reinforce this point,
with units positively and negatively correlated with locomotor
speed (Carvalho et al., 2020). Finally, a recent study using
a combination of gCaMP6 recording and loss or gain of
function experiments provides evidence for a PPN to spinal
cord projection involved in rearing (Ferreira-Pinto et al., 2021).
Notably, few cells within the PPN in this study were modulated
during locomotor activity.
PPN photostimulation associated with locomotor activity can
produce activity in the V1 neurons of the visual cortex (Lee
et al., 2014), through the basal forebrain bundle. So it is clear
that the MLR, as well as producing locomotion, can feedback
to cortical centers. Recently, a glutamatergic population was
identified that projects from the PPN to the substantia nigra
pars compacta (SNc), and which is involved in forelimb such as
grooming and handling of objects (Ferreira-Pinto et al., 2021).
This is interesting considering the newly discovered SNc to MLR
projection (Ryczko et al., 2016; Fougère et al., 2019), suggesting
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Noga and Whelan The Diversity of the MLR
a possible feedback mechanism. Combined with other work
establishing a role for complex forelimb movements within the
lateral rostral medulla (Esposito et al., 2014; Ruder et al., 2021), it
suggests the brainstem is an integral part of the coding of multiple
types of movements before the resultant command is relayed to
spinal cord structures.
While the role of the PPN in locomotion is under debate, there
is broad consensus that the CnF glutamatergic cells can initiate
locomotion and control speed (Caggiano et al., 2018; Josset et al.,
2018; Dautan et al., 2021). Low levels of stimulation promote
walking, and the stimulus can be tuned to elicit different gaits
(walk, trot, gallop, and bound). Extracellular recording confirms
the CnF spike activity is more correlated to higher speeds
compared to the PPN (Caggiano et al., 2018). This suggests that
although the CnF could be recruited during normal walking,
at higher levels of stimulation the locomotor activity patterns
observed resemble escape.
WHAT HAPPENS AT SLOW SPEEDS VS.
FAST SPEEDS—WHAT DO WE KNOW?
Locomotion elicited by stimulation of the MLR generally
falls into two categories. Stimulation of the PPN can elicit
slow-walking movements, along with arrest, while stimulation
of the CnF can elicit locomotion across a much greater range.
This difference was first reported by Orlovsky and colleagues, as
discussed, and was later associated with vGlut2 positive neurons
in both nuclei (Roseberry et al., 2016; Caggiano et al., 2018).
When we examine the firing properties of vGlut2 neurons,
they display diversity from rapidly adapting to non-adapting,
while CnF neurons are mainly fast adapting. This may be
associated with the heterogeneity of behaviors produced by PPN
stimulation (Ferreira-Pinto et al., 2021) and the more diverse
inputs to and projections of PPN vs. CnF (Caggiano et al., 2018;
Dautan et al., 2021). However, we are at the beginning of a
long journey to examine speed and other metrics of locomotion
as they relate to the behaving animal. The activity of the
MLR is related to the behavioral state. Reciprocal connectivity
with structures throughout the brain ensures that the control
exerted by the MLR fits the requirements of the behavioral
state in conjunction with afferent feedback, limb coordination,
and postural control (Mori, 1987; Mori et al., 1989). The
CnF forms part of the defense circuit in rodents and other
species (Mitchell et al., 1988). Glutamate and GABAergic cells
have higher firing rates during cortical arousal. The PPN has
extensive connectivity with dopaminergic and thalamic areas.
Therefore, the idea has been proposed that the PPN could be a
comparator region comparing expected and real situations and
causing upstream state changes through the cholinergic system.
The non-cholinergic system may contribute a different role in
executing these changes to the motor system.
MLR-WHAT HAVE WE BEEN
STIMULATING?
As mentioned, the MLR comprises mainly the CnF and the
PPN. Still, since, especially in early work, it was based on
electrical stimulus thresholds, it has not always been clear what
structures have been stimulated. Indeed, the proximity of the
two nuclei and the fact that they share a common border have
often made it difficult to narrow the stimulation to one or the
other. While the debate over the function of PPN and CnF has
been ongoing for several decades (Whelan, 1996), even with
less advanced techniques, evidence was already pointing to the
cuneiform as being important for locomotor control (Inglis and
Winn, 1995; Jordan, 1998). But even with multiple approaches
including electrical, chemical, and optogenetic stimulation, there
is still much debate regarding functional roles within MLR nuclei.
