Neurotransmitter phenotypes of neurons in the spinal cord and their functional role in the mouse locomotor network

Abstract: Walking is a special case of movement because it is rhythmic. An important area in current neuroscience research, is to better understand the different organizational levels (molecular, ion channels, neurotransmitters, connectivity etc.) that implement the function of rhythmic movements. The isolated spinal cord from rodents can generate rhythmic patterns that resemble many aspects of walking. The networks that support this autonomous function are called central pattern generators or CPGs. In this thesis, we use the mouse spinal cord as a model system for studying walking. We describe the neurotransmitter phenotype, development and functional organization of populations directly involved in the normal and abnormal function of spinal locomotor networks. Work presented in this thesis show compelling evidence that motoneurons, the final output cell in the spinal cord, releases glutamate in addition to acetylcholine (ACh) at the central synapses but not at the neuromuscular junction. This discovery changed a long held view that motoneurons only release ACh and challenge Dales principle according to which the same neurotransmitter is released from all terminals in a single neuron (paper I). We also investigate the neurotransmitter phenotypes of the commissural interneurons (CINs) that have axons crossing in the midline. The CINs system is essential for walking because CINs are required to coordinate rhythmic activity between left and right side of the body. We used a number of transgenic mice to show that CINs located in the ventral spinal cord are glycinergic, GABAergic and glutamatergic and these neurotransmitters are expressed in almost equal numbers without any evident topographic segregation (paper II). The study of the murine CIN systems confirm the presence of a well-know strong glycinergic inhibitory crossing in vertebrates but also reveal the existence of important excitatory glutamatergic and inhibitory GABAergic components. During development a number of axon guidance pathways are active to set up the organization of the nervous system. One of these is the Eph and ephrin system. Knockout of one of the Eph receptors, the EphA4, or its ligand ephrinB3 leads to a hopping gait. We show that this hopping gait is due to a reconfiguration of the spinal CPG possibly caused by an increased over-crossing of excitatory spinal neurons (paper III). In continuation, we investigated candidate populations as source of the abnormal crossing. One of these are the V2 interneurons that we showed are all ipsilaterally projecting and composed of V2a excitatory neurons expressing the transcription factor Chx10 and V2b inhibitory neurons that express the transcription factor GATA2/3. Although many V2 interneurons express EphA4 we could not find evidence of abnormnal crossing in the V2a population in the EphA4 knockout mice (paper IV). Finally, we investigate the proportion of excitatory and inhibitory neurons crossing in the EphA4 knockout mice. We show that there is a significant increase in the proportion of excitatory crossing neurons accompanied by a decrease in proportion of inhibitory glycinergic crossing neuronsin homozygous EphA4 knockout mice (paper V). This study confirms that the hopping gait seen in the EphA4 mutants may be the consequence of an abnormal change in the balance between excitation and inhibition of crossing neurons. However, the study also showed how changing the assembly of the nervous system may have unpredictable consequences. The study furthermore suggests a much more complex regulation of axon guidance imposed by the EphA4 and ephrinB3 system than previously believed. The work presented in this thesis have used a panoply of different techniques, to reach a comprehensive description of the neurotransmitter phenotypes of a number of spinal neuronal populations and their role in the walking CPG.