Kiehn Lab

Physiological and molecular organization of neuronal networks controlling movements in mammals

Movement takes many forms. Among movements, locomotion is one of the most fundamental—used by all animals and humans for interaction with the surroundings. Locomotion is employed episodically in many daily activities, representing an output measure for integrated brain activity involved in exploring the environment, escaping predators, and searching for food. Its activity also directly influences the state of sensory information processing.

The planning and initiation of locomotion takes place in the brain and brainstem, while the execution—which involves precise timing and coordination—is to a large part accomplished by activity within neuronal networks of the spinal cord itself.

Early work from our lab has revealed aspects of the overall organization of spinal locomotor networks, the implication of cellular properties for rhythmicity, and the nature of neuronal spike coding.

Recent work has focused on the functional organization of key neuronal elements that characterize limbed locomotion in mammals: 1) rhythm generation, 2) coordination of flexors and extensors across the same or different joints in a limb or between limbs, and 3) left/right coordination.

Rhythm generating neurons set tempo within the network, and are an elementary component of all vertebrate locomotor networks. Experiments from our lab using genetically driven expression of light-sensitive channels in excitatory and inhibitory neurons have demonstrated that excitatory neurons in the mammalian spinal cord are both sufficient and necessary for initiation and maintenance of rhythmic locomotor pattern. Using intersectional mouse genetics in combination with electrophysiology, we have identified non-overlapping subpopulations of excitatory neurons in the spinal cord that participate in rhythm generation.

We have also characterized networks involved in left-right coordination, which include commissural interneurons (CINs) whose axons cross the midline, in detail using anatomical, electrophysiological, and targeted genetic ablation techniques for molecularly defined subpopulations of CINs.

These studies have converged on a common organizational principle for circuits controlling left-right alternation in mammals, which consists of a modular organization for left-right alternating gaits (walk and trot) and synchronous gaits (gallop/bound). Moreover, we have addressed the organization of circuits controlling flexor-extensor coordination. We are now addressing the functional integration of these diverse circuit elements using both in vitro and in vivo studies.

In a general scheme of motor control, we study how spinal locomotor circuits are activated and controlled by descending command signals. Decision-making signals to locomote are conveyed from the brain to locomotor regions in the midbrain. The locomotor regions in the midbrain include the mesencephalic locomotor region (MLR)—a complex structure—that in turn is thought to activate neurons in the reticular formation (RF) in the lower brainstem, which project to locomotor networks in the spinal cord. In the first optogenetics experiments in the mammalian locomotor system, we provided direct evidence that glutamatergic neurons in the lower brainstem can provide a ‘go’- signal sufficient to activate spinal locomotor networks.

We have now implemented in vivo optogenetic and chemogenetic approaches to probe the involvement of locomotor promoting and locomotor arresting areas in the brainstem, and further explore how these brainstem circuits are selected by upstream circuitries. These experiments have defined ‘start’ neurons in the midbrain, confined to the cuneiform nucleus and pedunculopontine nucleus, which cooperate to set locomotor speed and context-dependent selection of locomotor gate.

We have also defined ‘stop’ neurons in nucleus gigantocellularis that arrest ongoing locomotion.

In future studies we will use Parkinson disease models to probe the role of different motor structures in development and encoding of disease-induced gait disturbances.

Cellular mechanism underlying spasticity after spinal cord injury
Severe muscle spasticity develops as a consequence of spinal cord injury or damage to motor pathways from the brain. We previously found evidence that the pathophysiology of spasticity after spinal cord injury is related to chronic expression of plateau properties in motor neurons. Plateau potentials in vertebrate motor neurons are caused by activation of prolonged sodium/calcium currents, and their expression is dependent on activation of noradrenergic and/or serotoninergic receptors. The normal function of motor neuron plateau potentials seems to be to maintain persistent motor output and amplify synaptic inputs during rhythmic motor activity.

To find possible targets for regulation of plateau potentials after spinal cord injury, we have performed global gene expression profiling from rat motor neurons isolated before or after injury to the cord. These studies have shown that the chronic expression of plateaux may be related to changes in genes coding for the regulatory units for persistent sodium and calcium channels. In ongoing experiments, we are investigating how changes in interneuronal network activity interact with plateaux to generate spasticity using mouse genetic, calcium imaging, and in vivo optogenetic experiments. Our long-term goal is to define new therapies for symptoms associated with spinal cord injury. 

Our lab has developed a broad experimental repertoire, that includes:

1. Production of transgenic mice lines:  Vglut2^Cre (Borgius et al. 2010), Vglut2^GFP (Calderia et al. 2017), Vglut2^ChR2 (Hagglund et al. 2010), Hoxb8^Flipo, R26CAG^{frt/stop/flxstopGaMP3} tailored for the locomotor experiments.
2. Application of genetically driven tracing: trans-synaptic tracing with attenuated rabies/AAV.
3. mRNA seq of interneurons in the spinal cord.
4. In vitro electrophysiology in isolated spinal cord-brainstem, including calcium- imaging and optogenetics.
5. Gait analyses.
6. In vivo electrophysiology, optogenetic, chemogenetic, and motor neurobehavioral analyses of acute and chronic perturbations (MotoRater; open  field, treadmill etc).
7. Clarity-clearing of tissue and imaging.
8. In situ hybridization.

These tools provide a solid basis for the sophisticated functional and network studies needed to understand the principal mode of operation of a large-scale mammalian motor circuits.