Electrical stimulation of the MLR in cats can produce different
latencies for evoked movement a fact noted for optogenetic
stimulation of the PPN compared to CnF (Caggiano et al., 2018).
Notably, electrode position, frequency, and current delivered are
critical factors across different terrestrial species, including the
cat, rat, mouse, and pig (Orlovsky et al., 1966; Garcia-Rill and
Skinner, 1987; Noga et al., 2003; Chang et al., 2021b).
Latency of the response to electrical stimulation can vary,
and this can be a function of the state, type of stimulus. In
the unanesthetized pig, for a well-placed electrode, latency to
onset of locomotion with electrical stimulation varies depending
upon the amount of current injected (Chang et al., 2021b).
Stimulation well above electrical thresholds for any particular
frequency will evoke locomotion quicker and produce faster
locomotion than if you stimulate at threshold strengths (Noga
et al., 2017a). Furthermore, faster locomotion at onset is
observed at threshold strengths with increasing stimulation
frequency, suggesting that optimal stimulation frequency to
engage the full range of walking gaits is lower, rather than
higher. Stimulation history may also contribute to these
effects. While the state of awake behaving animals can affect
MLR stimulation, decerebrate animals also show diminished
effectiveness with electrical stimulation related to whether the
MLR has been stimulated for a long time, with repeated
phasic stimulation inducing an inhibitory effect (Noga et al.,
2009, 2011; Opris et al., 2019). Both photo and electrical
stimulation have been used to examine MLR function, but
there are differences in the activation mechanism between
electrical and photostimulation. While optogenetics is useful
for directly activating cell types, it is often compared with
electrical stimulation. But it is important to note that electrical
stimulation can activate fibers much more easily than cell
bodies. That depends on stimulation configuration (whether
cathodal, anodal, monopolar, or bipolar), the type of electrode
(sharp or cylindrical) and the fiber orientations relative to
the electrode. Electrical stimulation may activate diverse fibers
leading to a mixture of monoamines, fast neurotransmitters, and
neuropeptides being released onto target neurons. So, effects
will depend on the various neurotransmitters’ combinatorial
actions. This is compared to opsins that have been inserted into
the membrane of neurons and which are then photostimulated
or inhibited (Boyden et al., 2005). One can modulate specific
circuits or specific neuronal phenotypes with the right tools.
ChR2, the standard excitatory opsin activated by blue light,
can follow stimulation frequencies up to approximately 30 Hz
due to the kinetics of the opsin channel. However, fast
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Noga and Whelan The Diversity of the MLR
FIGURE 1 | Connectivity of the MLR for (A) Motor System, (B) Respiratory System, and (C) Cardiovascular system. Abbreviations. CnF, Cuneiform Nucleus; LC,
Locus Coeruleus; PPN, Pedunculopontine Nucleus; SC, Superior Colliculus; SNc, Substantia Nigra compacta; PAG, Periaqueductal Gray; RfN, Raphe Nucleus;
MRF, Medullary Reticular Formation; NTS, nucleus tractus solitarii; RVLM, rostral ventrolateral medulla; VRG, Ventral Respiratory Group; V1, primary visual cortex.
opening channels, that would drive higher frequencies require
stronger light. Red-shifted opsins, such as Chrimson, balance
the need for stronger light for fast opening channels with
greater channel expression at the membrane (Mager et al.,
2018). From reported values for CnF and PPN unit frequencies,
units generally fire between 5 and 50 Hz for most behaviors
(Simon et al., 2010; Caggiano et al., 2018; Goetz et al., 2019;
Carvalho et al., 2020). For work using ChR2 opsins, even
when higher photostimulation frequency ranges (30–50 Hz)
are reported neurons may not follow faithfully with spikes.
Thus, the high firing frequency range of PPN and CnF
neurons has not been fully probed and awaits work with
opsins such as Chrimson and Cheta that follow higher
stimulation frequencies.
