Neural networks in the spinal cord known as central pattern generators produce the sequential activation of muscles needed for locomotion. The overall locomotor network architectures in limbed vertebrates have been much debated, and no consensus exists as to how they are structured. Here, we use optogenetics to dissect the excitatory and inhibitory neuronal populations and probe the organization of the mammalian central pattern generator. We find that locomotor-like rhythmic bursting can be induced unilaterally or independently in flexor or extensor networks. Furthermore, we show that individual flexor motor neuron pools can be recruited into bursting without any activity in other nearby flexor motor neuron pools. Our experiments differentiate among several proposed models for rhythm generation in the vertebrates and show that the basic structure underlying the locomotor network has a distributed organization with many intrinsically rhythmogenic modules.channelrhodopsin-2 | halorhodopsin | motor neurons | interneurons S pinal cord networks that drive and coordinate walking are, to a large extent, innate. Even in species that do not walk at birth, such as rodents and humans, the networks that generate walking are already present (1, 2). The early development of functional locomotor networks has been exploited and studied in the neonatal rodent spinal cord in vitro preparation in which locomotor-like activity can be induced pharmacologically or by electrical activation of descending or afferent fibers (3-6). The mammalian locomotor networks have also been studied by using the adult cat where locomotor-like activity similarly can be induced pharmacologically or electrically (7-9). A general notion from these studies is that forelimb and hindlimb locomotion are controlled by independent limb-controlling circuits (10) and that rhythm-generating excitatory neurons, and pattern-generating neurons, interact to produce the coordinated motor output (11)(12)(13)(14). The network layout within these limb-controlling circuits has, however, been debated. A number of conceptual models have been advanced. The classical half-center model asserts that flexor and extensor bursting is generated by two reciprocally coupled half-centers driving all flexors and extensors (8, 15). The flexor burst generator model is asymmetric and consists of a flexor burst generator that provides active excitation of flexor motor neurons and inhibition of extensor motor neurons that are otherwise tonically active (14,16,17). In response to evidence that the central pattern generator (CPG) could produce a more complex motor output than just a mere flexor-extensor alternation (9), the unit burst generator (UBG) model was proposed (18). According to this theory, separate modules can generate a rhythm in close muscle synergies and are distributed around each joint (18,19), or in the swimming network in each hemisegment (20). The UBGs therefore generate a local rhythmic activity that during locomotion will be recruited so that they form an interconnected n...

Central pattern generators (CPGs) are spinal neuronal networks required for locomotion. Glutamatergic neurons have been implicated as being important for intrinsic rhythm generation in the CPG and for the command signal for initiating locomotion, although this has not been demonstrated directly. We used a newly generated vesicular glutamate transporter 2-channelrhodopsin2-yellow fluorescent protein (Vglut2-ChR2-YFP) mouse to directly examine the functional role of glutamatergic neurons in rhythm generation and initiation of locomotion. This mouse line expressed ChR2-YFP in the spinal cord and hindbrain. ChR2-YFP was reliably expressed in Vglut2-positive cells and YFP-expressing cells could be activated by light. Photo-stimulation of either the lumbar spinal cord or the caudal hindbrain was sufficient to both initiate and maintain locomotor-like activity. Our results indicate that glutamatergic neurons in the spinal cord are critical for initiating or maintaining the rhythm and that activation of hindbrain areas containing the locomotor command regions is sufficient to directly activate the spinal locomotor network.

Muscle atrophy in many conditions share a common mechanism in the upregulation of the muscle-specific ubiquitin E3-ligases atrophy gene-1/muscle atrophy F-box (Atrogin-1/MAFbx) and muscle ring-finger protein 1 (MuRF1). E3-ligases are part of the ubiquitin proteasome pathway utilized for protein degradation during muscle atrophy. In this study, we provide new data to show that this is not the case in age-related loss of muscle mass (sarcopenia). On the contrary, Atrogin-1/MAFbx and MuRF1 are downregulated in skeletal muscle of 30-month-old rats, and our results suggest that AKT (protein kinase B)-mediated inactivation of forkhead box O 4 (FOXO4) underlies this suppression. The data also suggest that activation of AKT is mediated through the insulin-like growth factor-1 (IGF-1) receptor, signaling via ShcA-Grb2-GAB. Using dietary restriction, we find that it impedes sarcopenia as well as the effects of aging on AKT phosphorylation, FOXO4 phosphorylation, and Atrogin-1/MAFbx and MuRF1 transcript regulation. We conclude that sarcopenia is mechanistically different from acute atrophies induced by disuse, disease, and denervation.

Maintaining constant CO2 and H+ concentrations in the arterial blood is critical for life. The principal mechanism through which this is achieved in mammals is the respiratory chemoreflex whose circuitry is still elusive. A candidate element of this circuitry is the retrotrapezoid nucleus (RTN), a collection of neurons at the ventral medullary surface that are activated by increased CO2 or low pH and project to the respiratory rhythm generator. Here, we use intersectional genetic strategies to lesion the RTN neurons defined by Atoh1 and Phox2b expression and to block or activate their synaptic output. Photostimulation of these neurons entrains the respiratory rhythm. Conversely, abrogating expression of Atoh1 or Phox2b or glutamatergic transmission in these cells curtails the phrenic nerve response to low pH in embryonic preparations and abolishes the respiratory chemoreflex in behaving animals. Thus, the RTN neurons expressing Atoh1 and Phox2b are a necessary component of the chemoreflex circuitry.DOI: http://dx.doi.org/10.7554/eLife.07051.001

Neural networks in the spinal cord control two basic features of locomotor movements: rhythm generation and pattern generation. Rhythm generation is generally considered to be dependent on glutamatergic excitatory neurons. Pattern generation involves neural circuits controlling left-right alternation, which has been described in great detail, and flexor-extensor alternation, which remains poorly understood. Here, we use a mouse model in which glutamatergic neurotransmission has been ablated in the locomotor region of the spinal cord. The isolated in vitro spinal cord from these mice produces locomotor-like activity-when stimulated with neuroactive substances-with prominent flexor-extensor alternation. Under these conditions, unlike in control mice, networks of inhibitory interneurons generate the rhythmic activity. In the absence of glutamatergic synaptic transmission, the flexor-extensor alternation appears to be generated by Ia inhibitory interneurons, which mediate reciprocal inhibition from muscle proprioceptors to antagonist motor neurons. Our study defines a minimal inhibitory network that is needed to produce flexor-extensor alternation during locomotion.