My greatest highlight of monday at Neuroscience 2017 has been this minisymposium, centered in the modulation of locomotion at the level of the spinal cord, which was chaired by Dr. Patrick J. Whelan.
Activity-dependent regulation of spinal motor networks by sodium-potassium pumps. By Dr. Gareth Miles.
The first talk of the minisymposium was Activity-dependent regulation of spinal motor networks by sodium-potassium pumps, by Dr. Gareth Miles, from the University of St. Andrews (Scotland).
The motor system is capable of generating locomotor patterns with different velocities, for example when walking or running. This depends partially on the excitability of the neurons that integrate this network, which depends on their activity. However, what controls this excitability, has not yet been elucidated.
Sodium-potassium pumps can express different alpha subunits in the central nervous system: mostly α1 or α3. α3 sodium-potassium pumps have a lower affinity for intracellular sodium, and a higher affinity for ouabain, an inhibitor of the sodium-potassium pump. They are only recruited after intense neuronal firing.
α3 sodium pumps mediate ultra slow afterhyperpolarizations (usAHP, hyperpolarizations that last for up to 60 seconds after intense neuronal activity). These are present in about 50% of rodent motoneurons, 5% of all interneurons and 50% of Pitx2 interneurons.
The usAHP in motoneurons is blocked by tetrodotoxin and ouabain (therefore dependent on sodium, and the sodium-potassium pump), but enhanced by dopamine.
In the preparation of spinal cord in vitro, blocking the sodium-potassium pump with ouabain increases the duration and frequency of fictive locomotion. Inversely, the sodium ionophore monensin, which increases the function of sodium-potassium pump, decreases the frequency of fictive locomotion.
In the same preparation, when Dr. Miles’ team stimulated the spinal cord in control animals, and produce a second sensory stimulation 100 seconds later (after the 60 seconds of usAHP), they found no difference in the amplitude of the second response. However, when the second stimulation took place only 15 seconds after the first one, the second response was reduced. The interesting news are that, when this second stimulation 15 seconds after the first one takes place in the presence of ouabain, the second response is not diminished. So, the α3 sodium-potassium pump would be encoding a certain “short-term memory” of the first pulse, which would have an influence over the amplitude of the second one. This mechanism, acting at the motoneurons and Pitx2 interneurons influences the output of the locomotor network, modifying its output according to recent activity.
This mechanism is phylogenetically conserved, being present in Drosophila larvae, in Xenopus, and, as it has been shown here, in neonatal mice.
To conclude with his intervention, Dr. Miles’ group has also found this mechanism on human induced pluripotent stem cells, so be attentive, for these results will soon be published.
Contribution of DSCAM in the normal development of motor circuits. By Dr. Frédéric Bretzner.
The second talk of the minosymposium was Contribution of DSCAM in the normal development of motor circuits, by Dr. Frédéric Bretzner, from Université Laval (Canada).
Down syndrome cell-adhesion molecule (DSCAM) is expressed during development, it is implicated on axonal guidance, axonal fasciculation, dendritic arborization and on the formation and maintenance of a synapse. Although there is evidence that DSCAM is important for the normal development of neural circuits, little is known about its functional contribution to spinal motor circuits.
Dr. Bretzner’s team found that adult DSCAM2J mice –lacking DSCAM- have an aberrant posture, an aberrant gait, and a higher variability of locomotor pattern and rhythm on treadmill locomotion. Its performance in treadmill locomotion is more varied, but maladaptive: the wild type starts increasing walking velocity until it changes gait to run, while the DSCAM mice, at first does not walk normally, and when increasing velocity, instead of running, it performs other types of gait, less efficient.
On fictive locomotion, DSCAM2J mice show a reduced left-right coordination. Through a retrograde tracing study, the team found that commissural interneurons are increased in DSCAM mice compared to controls, which likely contributes to reducing left-right alternation, affecting locomotion.
The work from Dr. Bretzner’s lab points to DSCAM as a necessary protein for the normal development of spinal locomotor and sensorimotor circuits.
On their latest work, Dr. Bretzner’s lab produced two more mice strains, each with a conditional DSCAM mutation, either on vGluT2 or vGAT neurons. They found a slower locomotor rhythm, reduced ipsilateral inhibition and reduced number of V1 neurons on vGluT2 mutant, and a faster locomotor rhythm, increased ipsilateral inhibition, and V1 on vGAT mutant. All these results and much more, will soon be published.