PAG—INPUTS FOR DEFENSIVE BEHAVIOR
While not traditionally considered part of the MLR the proximity
and importance in defensive behaviors warrant a discussion of
this area. In prey species, defensive behaviors comprise both
a flight and a freezing response. The decision to evoke flight
or freezing responses is dependent on a combination of visual,
auditory, and somatosensory inputs, and depends on subcortical
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Noga and Whelan The Diversity of the MLR
systems. In this context, the PAG is an important mediator
of defensive behavior (Figure 1A) (Bandler, 1982; Bandler and
Carrive, 1988), including freezing or flight in response to threat
(Kim et al., 2017; Koutsikou et al., 2017). The PAG is a complex
structure and has four distinct regions: these comprise the
dorsomedial (dmPAG), dorsolateral (dlPAG), lateral (lPAG),
and ventrolateral (vlPAG) subdivisions (Carrive, 1993; Linnman
et al., 2012; Dampney et al., 2013). Freezing responses are
induced by neurons of the vlPAG (Bandler and Depaulis,
1988; Depaulis et al., 1989, 1992), which are glutamatergic and
generally under control by local GABAergic neurons. Freezing is
induced by projections to the pontomedullary (magnocellularis)
reticular formation (Tovote et al., 2016). With short reorienting
freezing responses, flight responses are induced by activation
of the glutamatergic neurons within the dlPAG or the lPAG
(Deng et al., 2016; Tovote et al., 2016). This response is likely
mediated by indirect activation of reticulospinal (RS) neurons via
an intermediary pathway to the MLR (Ferreira-Netto et al., 2005;
Dampney et al., 2013) although a direct pathway has also been
described for the monkey (Mantyh, 1983). Interestingly, dl/lPAG
glutamatergic neurons inhibit vlPAG glutamatergic neurons
by activating vlPAG GABAergic neurons, thus inhibiting the
freezing response (Tovote et al., 2016). Based upon these results,
MLR stimulation most likely activates (Opris et al., 2019)
glutamatergic neurons of the dl/lPAG and GABAergic neurons
of the vlPAG, facilitating locomotor activity and inhibiting
freezing responses, respectively. The lateral hypothalamus (LH)
provides an important projection to PAG, and photostimulation
of GABAergic LH neurons contributes to prey detection and
capture. In contrast, glutamatergic LH projections contribute
to defensive responses (Rossier et al., 2021). The anatomical
and functional linkage between the CnF, PAG, and limbic
system (Edwards and de Olmos, 1976; Mantyh, 1983; Steeves
and Jordan, 1984; Sotnichenko, 1985; Meller and Dennis, 1986;
Ferreira-Netto et al., 2005; Dampney et al., 2013; Koutsikou
et al., 2017; Caggiano et al., 2018) points to the important
role played by the MLR in the integration of complex motor
behaviors related to defensive behavior. The PAG receives other
inputs associated with locomotion, such as the core of the
nucleus accumbens, associated with reward-based locomotion,
and the amygdala (Gross and Canteras, 2012; Tovote et al., 2016),
which while traditionally associated with defensive responses is
also associated with approach behaviors. Indeed, the amygdala
also projects to the MLR, suggesting parallel pathways to
initiate escape. While the connectivity between PAG and CnF
is known, further studies regarding the interaction between
these regions are required. Furthermore, it is possible that
the PAG via MLR can influence visual processing through
direct and indirect connectivity to the visual cortex. This
may be another route whereby PAG can affect brain state
(Lee et al., 2014).
MONOAMINERGIC MODULATION
Dopamine
Dopamine modulation of motor pathways was thought to
be primarily indirect through the nigrostriatal pathway that
projects via the basal ganglia to cortical and to brainstem
regions (Figure 1A). The basal ganglia canonical circuit
is highly conserved across vertebrates from lamprey to
primates (Robertson et al., 2014). The direct D1R and
indirect D2R pathways modulate output from the SNr and
the globus pallidus internal onto motor centers. The SNr
has GABAergic projections to the MLR, consistent with the
indirect projections of the dopaminergic system (Roseberry
et al., 2016). Thus, activating the dopaminergic nigrostriatal
pathways leads to a removal of inhibition to the MLR
through the D1mediated direct and the D2mediated
indirect basal ganglia pathways. However, work demonstrates
that the SNc A9 dopamine region or its analogs projects
directly to the MLR in rat, salamander, lamprey, and mouse
(Ryczko et al., 2013, 2016; Roseberry et al., 2016; Caggiano
et al., 2018). Stimulation of the SNc increases extracellular
dopamine concentrations in the MLR, and these effects
are attenuated with D1R antagonists and potentiated with
amphetamine. Interestingly there is also an ascending projection
from the PPN to the SNc (Futami et al., 1995; Charara
et al., 1996; Martinez-Gonzalez et al., 2011), which has been
postulated to be involved in arousal, but equally could form
a recurrent excitatory feedback loop that reinforces ongoing
behavior. In mice, activity patterns within the SNc precede
and are associated with locomotor activity, indicating that
the direct projection to the MLR may increase activity
within locomotor-related brainstem neurons (da Silva et al.,
2018; Fougère et al., 2019). What additional role would
a direct SNc link to the MLR have? The nigrostriatal
pathway is one of massive convergence and compared to
the SNr there are 800 times more projection neurons onto
the striatum than the SNr projection neurons (Zheng and
Wilson, 2002; Dudman and Krakauer, 2016). The direct SNc
projections could be important for movement initiation or
to integrate subcortical inputs with more fidelity than when
filtered through the basal ganglia. Interestingly, the descending
dopaminergic cells appear to co-localize glutamate, which
provides a mechanism for fast excitatory activation of the MLR
from the basal ganglia.
