We have developed a highly effective method for in vivo gene silencing in the spinal cord and dorsal root ganglia (DRG) by a cationic lipid facilitated delivery of synthetic, small interfering RNA (siRNA). A siRNA to the delta opioid receptor (DOR), or a mismatch RNA, was mixed with the transfection reagent, i-FectTM (vehicle), and delivered as repeated daily bolus doses (0.5 mug to 4 mug) via implanted intrathecal catheter to the lumbar spinal cord of rats. Twenty-four hours after the last injection, rats were tested for antinociception by the DOR selective agonist, D-Ala2, Glu4]deltorphin II (DELT), or the mu opioid receptor (MOR) selective agonist, D-Ala2, N-Me-Phe4, Gly-ol5]enkephalin (DAMGO). Pretreatment with the siRNA, but not the mismatch RNA or vehicle alone, blocked DELT antinociception dose-dependently. The latter was concomitant with a reduction in the spinal immunoreactivity and receptor density of DOR, and in DOR transcripts in the lumbar DRG and spinal dorsal horn. Neither siRNA nor mismatch RNA pretreatment altered spinal immunoreactivity of MOR or antinociception by spinal DAMGO, and had no effect on the baseline thermal nociceptive threshold. The inhibition of function and expression of DOR by siRNA was reversed by 72 hr after the last RNA injection. The uptake of fluorescence-tagged siRNA was detected in both DRG and spinal cord. The low effective dose of siRNA/i-FectTM complex reflects an efficient delivery of the siRNA to peripheral and spinal neurons, produced no behavioral signs of toxicity. This delivery method may be optimized for other gene targets.

In contrast to mammals, adult zebrafish display cellular regeneration of lost motor neurons and achieve functional recovery following a complete spinal cord transection. Using adult zebrafish as a model to study how key developmental pathways can be re-activated to regulate neuroregeneration in cellular recovery, I addressed the following questions: 1) What is the role of Notch signalling during regenerative mechanisms in the lesioned spinal cord of the adult zebrafish? 2) What is the role of Notch overexpression in neurogenesis in the adult zebrafish retina? 3) Which additional signalling pathways are involved in the generation of motor neurons during spinal cord regeneration in adult zebrafish? 1) In the main part of my thesis I have investigated the role of Notch signalling during spinal cord regeneration. The Notch pathway has been shown to regulate neural progenitor maintenance and inhibit neuronal differentiation in the vertebrate nervous system. In the injured mammalian spinal cord, increased Notch signalling is held partly responsible for the low regenerative potential of endogenous progenitors to generate new neurons. However, this is difficult to test in an essentially non-regenerating system. We show that in adult zebrafish, which exhibit lesion-induced neurogenesis, e.g. of motor neurons from endogenous spinal progenitor cells, the Notch pathway is also reactivated. I over-activated the Notch pathway by forced expression of a heat-shock inducible active domain of notch in spinal progenitor cells. I observed that although apparently compatible with functional regeneration in zebrafish, forced activity of the pathway significantly decreased progenitor proliferation and motor neuron generation. Conversely, pharmacological inhibition of the pathway increased proliferation and motor neuron numbers. Thus in summary our work demonstrates that Notch is a negative signal for regenerative neurogenesis in the spinal cord. Importantly, we show for the first time that spinal motor neuron regeneration can be augmented in an adult vertebrate by inhibiting Notch signalling. 2) While in the lesioned spinal cord, over-activation of Notch attenuated neurogenesis, I observed that in the unlesioned retina the same manipulation led to strong proliferation of cells in the inner nuclear layer, presumable Müller glia cells which are the retinal progenitor cells. This coincided with an increase in eye size in adult zebrafish. These preliminary findings provide the first hint that the role of Notch may differ for different adult progenitor cell pools and will lead to future investigations of Notch induced neurogenesis in the retina. 3) We have evidence from previous studies that the dopamine and retinoic acid (RA) signalling pathways may be involved in the generation of motor neurons in the adult lesioned spinal cord. Using in situ hybridisation, I assessed the gene expression patterns a) for all D2-like receptors and b) candidate genes that relate to the RA pathway in the adult lesioned spinal cord to identify the signalling components. a) I found that only the D4a receptor was upregulated in spinal progenitor cells in the ventricular zone rostral to the lesion site, but not caudal to it. This correlates with other results showing that dopamine agonists increase motor neuron regeneration rostral, but not caudal to a spinal lesion site. b) I observed a strong increase in the expression of Cyp26a, a RA catabolising enzyme, in the ventricular progenitor zone caudal to the lesion site, in contrast to the weak expression rostrally. Crabp2a, a cellular retinoic acid binding protein, was also upregulated rostral and in close proximity to the lesion site in a subpopulation of neurons located ventrolaterally in the spinal cord. In summary, we show that the Notch pathway negatively regulates neurogenesis in the spinal cord in contrast to the retina and provide evidence that dopamine from the brain signals via the D4a receptor to promote the generation of motor neurons in addition to RA, which may also play a role in this process. These insights into adult neural progenitor cell activation in zebrafish may ultimately inform therapeutic strategies for spinal cord injury and neurodegenerative diseases such as motor neuron disease.