Contribution of non-linear firing behaviors in locomotor function and dysfunction. By Dr. Frédéric Brocard.
The third talk of the minisymposium was Contribution of non-linear firing behaviors in locomotor function and dysfunction, by Dr. Frédéric Brocard, from Aix-Marseille Université (France).
Spinal motoneurons are not simply the final common pathway for movement, passively driven by synaptic input. These neurons possess a number of important nonlinear properties that can be triggered by synaptic input, but they themselves shape their own output. The most distinctive nonlinear firing property is the generation of self-sustained tonic spiking evoked by a brief excitation, that arises in the form of a “plateau potential” which might be important for the maintenance of a postural tone. Another distinctive nonlinear firing has been found on interneurons named “pacemaker cells”, from the locomotor rhythm generating network. They are characterized by the ability to generate repetitive bursting discharges that might contribute to generate the locomotor rhythm.
Both nonlinear patterns (plateau and pacemaker) are dependent on a persistent sodium current. Dr. Frédéric Brocard’s laboratory studies sodium channels mediating these currents on rodents, and their contribution to the normal and abnormal function of the motor system.
Persistent sodium currents are necessary for the locomotor rhythm: when adding riluzole (a compound that reduces persistent inward sodium currents) to the bath on a model of fictive locomotion, the locomotor rhythm disappears.
KO mice for voltage-activated sodium channel NaV1.6 show motor deficits. These mice do not show an impairement neither on their pacemaker properties nor on the fictive locomotor rhythm. Also, NaV1.6 is not highly expressed in interneurons, so NaV1.6 does not seem to be critical for fictive locomotion and for the function of locomotor CPG.
However, this channel is necessary for plateau properties of motoneurons. Accordingly, NaV1.6 is strongly expressed in the initial segment of the axon of motoneurons.
After spinal cord injury, an upregulation of the persistent sodium current contributes to an overexpression of plateau properties on motoneurons, which is related to a hyperexcitability of the motor response. Spasticity -increased, involuntary, velocity-dependent muscle tone that causes resistance to movement- can arise after a spinal cord injury, and it is considered to be caused by a hyperexcitability of the motor response. Consequently, the group of Dr. Brocard has investigated whether there would be an alteration of NaV1.6 related to this condition.
NaV1.6 membrane expression is increased after a spinal cord injury in adult rats, and the Western blot analysis shows that NaV1.6 is cleaved on this condition. The team of Dr. Brocard demonstrated that NaV1.6 is sensitive to the calcium-activated protease calpain, which cleaves the inactivation gate of NaV1.6. Therefore, NaV1.6 channels remain open for a longer period causing an increase of the persistent sodium current.
Adult rats with a spinal cord transection, chronically injected with an inhibitor of calpain show a lower persistent sodium current, and a reduced cleavage of NaV1.6.
Currently, there is no treatment to reduce calpain function in human patients. However, the above mentioned riluzole, is approved for the treatment of amyotrophic lateral sclerosis. The group of Dr. Brocard has started a clinical essay to test the effects of Rilutek (the commercial name of riluzole) in reducing spasticity and hyperreflexia on spinal cord injury patients. Be attentive to avoid missing their next results!
Light on a sensory interface linking cerebrospinal fluid to motor circuits in vertebrates. By Dr. Claire Wyart.
The fourth talk of the minisymposium was Light on a sensory interface linking cerebrospinal fluid to motor circuits in vertebrates, by Dr. Claire Wyart, from Institut du Cerveau et de la Moelle Épinière (France).
The research of Dr. Claire Wyart has a slightly different center of interest than the previous talks, focusing on the sensory interface between motor systems and cerebrospinal fluid: the cerebrospinal fluid contacting neurons (CSF-cNs).
CSF-cNs have been identified in more than 200 species, so, as usAHP, they are phylogenetically conserved. CSF-cNs have an apical extension containing microvilli and one motile cilium –which beats spontaneously-. They are GABAergic, and they are characterized by the expression of a specific marker: PKD2L1.
CSF-cNs respond to mechanical stimuli: they can detect the curvature of the central canal of the spinal cord. These mechanosensory responses depend on their apical extension. The function of CSF-cNs seems to be a regulation of locomotion: when the fish are static, the optogenetic activation of CSF-cNs induces locomotion, however, when fish are engaged on a fast swimming movement, the activation of CSF-cNs reduces the speed of locomotion. This modulation of the speed of locomotion is mediated by the synapses of CSF-cNs on excitatory interneurons that are part of the locomotor CPG.