The other newly discovered pathway is the A13, a small
nucleus within the medial zona incerta that projects to the
CnF and, to a lesser extent, the PPN (Sharma et al., 2018,
2019). The A13 also projects to the superior colliculus and
appears to be part of a defensive behavior circuit (Bolton et al.,
2015). In contrast to the SNc descending circuit, A13 neurons
co-localize GABA (Venkataraman et al., 2021), suggesting a dual
fast inhibitory and a modulatory dopaminergic control of MLR
function. The A13 also projects to the superior colliculus where
there are D1receptors located predominantly on GABAergic
superficial neurons while D2receptors are located in the deep
layers (Bolton et al., 2015). This suggests both modulations
of visual input and motor responses by the A13 within the
superior colliculus (SC), which has been suggested to contribute
to the salience of a visual object (Woolrych et al., 2021).
An additional dopaminergic circuit can potentially modulate
the motor function and is contiguous with the A13, but
the A11 cell somas are noticeably larger and multipolar
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(Sharma et al., 2018). This is the A11 nucleus, contained within
the posterior hypothalamus, which projects to all segments of
the spinal cord (Björklund and Skagerberg, 1979; Skagerberg
et al., 1982; Qu et al., 2006; Koblinger et al., 2014). Directly
applied exogenous dopamine increases the excitability of lumbar
motoneurons and interneurons and can potentiate locomotor
activity (Humphreys and Whelan, 2012; Sharples et al., 2015,
2020). Optogenetic stimulation of the A11 led to an increase in
bouts of locomotor activity suggesting a possible motor function
for the A11 descending projection (Koblinger et al., 2018). The
A11 and its descending spinal projections are discussed in more
detail in other reviews (Sharples et al., 2014). In brief, it appears
to be the sole source of spinal dopamine in rodents. Spinally
applied dopamine can evoke diverse rhythms within the spinal
cord, including episodic and locomotor activity (Humphreys and
Whelan, 2012; Sharples et al., 2015, 2020, 2022).
There are other dopaminergic areas of importance for motor
function and there is evidence that the PPN projections can
alter the firing patterns of dopamine neurons, changing patterns
from burst to tonic firing. The rostral PPN cholinergic and
non-cholinergic neurons project widely to the dorsal striatum
and can affect dopamine presynaptic release along with striatal
neuronal firing (Dautan et al., 2014). Along with the direct
dorsal striatal connections, indirect PPN connections to the
dorsal striatum via the thalamus and ventral tegmental area
(VTA) have been reported. Glutamatergic PPN neurons appear
to project preferentially to the striatum compared to the
CnF (Dautan et al., 2021). Neurons within the VTA show
an increase in activity-dependent cFos activity during fictive
locomotion produced by stimulation of the CnF (Opris et al.,
2019). The VTA contains dopaminergic neurons involved
in goal-directed behavior and reinforcement learning (Wise,
2004). It receives direct input from non-catecholaminergic
neurons of the vlPAG (Suckow et al., 2013) and from
cholinergic and glutamatergic neurons of the PPT and
laterodorsal tegmental nucleus (LDT; Mena-Segovia and Bolam,
2017). Stimulation of cholinergic PPT terminals within the
VTA activates dopaminergic neurons and transiently increases
locomotor activity (Dautan et al., 2016). In contrast, LDT
cholinergic neuron activation decreases locomotion (Dautan
et al., 2016) and results in reward reinforcement (Xiao et al.,
2016). These differential effects are likely due to actions
on different neurons within the VTA. PPT glutamatergic
neurons also increase arousal and drive motivated behavior via
ascending projections, in part to the VTA (Kroeger et al., 2017;
Yoo et al., 2017).
