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Zebrafish, in contrast to mammals, are capable of functional regeneration after complete transection of the spinal cord. In this system I asked: (1) Which spinal cell types regenerate in the lesioned spinal cord? (2) To what extent do the dopaminergic and 5-HT systems regenerate and (3) do dopaminergic axons from the brain influence cellular regeneration in the spinal cord? (1) Lost motor neurons are replaced by newly born motor neurons that mature and are integrated into the spinal circuitry after a spinal lesion in adult zebrafish. Using immunohistochemical and transgenic markers in combination with BrdU labeling, we showed that also 5-HT, parvalbuminergic, Pax2+ and Vsx1+ cells are newly born after lesion. Thus, my work shows that diverse cell types are newly generated in the lesioned spinal cord of adult zebrafish. (2) After spinal cord lesion, zebrafish completely recover locomotion within six weeks. Previous work suggested that axonal regeneration is crucial for functional recovery. Here I analyzed changes in the density of 5-HT and dopaminergic axon terminals in the lesioned spinal cord during recovery. Rostral to the lesion site, I observed die-back and sprouting of dopaminergic axons within two weeks post-lesion. Caudal of the lesion, axons are lost indicating Wallerian degeneration. At six weeks post-lesion I tested functional recovery with a behavioral swim test. In recovered fish, a third of the axonal density was restored just caudal of the lesion site, but not at far caudal levels. In contrast, in fish that had non-recovered, only few axons had bridged the lesion site. Thus dopaminergic axon regrowth correlates with functional recovery. Re-transection of the spinal cord in recovered animals abolished re-gained swimming capability, suggesting that behavioral recovery critically depends on axons that crossed the spinal lesion site and not on an intraspinal circuit. 5-HT axon terminals are of both intra- and supraspinal origin. The overall time course of changes in axon terminal density during recovery is similar to that of dopaminergic axon terminals and also correlates with functional recovery. Overall, the organization of the spinal dopaminergic and 5-HT systems, consisting of neuronal somata in the spinal cord and descending axons, differs significantly from their unlesioned organization. I observe sprouting rostral to the lesion site and limited innervation of the caudal spinal cord, as axons do not regrow into the far distal spinal cord. (3) We further hypothesized that signals released by descending axons are involved in cellular regeneration around the lesion site. Dopaminergic axons of supraspinal origin sprout rostral, but are almost completely absent caudal to the lesion site at two weeks post-lesion. Moreover, we observe that expression of the dopamine receptor drd4a is only increased rostral to the lesion site in the ventricular zone of progenitor cells, including olig2 expressing motor neuron progenitor cells. Correlated with these rostro-caudal differences, numbers of regenerating motor neurons are almost two-fold higher rostral than caudal of the lesion site. To functionally test whether dopamine is involved in motor neuron regeneration, we ablated tyrosine hydroxylase positive, mostly dopaminergic axons by injecting the toxin 6-hydroxydopamine. This treatment significantly reduced motor neuron numbers only rostral to the lesion site. As a gain-of-function experiment, we injected the dopamine agonist NPA after spinal lesion, which increased motor neuron numbers only rostral to the lesion site at two weeks post-lesion. These results suggest that dopamine released by descending axons, augments the generation of motor neurons in the lesioned spinal cord of adult zebrafish. In summary, during spinal cord regeneration I observe generation of various cell types and plastic changes of descending axonal projections. Dopamine released by descending axons is able to increase motor neuron regeneration, showing for the first time that signals from descending axons influence cellular regeneration in the spinal cord.

Long projections from the brainstem to the lumbar cord activate locomotion. Using in vitro neonatal rats our laboratory showed that relay (propriospinal - PS) neurons also contribute to transmission of the locomotor signal. This thesis examines whether locomotor-related PS neurons exist in adult mammals, which has important clinical implications. The brainstem of adult decerebrate rats was stimulated to elicit stepping. The following manipulations were performed: 1) suppression of synaptic transmission to PS neurons, 2) lesioning of direct bulbospinal projections to lumbar segments, and 3) neurochemical excitation of PS neurons. In addition, in the absence of brainstem stimulation, the ability of neurochemically excited PS neurons to induce stepping was examined. Brainstem-evoked locomotion was suppressed by synaptic blockade, enhanced by PS neuron excitation, persists after lesioning of long-direct projections, and hindlimb stepping was elicited by PS neuron excitation alone. The findings support the existence of a locomotor-related PS system in adult mammals. / October 2016

