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.
Subscribe to Journal
Get full journal access for 1 year
We are sorry, but there is no personal subscription option available for your country.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Grillner, S. The motor infrastructure: from ion channels to neuronal networks. Nat. Rev. Neurosci. 4, 573–586 (2003).
Feldman, J.L. & Del Negro, C.A. Looking for inspiration: new perspectives on respiratory rhythm. Nat. Rev. Neurosci. 7, 232–242 (2006).
Kiehn, O. Locomotor circuits in the mammalian spinal cord. Annu. Rev. Neurosci. 29, 279–306 (2006).
Dubuc, R. et al. Initiation of locomotion in lampreys. Brain Res. Rev. 57, 172–182 (2008).
Jordan, L.M., Liu, J., Hedlund, P.B., Akay, T. & Pearson, K.G. Descending command systems for the initiation of locomotion in mammals. Brain Res. Rev. 57, 183–191 (2008).
Schmidt, B.J. & Jordan, L.M. The role of serotonin in reflex modulation and locomotor rhythm production in the mammalian spinal cord. Brain Res. Bull. 53, 689–710 (2000).
Kiehn, O., Kjaerulff, O., Tresch, M.C. & Harris-Warrick, R.M. Contributions of intrinsic motor neuron properties to the production of rhythmic motor output in the mammalian spinal cord. Brain Res. Bull. 53, 649–659 (2000).
Nishimaru, H. & Kudo, N. Formation of the central pattern generator for locomotion in the rat and mouse. Brain Res. Bull. 53, 661–669 (2000).
Clarac, F., Pearlstein, E., Pflieger, J.F. & Vinay, L. The in vitro neonatal rat spinal cord preparation: a new insight into mammalian locomotor mechanisms. J. Comp. Physiol. A Neuroethol. Sens. Neural Behav. Physiol. 190, 343–357 (2004).
Kullander, K. et al. Role of EphA4 and EphrinB3 in local neuronal circuits that control walking. Science 299, 1889–1892 (2003).
Lanuza, G.M., Gosgnach, S., Pierani, A., Jessell, T.M. & Goulding, M. Genetic identification of spinal interneurons that coordinate left-right locomotor activity necessary for walking movements. Neuron 42, 375–386 (2004).
Gosgnach, S. et al. V1 spinal neurons regulate the speed of vertebrate locomotor outputs. Nature [see comment] 440, 215–219 (2006).
Crone, S.A. et al. Genetic ablation of V2a ipsilateral interneurons disrupts left-right locomotor coordination in mammalian spinal cord. Neuron 60, 70–83 (2008).
Zhang, Y. et al. V3 spinal neurons establish a robust and balanced locomotor rhythm during walking. Neuron 60, 84–96 (2008).
Zhang, F., Aravanis, A.M., Adamantidis, A., de Lecea, L. & Deisseroth, K. Circuit-breakers: optical technologies for probing neural signals and systems. Nat. Rev. Neurosci. 8, 577–581 (2007).
Adamantidis, A.R., Zhang, F., Aravanis, A.M., Deisseroth, K. & de Lecea, L. Neural substrates of awakening probed with optogenetic control of hypocretin neurons. Nature 450, 420–424 (2007).
Huber, D. et al. Sparse optical microstimulation in barrel cortex drives learned behavior in freely moving mice. Nature 451, 61–64 (2008).
Gradinaru, V., Mogri, M., Thompson, K.R., Henderson, J.M. & Deisseroth, K. Optical deconstruction of parkinsonian neural circuitry. Science 324, 354–359 (2009).
Cardin, J.A. et al. Driving fast-spiking cells induces gamma rhythm and controls sensory responses. Nature 459, 663–667 (2009).
Sohal, V.S., Zhang, F., Yizhar, O. & Deisseroth, K. Parvalbumin neurons and gamma rhythms enhance cortical circuit performance. Nature 459, 698–702 (2009).