Inflammatory pain is a debilitating condition that can occur following tissue injury or inflammation and results in touch evoked pain (allodynia), exaggerated pain (hyperalgesia) and spontaneous pain, yet the neural plasticity underlying these symptoms is not fully understood. However, it is known that lamina I neurokinin 1 receptor expressing (NK1R+) spinal cord output neurons are crucial for the manifestation of inflammatory pain. There is also evidence that the afferent input to and the postsynaptic response of these neurons may be altered in inflammatory pain, which could be relevant for inflammatory pain hypersensitivity. Therefore, the aim of this thesis was to study inflammatory pain spinal plasticity mechanisms by investigating the synaptic input to lamina I NK1R+ neurons. In ex vivo spinal cord and dorsal root preparations from the rat, electrophysiological techniques were used to assess inflammation-induced changes in and pharmacological manipulation of the primary afferent drive to lamina I NK1R+ neurons. The excitatory input to lamina I NK1R+ neurons was examined and it was found that inflammation did not alter the relative distribution of the type of primary afferent input received and did not potentiate monosynaptic A δ or monosynaptic C-fibre input, the predominant input to these neurons. Spontaneous excitatory input was significantly elevated in the subset of neurons that received monosynaptic A δ-fibre input only, regardless of inflammation. It has recently been shown that the chemerin receptor 23 (ChemR23) represents a novel inflammatory pain target, whereby ChemR23 agonists can decrease inflammatory pain hypersensitivity, by a mechanism that involves the attenuation of potentiated spinal cord responses. This study has found that the ChemR23 agonist, chemerin, attenuated capsaicin potentiation of excitatory input to lamina I NK1R+ neurons and significantly reduced monosynaptic C-fibre input to a subset of these neurons in inflammatory pain. However, chemerin was without effect in non-potentiated conditions. In exploring potential inflammatory pain spinal plasticity mechanisms, I have investigated a phenomenon called activity-dependent slowing (ADS), whereby repetitive stimulation of C-fibres at frequencies of 1Hz or above results in a progressive slowing of action potential conduction velocity, which manifests as a progressive increase in response latency. This is proposed to limit nociceptive input to the spinal cord, thus regulating plasticity. Results demonstrate that inflammation significantly attenuated C-fibre ADS in isolated dorsal roots. Furthermore, ADS in monosynaptic C-fibre input to lamina I NK1R+ neurons was significantly reduced in inflammatory pain, which could facilitate nociceptive drive to these key spinal cord output neurons and promote inflammatory pain spinal cord plasticity. In conclusion, the major novel findings of this thesis are firstly, that chemerin can attenuate primary afferent input to lamina I NK1R+ neurons in potentiated conditions, which supports recent studies that suggest ChemR23 is a potential target for the development of new analgesics. Secondly, it was discovered that ADS in monosynaptic C-fibre inputs to lamina I NK1R+ neurons is altered in inflammatory pain, which could be relevant for inflammatory pain spinal plasticity. The findings presented in this thesis could contribute to the development of novel inflammatory pain treatments.

