Spinal cord networks that control motor output are dominated by a large variety of spinal interneurons with different properties and network roles. These interneurons were defined in the past by their function in reflex pathways, however a more modern view is based on developmental biology. We now define them by their developmental origins and genetic makeup.

Pioneering work in the early 2000’s defined eleven progenitor groups in the early neural tube giving rise to a limited set of cardinal neurons in the spinal cord. In the motor ventral horn, one progenitor domain generates all motoneurons and four other progenitor domains give rise to four classes of interneurons. These are defined by their early genetic makeup and transcription factor expression. Each cardinal group shares a similar neurotransmitter phenotype and the initial trajectory and targeting of their axons (ipsilateral, contralateral, ascending or descending). They greatly diversify and acquire novel functions during development and also throughout the vertebrate phylogeny to adapt to the different demands of the motor system as species evolved. We focus on one ventral interneuron class that forms ipsilateral inhibitory connections known as V1 interneurons. The current work in the lab is defining the subclasses of interneurons derived from the V1 group, what are their distinctive neurochemical and electrophysiological properties, how they become integrated in the ventral horn synaptic network, acquired their synaptic inputs and electrophysiological properties and what is their impact in motor behaviors after silencing or deleting them using cell-specific genetic approaches.

Techniques: In this project we use intersectional genetic approaches in transgenic mice to lineage label subclasses of V1 interneurons and follow them from neurogenesis to maturation to study the emergence of their distinct properties and connectivity. These are tested by a variety of neuroanatomical, developmental biology, in vitro electrophysiology (patch-clamp whole cell recording) and in vivo motor behaviors (kinematics and chronic EMGs during rhythmic locomotion).

Following injury to peripheral nerves, motor and sensory axons actively regenerate in the periphery. These axons eventually reinnervate muscles such that motor output, muscle force and sensory encoding of muscle properties return. However, motor function recovery is usually disappointing. We are interested in the synaptic plasticity that occurs inside the spinal cord triggered by nerve injuries and how it affects motor function after peripheral nerve regeneration.

We found that many changes in the ventral spinal cord motor circuits are related to the neuroinflammatory response that occurs centrally after peripheral nerve injuries. In particular we are interested in revealing interactions between microglia, peripheral immune cells and motoneurons and central synapses of axons injured in the periphery (sensory and motor axons). We investigate the neuroinflammatory response using genetic tools to label or modify specfic elements and test hypothesis on mechanisms that result in either protection or deletion of synapses and/or neurons after their axotomy in the peripheral nerve.

Techniques: This work involves analyzing synaptology on 3D neuron reconstructions, following with genetic tagging the demise of specific synapses in the ventral horn, studying the infiltration of peripheral immune cells and their communication with microglia resulting in synaptic deletions, directly observing using two-photon microscopy microglia behaviors and their relation with axotomized motoneurons and the central branches of peripehrally injured sensory afferents. Finally, we use physiologial tests to analyze motor function after interfering with specific elements of the neuroinflammatory response to improve synaptic preservation.

Motoneuron axotomy after nerve injuries induces a number of changes in the motoneuron. These include changes in synaptic composition, increased excitability and we also found that the isoform two of the potassium chloride co-transporter, KCC2, is downregulated. We are trying to elucidate mechanisms that remove KCC2 from the motoneuron surface and its functional significance.

KCC2 extrudes chloride and sets the low intracellular concentration of chloride characteristic of adult neurons. Since chloride carries most of the current at inhibitory GABAergic and glycinergic synapses, it is well known that KCC2 downregulation shifts these synapses towards more depolarizing and excitatory. The significance of this downregulation is unknown, but we hypothesize that in conjunction with the changes in synaptic composition and excitability it might regulate motoneuron activity in a way that promotes axon regeneration.

Techniques: Confocal microscopy, ICC and RNAscope to study KCC2 gene expression and localization to the membrane after interfering with specific signaling pathways. Two photon microscopy of chloride content in the cell body and dendrites of axotomized motoneurons. Electrophysiological and electron microscopy analyses of GABA synapses on the cell body of axotomized motoneurons. Testing hypothesis on axon regeneration by analyzing the recovery of EMG M responses and the reinnervation of neuromuscular junctions after interfering with either KCC2 downregulation or the activity of inhibitory synapses targeting the cell body of axotomized motoneurons.