Pulver, S.R., Pashkovski, S.L., Hornstein, N.J., Garrity, P.A. & Griffith, L.C. Temporal dynamics of neuronal activation by Channelrhodopsin-2 and TRPA1 determine behavioral output in Drosophila larvae. J. Neurophysiol. 101, 3075–3088 (2009).
Liewald, J.F. et al. Optogenetic analysis of synaptic function. Nat. Methods 5, 895–902 (2008).
Douglass, A.D., Kraves, S., Deisseroth, K., Schier, A.F. & Engert, F. Escape behavior elicited by single, channelrhodopsin-2–evoked spikes in zebrafish somatosensory neurons. Curr. Biol. 18, 1133–1137 (2008).
Arenkiel, B.R. et al. In vivo light-induced activation of neural circuitry in transgenic mice expressing channelrhodopsin-2. Neuron 54, 205–218 (2007).
Wang, H. et al. High-speed mapping of synaptic connectivity using photostimulation in Channelrhodopsin-2 transgenic mice. Proc. Natl. Acad. Sci. USA 104, 8143–8148 (2007).
Fremeau, R.T. Jr. et al. The expression of vesicular glutamate transporters defines two classes of excitatory synapse. Neuron 31, 247–260 (2001).
Fremeau, R.T. Jr., Voglmaier, S., Seal, R.P. & Edwards, R.H. VGLUTs define subsets of excitatory neurons and suggest novel roles for glutamate. Trends Neurosci. 27, 98–103 (2004).
Wilson, J.M. et al. Conditional rhythmicity of ventral spinal interneurons defined by expression of the Hb9 homeodomain protein. J. Neurosci. 25, 5710–5719 (2005).
Lundfald, L. et al. Phenotype of V2-derived interneurons and their relationship to the axon guidance molecule EphA4 in the developing mouse spinal cord. Eur. J. Neurosci. 26, 2989–3002 (2007).
Lein, E.S. et al. Genome-wide atlas of gene expression in the adult mouse brain. Nature 445, 168–176 (2007).
Jessell, T.M. Neuronal specification in the spinal cord: inductive signals and transcriptional codes. Nat. Rev. Genet. 1, 20–29 (2000).
Crone, S.A., Zhong, G., Harris-Warrick, R. & Sharma, K. In mice lacking V2a interneurons, gait depends on speed of locomotion. J. Neurosci. 29, 7098–7109 (2009).
Restrepo, C.E. et al. Transmitter-phenotypes of commissural interneurons in the lumbar spinal cord of newborn mice. J. Comp. Neurol. 517, 177–192 (2009).
Nagel, G. et al. Channelrhodopsin-2, a directly light-gated cation-selective membrane channel. Proc. Natl. Acad. Sci. USA 100, 13940–13945 (2003).
Boyden, E.S., Zhang, F., Bamberg, E., Nagel, G. & Deisseroth, K. Millisecond-timescale, genetically targeted optical control of neural activity. Nat. Neurosci. 8, 1263–1268 (2005).
Kiehn, O. & Kjaerulff, O. Distribution of central pattern generators for rhythmic motor outputs in the spinal cord of limbed vertebrates. Ann. NY Acad. Sci. 860, 110–129 (1998).
Zhong, G., Diaz-Rios, M. & Harris-Warrick, R.M. Serotonin modulates the properties of ascending commissural interneurons in the neonatal mouse spinal cord. J. Neurophysiol. 95, 1545–1555 (2006).
Bracci, E., Ballerini, L. & Nistri, A. Spontaneous rhythmic bursts induced by pharmacological block of inhibition in lumbar motoneurons of the neonatal rat spinal cord. J. Neurophysiol. 75, 640–647 (1996).
Zaporozhets, E., Cowley, K.C. & Schmidt, B.J. Propriospinal neurons contribute to bulbospinal transmission of the locomotor command signal in the neonatal rat spinal cord. J. Physiol. (Lond.) 572, 443–458 (2006).