Previous work has demonstrated that rhythmic motor outputs, such as locomotion and scratch induce a hyperpolarization of the voltage threshold (Vth) for action potential initiation in spinal motoneurons, enhancing their excitability. Descending monoamines were implicated in mediating this effect; however, the recent observation that changes in Vth persist during fictive scratch in cats following acute cervical transection revealed that intraspinal systems, of unknown neuromodulatory identity, also have the ability to regulate motoneuron excitability during motor behaviour. This thesis addresses: 1) whether acetylcholine (ACh) is able to modulate spinal motoneuron Vth, and 2) whether endogenous ACh modulates motoneuron excitability during motor activity without intact descending modulation. Our first study investigates whether ACh from exogenous and/or endogenous sources alters motoneuron Vth. We made intracellular recordings of lumbar motoneurons from neonatal rats to pharmacologically manipulate muscarinic and nicotinic receptor activity. Results show that ACh induces either Vth hyperpolarization, Vth depolarization or no change in Vth depending on the activity state of the network, the ACh concentration, and influences from other systems. Our second study investigates whether intraspinal cholinergic inputs induce Vth hyperpolarization during rhythmic motor output when descending projections are disrupted. For this we developed an in vitro neonatal rat spinal cord preparation to elicit rhythmic activity independently of brainstem or lumbar cord stimulation. Intracellular recordings from motoneurons allowed comparison of the Vth prior to and during rhythmic output, both in the absence and presence of cholinergic antagonists in the lumbar cord. Results show that intraspinal cholinergic mechanisms are active and importantly contribute to modulation of motoneuron Vth during motor output. We suggest that in addition to descending modulation, the spinal cholinergic system regulates motoneuron Vth to either facilitate or inhibit recruitment according to the motor network state. Motoneuron excitability regulation by modification of distinct membrane properties resulting from separate modulatory systems activation during diverse motor behaviours is discussed. This work is the first to demonstrate the role of cholinergic mechanisms in regulating motoneuron excitability through modulation of Vth in an activity based context, and therefore outlines a spinal modulatory system that would contribute to motor control in both normal and pathological states. / May 2016

Following cervical or upper thoracic level spinal cord injury (SCI), motor, sensory and autonomic systems are disrupted. One form of this autonomic dysfunction is the condition autonomic dysreflexia (AD), which is characterised by episodes of high blood pressure in response to afferent input from regions below the injury level. An animal model of autonomic dysreflexia, the T4 transected rat, was used in this thesis to gain insight into the cardiovascular and temperature components of the disorder, possible peripheral mechanisms and interventions to prevent its development. Chapter 2 of the thesis includes the charaterisation of a T4 transection rat model of spinal cord injury. This characterisation includes confirmation of decreased baseline mean arterial pressure (MAP, 71 down from 117 mmHg) and elevated heart rate (HR, 431 bpm from 366 bpm) for 6 weeks post injury (p.i.). Documentation of the development of AD found that hypertensive responses were fully developed (+20 mmHg) by 4 weeks p.i. Further, during episodes of AD at Weeks 4 and 5 p.i., tail surface temperatures decreased significantly (mid-tail, -1.7oC), indicative of extensive vasoconstriction. Comparison of vascular responses of intact and SCI animals to adrenergic agonists (phenylephrine, PHE and methoxamine, METH) following ganglionic blockade in vivo found that SCI animals experienced prolonged vasoconstriction in blood vessels above and below injury level in response to PHE but not METH. Possible mechanisms of this change included decreased neuronal reuptake of PHE (METH is not a substrate for neuronal reuptake). The presence of prolonged vasoconstriction in blood vessels throughout the body, not just regions below injury level, suggests a widespread mechanism for the change, such as the decreased basal MAP, norepinephrine levels or neural activity present following injury. Thus, it was hypothesised that increased activity from an early stage post injury may prevent the peripheral adaptation and perhaps hinder development of AD. For this, the common rehabilitation technique, treadmill training, was used. Surprisingly, rather than preventing AD, the training actually accelerated its development, producing exaggerated hypertensive responses to colorectal distension (CRD) at Weeks 3 and 4 post-injury (Week 4, Trained: +38.5 ?? 1.5 mmHg; Sedentary: 23.4 ?? 3.1mmHg). Comparison of vascular responses of both groups to PHE injection found no significant difference indicating that the enhanced responses were not a result of peripheral vascular changes. Investigation of the central morphology following SCI, made via immunohistochemical processing of the post-mortem spinal cords, found that Treadmill Trained SCI animals had elevated calcitonin gene related peptide (CGRP) immunoreactivity within lamina III/IV of lumbar segments, compared to intact cords. It is possible that this finding indicates afferent sprouting that may have accelerated the development of AD in Treadmill Trained animals. The results within this thesis highlight the importance of awareness and examination of autonomic function in SCI patients, especially those undergoing rehabilitative training.

Thesis (Ph. D.)--Drexel University, 2007. / Includes abstract and vita. Includes bibliographical references (leaves 111-123).

Thesis (M.A.(Psychology))--University of Pretoria, 2001. / Includes bibliographical references (leave. 175-185).

Magnetic Resonance Imaging (MRI) has been applied in diagnosing Cervical Spondylosis Myelopathy (CSM) clinically. However, morphometric and signal change of MRI have not shown consistent relations with neurological function or outcome after surgical intervention. Diffusion Tensor Imaging (DTI) is an advanced MRI technology, which uses the principle of anisotropic water diffusion property. Recent studies indicated that DTI could be used as diagnostic tools for Cervical Spondylosis Myelopathy (CSM). The study aims to establish a Region of Interest (ROI)-based database. 65 healthy Chinese subjects were recruited for functional MR scanning. The effects on age and gender would also be investigated. Whole cord FA values decreased from upper cord level to lower cord level. White matter FA and AD values are significant higher than grey matter. White matter RD values are significant lower than grey matter. MD values of whole cord, white matter and grey matter are similar. There are no significant differences (P>0.05) of DTI metrics between males and females. There are significant differences (P<0.05) of DTI metrics in cervical spinal cord white matter in advancing age. / published_or_final_version / Orthopaedics and Traumatology / Master / Master of Medical Sciences

published_or_final_version / Physiology / Doctoral / Doctor of Philosophy