Noradrenaline and Serotonin
The first demonstration of a key role for monoamines in
activating spinal locomotor networks was the observation that
intravenous noradrenaline and serotonin precursors produced
reflex discharges in the spinal cat or rabbit that resembled
locomotion (Jankowska et al., 1967; Viala and Buser, 1969). Soon
after that, based on the resemblance of MLR evoked locomotion
to the activity seen following l-3,4-dihydroxyphenylalanine (L-
DOPA), Grillner and Shik (1973) postulated that the MLR
activated a descending noradrenergic pathway controlling
the spinal locomotor network. This idea was supported by
the demonstration of noradrenergic neurons near the MLR,
and that descending MLR projections included noradrenergic
and serotonergic nuclei (Jordan and Steeves, 1976; Steeves
and Jordan, 1984; Sotnichenko, 1985). Since then, multiple
studies have shown that the spinal application of monoamines
can initiate and modulate ongoing locomotor activity (e.g.,
Barbeau and Rossignol, 1991; Chau et al., 1998; Brustein and
Rossignol, 1999; Musienko et al., 2011; Perrier and Cotel,
2015; Sharples et al., 2015, 2020). In spinal cord injured
patients, a marked improvement in locomotor function and
marked reductions in stretch reflexes and clonus may be
obtained following oral administration of the noradrenergic
alpha-agonist clonidine (Fung et al., 1990; Stewart et al.,
1991). The neuromodulatory potential of monoamines varies
by species. For example, in chronic spinal cats, noradrenergic
agonists are most effective for enabling the initiation of
locomotion (Barbeau and Rossignol, 1991; Marcoux and
Rossignol, 2000) whereas, in the spinal rat and in vitro neonatal
rat preparation, spinal application of serotonin or dopamine,
with or without the co-application of N-methyl-D, L-aspartate,
is most effective in eliciting stepping (Atsuta et al., 1991; Cowley
and Schmidt, 1997; Sharples et al., 2015). In the cat, locomotor-
activated neurons are innervated by monoaminergic fibers
and express the serotonergic and noradrenergic postsynaptic
receptors that mediate such effects (Noga et al., 2009, 2011).
Spinal monoaminergic receptors are also found presynaptically
on primary afferent and central terminals (Stone et al.,
1998; Riedl et al., 2009), acting either as autoreceptors or
heteroreceptors regulating transmitter release (Umeda et al.,
1997; Li et al., 2000). Manipulation of endogenously released
serotonin was shown to modulate the locomotor network in
the in vitro neonatal mouse (Dunbar et al., 2010). That and the
demonstration that cerulear and raphe neurons are activated
during voluntary locomotion (Rasmussen et al., 1986; Jacobs
and Fornal, 1999) suggested that monoaminergic modulatory
pathways are engaged during locomotion, even though their
activation is not obligatory for MLR evoked locomotion
(Steeves et al., 1980). Subsequently, MLR stimulation was
shown to activate noradrenergic and serotonergic nuclei of the
brainstem (Opris et al., 2019) and result in the widespread
release of noradrenaline and serotonin within the spinal
cord during evoked locomotion (Noga et al., 2017b). Thus,
descending monoaminergic pathways are activated in parallel
with reticulospinal pathways during MLR-evoked locomotion
and must be considered as a component of the descending
locomotor pathway (Figure 1A).
