Neuronal networks are the central functional units of the nervous system. Knowledge about the identity of participating neurons and the assembly of these during development is crucial for the understanding of CNS function. A promising system to dissect the development and functionalities of a neuronal network is the central pattern generator (CPG) for locomotion. We used screening approaches to identify spinal neuronal subpopulations by their specific gene expression, potentially involved in CPG function. Amongst others we found paired-like homeodomain transcription factor 2 (Pitx2) as a cholinergic interneuron marker for partition cells, with a possible role in the spinal network for locomotion. In addition, we present two genes, Chondrolectin (Chodl) and Estrogen-related receptor beta (ERRβ) as novel markers for fast and slow motor neurons, respectively. The neuronal components of the CPG integrate three key functions; rhythm generation, ipsilateral flexors/extensors coordination and bilateral coordination over the midline. Commissural interneurons (CINs) are considered to participate in the latter. During development axons are guided to their targets by the help of axon guidance molecules. Netrin-1 and its receptor DCC (Deleted in Colorectal Cancer) have been shown to play an important role for spinal cord neurons in axon-pathfinding and migration towards the midline. We show that loss of netrin-1 functionally results in a switch from alternating to synchronous left-right locomotor activity and deletion of DCC surprisingly leads to a different phenotype, best described as uncoordination. Thus, during development, netrin-1 and DCC play a critical role for the establishment of a functional balanced CPG. Further we show a selective loss of CINs, predominantly from dorsally originating subtypes, not affecting the ventral-most V3 subtype in netrin-1 mutant mice, but a loss of CINs from all progenitor domains in Dcc mutant mice. Together, our data suggest a netrin-1-independent mechanism for DCC in axon guidance and a role of the most ventral originating CINs as part of the neuronal network controlling synchronous activities over the midline. Another pair of axon guidance molecules, EphA4 and ephrinB3, has been shown to cooperate in preventing ipsilateral interneurons from crossing the spinal midline and if either molecule is deleted in mice, this will result in a defect in left-right coordination of locomotion. We provide in vivo and in vitro evidence that the GTPase-activating protein α2-chimerin, as a downstream molecule of EphA4 signaling, is essential in axon guidance decisions involved in the correct formation of the spinal circuitry for locomotion.

Acquiring knowledge of the morphological, molecular, and functional changes that occur to neurons following axotomy is a key step for a comprehensive understanding of the nervous system and how it reacts to injury. Propriospinal commissural interneurons (PCIs or CINs) are a class of neuron with axons that project through the ventral commissure to the contralateral spinal cord. My goal was to examine the morphological, molecular, and functional changes that occur to adult feline PCIs following a proximal axotomy. We first determined whether proximally axotomized PCIs develop de novo axons from their dendrites. C3 PCIs were proximally axotomized and several weeks later we stained PCIs and prepared the tissue for histological evaluation. Two primary classes of axotomized PCI were identified: those with a very short axon (called permanently axotomized) and those with an axon that projected across the injury site. Permanently axotomized PCIs had processes with morphological features typical of axons that emerged from their distal dendrites. These axonal processes of the distal dendrites also had GAP-43 (an axonal marker) and lacked MAP2a/b (a dendritic marker). We concluded that permanently axotomized PCIs develop de novo axons from distal dendrites. We then determined whether the axons that crossed the lesion site were representative of spontaneous functional regeneration. First, we showed that PCI axons regenerate through an environment that is typically highly inhibitory to regenerating axons. Second, we established that the regenerated axons conduct action potentials. Finally, we found that regenerated PCI axons form functional synaptic connections with neurons in the contralateral spinal cord. Collectively, these data indicated that spinal interneurons are capable of spontaneous functional regeneration through an injured spinal cord. PCI growth cones are complex and unlike growth cones previously described in the literature. The final study of the thesis examines the morphologies of PCI growth cones within spinal cord injury sites. We found that PCI growth cones have a wide range of morphologies that is independent of their location within the lesion site. Taken together, these data indicate that PCIs have a remarkable capacity for axonal elongation and contribute to remodelling of spinal circuitry following spinal injury. / Thesis (Ph.D, Physiology) -- Queen's University, 2009-12-07 11:21:47.036