The most recent work from Dr. Wyart’s lab shows that the ventral cilia in the spinal cord are more motile, and they beat cerebrospinal fluid on a rostral to caudal sense; conversely, on the dorsal side of the central canal where cilia are static, they beat cerebrospinal fluid on a caudal to rostral sense. Her team found also that the malfunction of CSF-cNs in pkd2l1 mutants is correlated with spine malformations in the adult. You will not have to wait for long to see these results (with more details) published!
Dopaminergic control of locomotion: uncovering parallel pathways for motor control. By Dr. Patrick Whelan.
The fifth talk of the minisymposium was Dopaminergic control of locomotion: uncovering parallel pathways for motor control, by Dr. Patrick Whelan, from the University of Calgary (Canada).
The lab of Dr. Patrick Whelan challenges the traditional vision of dopaminergic systems, which considers that dopamine effects on spinal cord locomotor networks are mostly indirect. There are dopaminergic projections and dopaminergic receptors in the spinal cord, and the absence of dopamine in this region generates locomotor deficits. Regarding these remarks, Dr. Whelan’s lab concluded that we had been missing something.
Dr. Whelan’s lab has defined the connectome of hypothalamic areas A11 and A13. Neurons in these two areas are non canonical dopaminergic neurons, expressing tyrosine hydroxilase, but not dopamine amino-transferase.
A13 neurons project to the cuneiform nucleus -which is directly implicated in locomotion-, and to the pedunculopontine nucleus –also implicated in locomotion, on a more indirect way-. A11 neurons project to the mesencephalic locomotor region (MLR), to the spinal cord, and also to the pedunculopontine nucleus.
When they stimulated these two regions by optogenetics, the stimulation of both areas increased the exploratory behavior of mice. In the case of A13, the movements were faster and more “robotic”, which suggested a more clear activation of locomotor networks –the activation of A11 produced also an increase in sniffing and other exploratory behaviors, unrelated to locomotion-.
The injection of dopamine in spinal cord injured rats augments extensor activity to improve weight bearing on treadmill locomotion. Considering this effect, together with the previous results on optogenetic stimulation, suggests that dopamine improves both locomotion and posture. But how can dopamine perform both effects?
Dr. Whelan’s team was interested by a paper by Marder et al. (2014) which showed that the effects of neuromodulators depend on the underlying state of the network they modulate. Following this research, the group of Dr. Whelan identified 4 states in the locomotor network, characterized by increasing excitability:
- A basal, submodulated state
- A tonic state
- A multi-rhythmic state
- The locomotor state
The team found that on an in vitro preparation (the decerebrated neonatal mouse model), the addition of 5-HT and NMA produced an alternating pattern of locomotion, however, the pattern was sometimes disturbed by other movements. They associate this pattern with the multi-rhythmic state of the network. The addition of dopamine to this preparation, allowed to move from a multi-rhythmic state to a clear, oscillatory, alternating locomotor rhythm.
Therefore, beyond improving posture, dopamine also allows the transition from the multi-rhythmic state, to the excitatory, alternating state that characterizes locomotion. Dopamine seems to provide an essential descending input to modulate the locomotor rhythm. This, together with its role in upregulating usAHP, from the talk of Gareth Miles, points to dopamine as a critical neuromodulator of the locomotor rhythm.
Brainstem descending cells involved in starting, maintaining and stopping locomotion. By Dr. Réjean Dubuc.
The last talk of the minisymposium was Brainstem descending cells involved in starting, maintaining and stopping locomotion, by Dr. Réjean Dubuc, from the Université du Québec à Montréal and Université de Montréal (Canada). For this last talk, we descended slightly, from the previous hypothalamus regions, to stay at the midbrain and the hindbrain. More specifically, at the mesencephalic locomotor region (MLR), which is very preserved phylogenetically, present in many vertebrates, including the lamprey.
For experiments, Dr. Dubuc’s lab used a preparation including the intact body of the lamprey, and the brain, including the MLR. The stimulation of MLR in this preparation elicits swimming in the lamprey body, and an increase in the intensity of stimulation, produces a more vigorous activity.