VISUAL SYSTEM—INTEGRATION FOR
ESCAPE
The MLR can influence primary visual cortex V1 activity,
via the basal forebrain, leading to increased gamma and
reduced low-frequency oscillations (Lee et al., 2014). The other
pathway is through the SC, which consists of an outer layer
associated with vision and a deeper zone associated with
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Noga and Whelan The Diversity of the MLR
motor and other functions (May, 2006). The SC is critical
for triggering appropriate locomotor behavior. For example,
when an approaching stimulus is presented to the upper visual
field it evokes escape-like behavior (Yilmaz and Meister, 2013);
in contrast, when approaching stimuli are displayed to the
lower visual field, exploratory movements are evoked (Comoli
et al., 2012). This is associated with predatory and appetitive
stimuli, respectively. The upper visual field maps onto the
medial SC, while the lower visual field projects onto the lateral
SC. The medial SC projects onto the ipsilateral CnF with a
smaller projection onto the contralateral PPN (Figure 1A) (Dean
et al., 1989). Stimulation of the medial SC evokes locomotor
movements similar to that evoked by the MLR, although the
response is mediated by a projection onto GABAergic cells
(Roseberry et al., 2016). It is not yet known what the transmitter
types are that project from the SC to the MLR. Thus, either
an excitatory or inhibitory projection onto CnF neurons is
technically feasible. On the other hand, stimulation of the
lateral SC produces contralateral orientating types of movements
followed by exploratory movements (Sahibzada et al., 1986). The
medial and lateral SC also project to the MRF. In contrast to
its effects on V1 neurons, locomotor activity does not have a
major effect on superficial SC neural activity, suggesting that the
SC responds more faithfully to visual stimuli during movement
(Savier et al., 2019).
AUTONOMIC NERVOUS
SYSTEM—PRIMING THE SYSTEM?
The MLR is intimately connected with the autonomic nervous
system (Figures 1B,C). The link between the cuneiform
and hemodynamic function was noted by Sirota and Shik
(Sirota et al., 1970; Shik and Orlovsky, 1976) and has
been confirmed since that period. Projections from the CnF
project to areas associated with a cardiovascular function
such as the rostral ventrolateral medulla (RVLM), PAG,
locus coeruleus (LC), nucleus tractus solitarii (NTS), lateral
paragigantocellular nucleus (LPGi) and parabrachial nucleus
(Korte et al., 1992; Verberne, 1995; Lam et al., 1997; Shafei et al.,
2012; Dampney et al., 2013; Netzer and Sévoz-Couche, 2021).
Electrical stimulation of the CnF produces pressor responses in
animals immobilized with neuromuscular blockers during fictive
locomotion, activating neurons within several nuclei regulating
blood pressure (Opris et al., 2019) and therefore is centrally
coupled to the sympathetic system. Efferent connectivity from
the CnF to the parabrachial and Kölliker-Fuse (KF) nucleus
appears to mediate the sympathetic arm of the CnF (Korte
et al., 1992). The PPN also contributes to the control of
cardiovascular function likely through projections to the RVLM
(Yasui et al., 1990). Interestingly, acetylcholine counteracts
the pressor effect of CnF stimulation (Shafei et al., 2013)
although elevations in sympathetic nerve activity, blood pressure,
and baroreflex have been noted with chemical stimulation
of the PPN in anesthetized rats (Padley et al., 2007). DBS
stimulation of the PPN in Parkinson’s patients produces an
elevation in blood pressure and baroreflex sensitivity (Hyam
et al., 2019), which was particularly evident when the caudal
PPN was targeted. On the other hand, CnF projections
to the motor nucleus of the vagus and the NTS could
be part of a proposed parasympathetic arm. Also, several
nuclei are known to produce hypotension (Dampney and
Horiuchi, 2003) and could be activated to counteract the
pressor effect of MLR stimulation. Such nuclei include the
nucleus ambiguous (Machado and Brody, 1988, 1990) and
the dorsal motor nucleus of the vagus, possibly via a direct
projection from the RVLM (DePuy et al., 2013). As part of a
coordinated autonomic response, MLR stimulation results in
cFos activation in the NTS (dorsal respiratory group), along
with the retrofacial and lateral reticular nuclei (LRN—ventral
respiratory group; Opris et al., 2019). Other nuclei associated
with respiratory function include the raphe/parapyramidal
region, LC/subcoeruleus, KF, PPT, and PAG (Kubin and Fenik,
2004; Gargaglioni et al., 2010; Dutschmann and Dick, 2012;
Dampney et al., 2013; Subramanian and Holstege, 2014; Opris
et al., 2019). Combined with previous work showing that
stimulation of the hypothalamic and MLR facilitate respiratory
rhythms and respiratory output (Sirota et al., 1971; Eldridge
et al., 1981; DiMarco et al., 1983; Millhorn et al., 1987; Kawahara
et al., 1989; Ezure and Tanaka, 1997), this points to an
important role for the MLR in controlling respiratory function.