Reed, W.R., Shum-Siu, A. & Magnuson, D.S. Reticulospinal pathways in the ventrolateral funiculus with terminations in the cervical and lumbar enlargements of the adult rat spinal cord. Neuroscience 151, 505–517 (2008).
Liu, J. & Jordan, L.M. Stimulation of the parapyramidal region of the neonatal rat brain stem produces locomotor-like activity involving spinal 5-HT7 and 5-HT2A receptors. J. Neurophysiol. 94, 1392–1404 (2005).
Kiehn, O. & Kjaerulff, O. Spatiotemporal characteristics of 5-HT and dopamine-induced rhythmic hindlimb activity in the in vitro neonatal rat. J. Neurophysiol. 75, 1472–1482 (1996).
Aravanis, A.M. et al. An optical neural interface: in vivo control of rodent motor cortex with integrated fiberoptic and optogenetic technology. J. Neural Eng. 4, S143–S156 (2007).
McLean, D.L., Masino, M.A., Koh, I.Y., Lindquist, W.B. & Fetcho, J.R. Continuous shifts in the active set of spinal interneurons during changes in locomotor speed. Nat. Neurosci. 11, 1419–1429 (2008).
Roberts, A., Soffe, S.R., Wolf, E.S., Yoshida, M. & Zhao, F.Y. Central circuits controlling locomotion in young frog tadpoles. Ann. NY Acad. Sci. 860, 19–34 (1998).
Magnuson, D.S. & Trinder, T.C. Locomotor rhythm evoked by ventrolateral funiculus stimulation in the neonatal rat spinal cord in vitro. J. Neurophysiol. 77, 200–206 (1997).
Marchetti, C., Beato, M. & Nistri, A. Alternating rhythmic activity induced by dorsal root stimulation in the neonatal rat spinal cord in vitro. J. Physiol. (Lond.) 530, 105–112 (2001).
Bonnot, A., Whelan, P.J., Mentis, G.Z. & O'Donovan, M.J. Locomotor-like activity generated by the neonatal mouse spinal cord. Brain Res. Brain Res. Rev. 40, 141–151 (2002).
Stornetta, R.L., Sevigny, C.P. & Guyenet, P.G. Vesicular glutamate transporter DNPI/VGLUT2 mRNA is present in C1 and several other groups of brainstem catecholaminergic neurons. J. Comp. Neurol. 444, 191–206 (2002).
Parkitna, J.R., Engblom, D. & Schutz, G. Generation of Cre recombinase-expressing transgenic mice using bacterial artificial chromosomes. Methods Mol. Biol. 530, 325–342 (2009).
We thank K. Deisseroth for providing us with the ChR2 construct, D. Engblom for technical assistance with BAC cloning and the Karolinska Center for Transgenic Technologies for pronuclear injection. This work was supported by grants from the US National Institutes of Health (R01NS040795-08), the European Union (SPINAL CORD REPAIR), the National Science Foundation (0701166 to K.J.D.), and by the Swedish Medical Research Council, the Karolinska Institutet's foundation for support of graduate students (M.H.) and postdocs (L.B.), Söderbergs Foundation, and Friends of Karolinska Intitutet.
About this article
Cite this article
Hägglund, M., Borgius, L., Dougherty, K. et al. Activation of groups of excitatory neurons in the mammalian spinal cord or hindbrain evokes locomotion. Nat Neurosci 13, 246–252 (2010) doi:10.1038/nn.2482
Myelinated axons and functional blood vessels populate mechanically compliant rGO foams in chronic cervical hemisected rats
Glutamatergic neurons of the gigantocellular reticular nucleus shape locomotor pattern and rhythm in the freely behaving mouse
PLOS Biology (2019)
Current Opinion in Physiology (2019)
Current Opinion in Physiology (2019)
Current Opinion in Physiology (2019)