The MLR controls locomotion through two pathways:
-Bilateral projections to reticulospinal cells
-Projections to muscarinoceptive neurons, present in the brainstem. When activated, they project back to the reticulospinal cells to boost the locomotor rhythm
Down in the hindbrain, the reticulospinal neurons display 3 types of response:
-Start cells: they spike at the beginning of the MLR stimulation, and this burst is time-linked with the beginning of locomotion
-Sustained cells: they present plateau potentials and are active throughout the whole locomotor bout
-Stop cells: they spike at the beginning of the MLR stimulation, and at the end, and the termination burst is time-linked with the end of locomotion
It is possible to activate or inactivate stop cells with pharmacological D-glutamate stimulation: when reticulospinal stop cells are activated in the presence of glutamate during swimming, the body movement stops immediately. However, when reticulospinal stop cells are activated in the presence of glutamate antagonists, there is a change in the stopping of locomotion: the lamprey is capable to stop its swimming, but does so very slowly.
The termination of locomotion is time-linked with the second burst of stop neurons after stimulation. It is also dependent on the intensity of stimulation of the MLR :
-If the intensity of stimulation is low and sub-threshold, there is no effect when the animal is at rest.
-If the MLR is stimulated with this low intensity while the animal is swimming, there is a stop of locomotion
-If the intensity of stimulation is high, there is a prolongation of ongoing locomotion
To summarize, as we have seen in the previous talks, the modulation of motor circuits is less linear than previously thought. In the lamprey, apart from the descending excitatory input of the MLR, we find the contribution of muscarinic brainstem neurons, which modulate the locomotor rhythm by increasing the excitability of reticulospinal neurons. Moreover, the group of Dr. Dubuc found that the MLR is capable of stopping locomotor activity; a gating mechanism might be involved in the capability of stopping locomotion on an efficient way.
Altogether, the take home message of the six talks is that the modulation of locomotor networks is more complex and finely-tuned than we had previously expected. It depends on intracellular, intercellular, and network processes. We are increasing our knowledge of these modulation processes, but the motor systems still have mysteries to unravel, which can have applications for human health.
Activity-dependent regulation of spinal motor networks by sodium-potassium pumps, by Dr. Gareth Miles:
Picton LD, Zhang H, Sillar KT (2017). Sodium pump regulation of locomotor control circuits. J Neurophysiol 118(2):1070-81.
Picton LD, Nascimento F, Broadhead MJ, Sillar KT, Miles GB (2017). Sodium pumps mediate activity-dependent changes in mammalian motor networks. J Neurosci 37(4):906-21.
Pulver SR and Griffith LC (2010). Spike integration and cellular memory in a rhythmic network from Na+/K+ pump current dynamics. Nat Neurosci 13(1):53-9.
Zhang HY and Sillar KT (2012). Short-term memory of motor network performance via activity-dependent potentiation of Na+/K+ pump function. Curr Biol 22(6):526-31.
Zhang HY, Picton L, Li WC, Sillar KT (2015). Mechanisms underlying the activity-dependent regulation of locomotor network performance by the Na+ pump. Sci Rep 5:16188.
Contribution of DSCAM in the normal development of motor circuits, by Dr. Frédéric Bretzner:
Lemieux M, Laflamme OD, Thiry L, Boulanger-Piette A, Frenette J, Bretzner F (2016). Motor hypertonia and lack of locomotor coordination in mutant mice lacking DSCAM. J Neurophysiol 115(3):1355-71.
Thiry L, Lemieux M, Bretzner F (2017). Age- and speed-dependent modulation of locomotor gaits in DSCAM2J mutant mice. J Neurophysiol [Epub ahead of print]
Thiry L, Lemieux M, Laflamme DO and Bretzner F (2016). Role of DSCAM in the development of the spinal locomotor and sensorimotor circuits. J Neurophysiol 115(3):1338-54.
Contribution of non-linear firing behaviors in locomotor function and dysfunction, by Dr. Frédéric Brocard:
Bouhadfane M, Tazerart S, Moqrich A, Vinay L, Brocard F (2013). Sodium-mediated plateau potentials in lumbar motoneurons of neonatal rats. J Neurosci 33(39):15626-41.
Brocard C, Plantier V, Boulenguez P, Liabeuf S, Bouhadfane M, Viallat-Lieutaud A, Vinay L, Brocard F (2016). Cleavage of Na(+) channels by calpain increases persistent Na(+) current and promotes spasticity after spinal cord injury. Nat Med 22(4):404-11.