Synchronized neural activity, in which the firing of neurons is coordinated in time, is an observed phenomenon in many neural functions. The conditions that promote synchrony and the dynamics of synchronized activity are active areas of investigation because they are incompletely understood. In addition, variability is intrinsic to biological systems, but the effect of neuron spike time variability on synchronization dynamics is a question that merits more attention. Previous experiments using a hybrid circuit of one biological neuron coupled to one computational neuron revealed that irregularity in biological neuron spike timing could change synchronization in the circuit, transitioning the activity between phase-locked and phase slipping. Simulations of this circuit could not replicate the transitions in network activity if neuron period was represented as a Gaussian process, but could if a process with history and a stochastic component were used. The phase resetting curve (PRC), which describes how neuron cycles change in response to input, can be used to construct a map that predicts if synchronization will occur in hybrid circuits. Without modification, these maps did not always capture observed network activity. I conducted long-term recordings of invertebrate neurons and show that interspike interval (ISI) can be represented as an autoregressive integrated moving average process, where ISI is dependent on past history and a stochastic component with history. Using integrate and fire model simulations, I suggest that stochastic activity in adaptation channels could be responsible for the history dependence and correlational structure observed in these neurons. This evidence for stochastic, history-dependent noise in neural systems indicates that our understanding of network dynamics could be enhanced by including more complex, but relevant, forms of noise. I show that cycle-by-cycle dynamics of the coupled system can be used to infer features of the dynamic map, even if it cannot be measured or is changing over time. Using this method, stable fixed points can be distinguished from ghost attractors in the presence of noise, networks with similar phase but different underlying dynamics can be resolved, and the movement of stable fixed points can be observed. The time-series vector method is a valuable tool for distinguishing dynamics and describing robustness. It can be adapted for use in larger populations and non-reciprocal circuits. Finally, some larger implications of neuroscience research, specifically the use of neural interfaces for national security, are discussed. Neural interfaces for human enhancement in a national security context raise a number of unique ethical and policy concerns not common to dual use research of concern or traditional human subjects research. Guidelines about which technologies should be developed are lacking. We discuss a two-step framework with 1) an initial screen to prioritize technologies that should be reviewed immediately, and 2) a comprehensive ethical review regarding concerns for the enhanced individual, operational norms, and multi-use applications in the case of transfer to civilian contexts.

In this thesis, experiments into artificially guiding neuronal growth cones using tightly focused laser beams were performed and evaluated. The experiments are performed by focusing a laser beam to the leading edge of a developing growth cone and attempting to change the direction of growth cone. These experiments were carried out using Gaussian, line and asymmetric line beam profiles. There was no noticeable change in the success rate with different beam profiles. Following this, I assisted my colleague Dr Michael Mazilu in the construction of a mathematical model of filopedia in an optical field in order to help explain the mechanism for optically guided neuronal growth which suggests that optical trapping forces on filopedia are responsible. Next, I set about implementing a system to automate the process of laser guided neuron growth by employing a spatial light modulator and a custom-built computer program. This allowed the computer to track a developing growth cone and automatically adjust the position of the laser beam as the growth cone developed. This program was successfully employed to artificially grow neuronal growth cones towards a user-inputted target point. The use of the spatial light modulator to beam shape was also demonstrated with the use of a Bessel beam being used to guide neurons for the first time. I also used a transgenic cell line of neurons to show for the first time that HSP70 is not involved in this phenomenon. This was accomplished by transfecting NG108’s with a plasmid containing HSP70 promoter tagged GFP. Under enough thermal or mechanical stress, the cells would express HSP70 which would produce a detectable GFP signal. No GFP was detected in cells after being exposed to laser irradiation of a power higher than would normally be used to guide neurons. Combined, these experiments show that the beam profile of the operating laser does not significantly affect the success of artificial growth and that the optical force on filopedia near the laser beam is likely to be the mechanism for this phenomenon. A possible heating effect of the laser has also been shown to not be strong enough to elicit a heat shock stress response from the cell. The demonstration of an automatic system which incorporates beam shaping has also been shown and such a system shows the potential to advance the investigation of artificial neuron growth using lasers.

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Physiology / Master / Master of Philosophy

published_or_final_version / abstract / Physiology / Master / Master of Philosophy