Activating specific calcium signals to control axon growth in vivo

During development of the central nervous system (CNS), circuit formation and connectivity between neurons begins with a process called axon pathfinding which is controlled by the specialised motile structure at the distal tips of axons called the growth cone. The CNS is permissive to axon pathfinding with an array of extrinsic guidance cues controlling growth cone trajectories. However, after injury or trauma, the environment within the CNS becomes inhibitory to growth, limiting any functional regeneration. Therefore, novel approaches to drive axon growth and enhance regeneration are a major focus for the neuroscience community.

Axon pathfinding is controlled by extrinsic guidance cues, contact mediated cues and neuronal activity which signal through the secondary messenger calcium. Rises in intracellular calcium, termed calcium signals or transients can regulate axon extension and directional motility of the growth cone. Calcium signals vary in frequency, amplitude, duration, and source, with each of these components capable of having specific effects on axon pathfinding. Specific calcium signals have been shown to control axonal growth in vitro, however, approaches to use calcium signals to control axon pathfinding in vivo remain limited. To demonstrate the requirement of a specific calcium signal on axon pathfinding in vivo, we disrupted store operated calcium entry which is controlled by the lumenal endoplasmic reticulum (ER) calcium sensing protein, stromal interaction molecule 1 (STIM1). We demonstrated that a reduction in STIM1 function within the developing zebrafish (Danio rerio) spinal motor circuit disrupted axon extension and pathfinding, suggesting that precise regulation of calcium signals from the ER is crucial for axon guidance. Given this initial finding, we hypothesised that precise spatial and temporal activation of specific calcium signals can be used to control axon pathfinding and be further used as an approach to enhance regeneration in vivo.

To activate specific calcium signals we used an optogenetic approach. The high spatial and temporal resolution of optogenetics allows for specific control of calcium signals in individual growth cones. To generate fast and large calcium signals we used the channelrhodopsin variant ChIEF. Given that STIM1 mediated calcium signals were crucial for regulating axon growth in vivo, we used OptoSTIM1 to activate an extracellular calcium signal with a small amplitude but long duration. Release of calcium from the ER was controlled by regulating tropomycin receptor kinase B (TrKB) signalling cascades using the photoactivatable TrKB (PhaT-B). Calcium signals elicited with these optogenetic tools were examined in vitro by fura-2 calcium imaging in cultured zebrafish spinal motor neurons. ChIEF elicited a large and fast calcium signal while OptoSTIM1 elicited a small and slow calcium rise. However, PhaT-B failed to elicit any calcium signal in zebrafish motor neurons. These data suggest that activation of either ChIEF or OptoSTIM1 could be used to control fast and slow calcium signals respectively, and hence could be used as an approach to control axon pathfinding in vivo.

The zebrafish spinal motor circuit was used to examine whether controlling specific calcium signals could alter axon pathfinding in vivo. We expressed each optogenetic construct using tol2 transgenesis in the stereotypic caudal primary (CaP) motor neurons. Axon extension and changes to exit angle at the intermediate checkpoint, horizontal myoseptum were analysed. Activation of STIM1 mediated calcium signals at either 1.1mHz or 3.3mHz for 3hrs altered the angle of exit from the horizontal myoseptum and decreased axon extension, respectively. Activation of ChIEF at 3.3mHz decreased axon extension but not angle of exit from the horizontal myoseptum. Given that activation of OptoSTIM1 could alter directional motility but not axon extension at low frequency stimulation, we investigated whether activation of OptoSTIM1 can directly control growth cone motility in vivo. Activation of OptoSTIM1 on the leading edge of CaP motor neuron growth cones stalled axon extension. Whereas asymmetric activation of OptoSTIM1 in CaP motor neuron growth cones could potentially control directional motility towards the stimulated side. Taken together, these data suggest that large amplitude and rapid duration calcium signals can slow axon extension, but activation of small amplitude and long duration calcium signals can be potentially used to control the direction of axon growth in vivo.

The final set of experiments sought to determine whether the activation of OptoSTIM1 mediated calcium signals could enhance zebrafish spinal cord regeneration. The zebrafish spinal cord was lesioned at 3 days post fertilisation (dpf) and stimulated with 1.1mHz blue light for 12hrs either at 1 hour post injury (hpi) or at 12hpi. Activation of OptoSTIM1 did not increase spinal motor function in lesioned zebrafish as measured with a startle reflex at 1- and 2-days post injury (dpi). To determine connectivity of the spinal cord we measured the proportion of zebrafish with an axon bridge across the lesion site at 1- and 2dpi. Activation of OptoSTIM1 did not increase the proportion of zebrafish with an axonal bridge, suggesting that the parameters used to activate OptoSTIM1 does not enhance regeneration.

The data described in this thesis demonstrate that circuit wide activation of slow or fast calcium signals can differentially alter axon pathfinding in vivo. At the single cell level, we used specific control of OptoSTIM1 mediated calcium signals in the growth cone to control axon extension and potentially control directional motility in vivo. This data supports a possible future use for regulating specific calcium signals to alter axon pathfinding and growth. In this thesis, preliminary experiments aimed at activating STIM1 in a regeneration setting were not successful. However, future experiments that couple optogenetic activation of select calcium signals with new technology, for example devices that deliver light to the mammalian CNS, may improve efforts to enhance CNS regeneration.