Interestingly, respiratory activity increases before locomotor
onset (Eldridge et al., 1981) indicating the preparatory nature
of this control. Furthermore, treadmill exercise also activates
neurons in many of the same areas (Iwamoto et al., 1996).
Neurons within the LRN receive input from central respiratory
and locomotor rhythms (Ezure and Tanaka, 1997) and are
thought to transmit information of linked motor components
to the cerebellum for eventual modulation of motor behaviors
(Alstermark and Ekerot, 2013). In addition to cardiovascular
and respiratory control, the PPN is reported to contribute to
renal sympathetic nerve activity (Fink et al., 2017), bladder
(Aviles-Olmos et al., 2011; Roy et al., 2018), In summary, we
need to consider the MLR as part of a central controlling
system that initiates locomotor and motor functions while
concomitantly activating appropriate arms of the sympathetic
and parasympathetic nervous systems. Furthermore, links with
the autonomic nervous system coupled with the locomotor
systems make the MLR an important target for coordinated
recovery of multiple spinal cord centers following spinal cord
injury. Notably, the pig, a valuable model for spinal cord injury
research, also increases heart rate following MLR stimulation
(Chang et al., 2021a). More research is required that carefully
examines links with the autonomic nervous system using modern
circuit-specific approaches and closed-loop feedback control
(Noga and Guest, 2021).
UNDERSTANDING HOW THE MLR IS
INTEGRATED FROM A COMPARATIVE
AND TRANSLATIONAL PERSPECTIVE
The MLR and specifically the PPN have been the focus of
DBS trials designed to address movement disorders in patients.
Primarily these patients have gait dysfunction (freezing-of-gait
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Noga and Whelan The Diversity of the MLR
or FOG) because of Parkinson’s disease (PD). Initial reports
were promising following DBS of the PPN, with motor scores
and Unified Parkinson’s Disease Rating Scale improvements of
57% and 53%, respectively (Plaha and Gill, 2005). However,
subsequent studies have shown mixed results as summarized
in a recent meta-analysis (Wang et al., 2017). One possible
issue is the dorsal MLR encompassing the CnF is critical and
small differences in targeting produce significant effects on
performance (Thevathasan et al., 2018; Goetz et al., 2019). These
results correspond to results in rodents discussed previously,
where PPN stimulation produces mixed effects while CnF
produces generally consistent locomotory results. That said the
type of cells and location within the PPN matter. Recent work
found that activating caudal glutamatergic PPN neurons was
particularly effective in rescuing locomotor activity (Masini and
Kiehn, 2022). This rescue was independent of CnF glutamatergic
neurons. Interestingly, activation of GABAergic PPN neurons
effectively restored slow locomotor activity, which may suggest a
combinatorial strategy in targeting neuronal populations within
the MLR (Masini and Kiehn, 2022). More recently, the CnF
has been promoted as an alternative target for FOG (Chang
et al., 2020, 2021a) and a recent study in a mouse model of PD
has shown that glutamatergic CnF neuron stimulation improves
the initiation of locomotion while reducing the time spent
immobile (Fougère et al., 2021). Preliminary results targeting the
CnF in a PD patient with levodopa-resistant FOG demonstrate
the procedure’s safety and show significant improvements in
many gait parameters during CnF DBS (Chang et al., 2021a).
Stimulation of the anterior CnF also showed significant increases
in step length and velocity over that seen with either sham-DBS
or PPN DBS (2-month period of DBS). Still, no significant
improvements in clinical outcomes were observed for either DBS
condition in PD patients with severe gait and balance disorders
(Bourilhon et al., 2022). In addition to Parkinson’s disease,
clinical trials are underway to determine if DBS of the MLR can
improve function in incomplete spinal cord injured individuals.
This work was a product of rodent work showing that MLR
stimulation in a model with 80% of the cord damaged produced
walking and swimming movements (Bachmann et al., 2013).