Brocard F, Shevtsova NA, Bouhadfane M, Tazerart S, Heinemann U, Rybak IA, Vinay L (2013). Activity-dependent changes in extracellular Ca2+ and K+ reveal pacemakers in the spinal locomotor-related network. Neuron 77(6):1047-54.
Brocard F, Tazerart S, Vinay L (2010). Do pacemakers drive the central pattern generator for locomotion in mammals? Neuroscientist 16(2):139-55.
Plantier V and Brocard F (2017). Spasticity, a new calpainopathy after a spinal cord injury. Med Sci (Paris) 33(6-7):629-36.
Tazerart S, Viemari JC, Darbon P, Vinay L, Brocard F (2007). Contribution of persistent sodium current to locomotor pattern generation in neonatal rats. J Neurophysiol 98(2):613-28.
Tazerart S, Vinay L, Brocard F (2008). The persistent sodium current generates pacemaker activities in the central pattern generator for locomotion and regulates the locomotor rhythm. J Neurosci 28(34):8577-89.
Light on a sensory interface linking cerebrospinal fluid to motor circuits in vertebrates, by Dr. Claire Wyart:
Böhm UL, Prendergast A, Djenoune L, Nunes Figueiredo S, Gomez J, Stokes C, Kaiser S, Suster M, Kawakami K, Charpentier M, Concordet JP, Rio JP, Del Bene F, Wyart C (2016). CSF-contacting neurons regulate locomotion by relaying mechanical stimuli to spinal circuits. Nat Commun 7:10866.
Fidelin K, Djenoune L, Stokes C, Prendergast A, Gomez J, Baradel A, Del Bene F, Wyart C (2015). State-dependent modulation of locomotion by GABAergic spinal sensory neurons. Curr Biol 25(23):3035-47.
Hubbard JM, Böhm UL, Prendergast A, Tseng PB, Newman M, Stokes C, Wyart C (2016). Intraspinal sensory neurons provide powerful inhibition to motor circuits ensuring postural control during locomotion. Curr Biol 26(21):2841-53.
Sternberg JR, Severi KE, Fidelin K, Gomez J, Ihara H, Alcheikh Y, Hubbard JM, Kawakami K, Suster M, Wyart C (2016). Optimization of a neurotoxin to investigate the contribution of excitatory interneurons to speed modulation in vivo. Curr Biol 26(17):2319-28.
Dopaminergic control of locomotion: uncovering parallel pathways for motor control, by Dr. Patrick Whelan:
Koblinger K, Füzesi T, Ejdrygiewicz J, Krajacic A, Bains JS, Whelan PJ (2014). Characterization of A11 neurons projecting to the spinal cord of mice. PLoS One 9(10):e109636.
Marder E, O’Leary T, Shruti S (2014). Neuromodulation of circuits with variable parameters: single neurons and small circuits reveal principles of state-dependent and robust neuromodulation. Annu Rev Neurosci 37:329-46.
Sharples SA, Humphreys JM, Jensen AM, Dhoopar S, Delaloye N, Clemens S, Whelan PJ (2015). Dopaminergic modulation of locomotor network activity in the neonatal mouse spinal cord. J Neurophysiol 113(7):2500-10.
Sharples SA, Koblinger K, Humphreys JM, Whelan PJ (2014). Dopamine: a parallel pathway for the modulation of spinal locomotor networks. Front Neural Circuits 8:55.
Sharples SA and Whelan PJ (2017). Modulation of rhythmic activity in mammalian spinal networks is dependent on excitability state. eNeuro 4(1).
Brainstem descending cells involved in starting, maintaining and stopping locomotion, by Dr. Réjean Dubuc:
Juvin L, Grätsch S, Trillaud-Doppia E, Gariépy JF, Büschges A, Dubuc R (2016). A specific population of reticulospinal neurons controls the termination of locomotion. Cell Rep 15(11):2377-86.
Smetana R, Juvin L, Dubuc R, Alford S (2010). A parallel cholinergic brainstem pathway for enhancing locomotor drive. Nat Neurosci 13(6):731-8.
Aix-Marseille Université, France
Universitat Autònoma de Barcelona, Spain
ICN PhD Program in Neuroscience: http://neuro-marseille.org/en/phd-program-en/le-phd-program/