Significant improvements in gait (stepping, electromyogram
amplitude, speed, interlimb coordination, and joint excursion)
are also observed following spinal contusion injuries in the pig
(Noga et al., 2020). A detailed analysis of the efficacy of the CnF
vs. the PPN has not been completed, but work in 6-OHDA mice
shows that the CnF also is effective in augmenting locomotion
(Fougère et al., 2021). The long translational timeframe since
Shik and Orlovsky’s initial discovery of the MLR may rest on the
necessity of stimulating the CnF, rather than the PPN. Ironically,
they pointed out that the CnF appeared to be a better target for
initiating locomotion more than 50 years ago. The MLR or its
analog is found in diverse species from lampreys, skates, rodents,
pigs, monkeys, and humans, and many similarities have been
observed. But there are limitations to the translation of findings.
For Parkinson’s disease, for example, no animal model to date
can recapitulate the chronic pathology observed in humans. Due
to bipedalism, differences in locomotor and postural control will
presumably affect MLR connectivity and function.
FUTURE DIRECTIONS
As we move forward, it will be critical to evaluate the
role of the MLR in downstream connectivity to motor
centers such as the MRF and spinal cord, connectivity to
hemodynamic areas within the brainstem and spinal cord,
and finally connectivity to cortical and limbic structures. To
accomplish this, we need to deploy tools such as multi-site
fiber photometry to record from these diverse areas. A
limitation of both electrical and photostimulation is that the
recruitment of populations tends to be synchronized and
does not match the asynchronous firing of units observed.
Overcoming this will likely take a combination of directed
optogenetic activation of individual elements in cell populations
coupled with closed-loop recordings (Shemesh et al., 2017).
Another tool that is finally maturing are voltage sensors allowing
all optical electrophysiology to be coupled with optogenetics.
This has the potential of significantly moving the field forward
since the spiking of populations of MLR neurons can be
monitored to examine connectivity patterns (Fan et al., 2020).
Cell-specific activation of the PPN and CnF will be critical in
this endeavor as will be tagging activity with different behavioral
states.
What is missing is the analysis of network connectivity,
such as graph theory, to examine functional connectivity
during the performance of different locomotor behaviors
(Bassett and Sporns, 2017). An open question is whether the
current behavioral tests provide a realistic portrayal of the
diversity of behavioral states. To achieve this, we will need
to develop more naturalistic testing environments. Finally,
using diverse species to study MLR function is crucial (Chang
et al., 2021b). This is critical not only for the inherent value
of comparative biology but also for translational research
leading to the development of new therapeutic approaches
(Noga and Guest, 2021). This type of research is critical
to explain the side effects of stimulation seen in the use
of DBS, and indeed may necessitate the development and
use of circuit-specific viral tools to ameliorate specific gait
abnormalities.
CONCLUSIONS
Our understanding of the MLR has evolved and while
locomotion is one of the most reported outputs it has been
clear for some time that it contributes to other functions. This
is especially true of the PPN, where multiple motor behaviors
have been reported such as rearing, grooming, and grasping. The
PPN modulates other functions such as sleep-wake, arousal, and
control of cardiovascular and respiratory function. On the other
hand, the CnF while contributing to cardiovascular function, is
more of a bona fide locomotor center. In line with PPN’s multiple
roles, it shows a greater diversity of inputs than the CnF. In
closing, the diversity of functions of the MLR should be kept
in mind and it is our hope that this review encourages more
collaborations with those from respiratory, cardiovascular, and
motor neuroscientists.
Frontiers in Neural Circuits | www.frontiersin.org 8May 2022 | Volume 16 | Article 884785

Noga and Whelan The Diversity of the MLR
AUTHOR CONTRIBUTIONS
Both BN and PW contributed to the writing and editing of the
manuscript. All authors contributed to the article and approved
the submitted version.
FUNDING
PW acknowledges funds from Canadian Institutes of Health
Research (CIHR) Project Operating Grant (PJT-173511),
Natural Sciences and Engineering Research Council of
Canada (NSERC) Discovery Grants (RGPIN/04394-2019),
and the Frank LeBlanc Chair in Spinal Cord Injury Research.
This work was supported by the U.S. Department of
Defense (DOD) awards W81XWH-21-1-0791 (SC200294),
W81XWH-15-1-0584 (SC140238) and the National Institutes
of Neurological Disorders and Stroke (NINDS) grant R01
NS089972 to BN.
ACKNOWLEDGMENTS
PW gratefully acknowledges Dr. Manuel Hulliger for useful
conversations regarding the early history of locomotion. Figures
constructed using BioRender.com.
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