Locomotion, including running, walking, and swimming, is a complex behavior enabling animals to interact with the environment. Vertebrate locomotion depends upon sets of interneurons in the spinal cord, known as the central pattern generator (CPG). The CPG performs multiple roles: pattern formation (left-right alternation and flexor-extensor alternation) and rhythm generation (the onset and frequency of locomotion). Many studies have begun to unravel the organization of the neuronal circuits underlying left-right and flexor-extensor alternation. However, despite pharmacologic, lesion, and optogenetic studies suggesting that the rhythm generating neurons are ispilaterally-projecting glutamatergic neurons, the precise cellular identification of rhythm generating neurons remains largely unknown. Traditionally, CPG networks (both pattern formation and rhythm generation) are thought to reside upstream of motor neurons, which serve as the output of the spinal cord. Recently however, it has been discovered that direct stimulation of lumbar motor neurons using the intact ex vivo neonate mouse spinal cord preparation can activate CPG networks to produce locomotor-like behavior. Furthermore, depressing motor neuron discharge decreases locomotor frequency, whereas increasing motor neuron discharge accelerates locomotor frequency, suggesting that motor neurons provide ongoing feedback to the CPG. However, the circuit mechanisms through which motor neurons can influence activity in the CPG in mammals remain unknown. Here, I used motor neurons as a means of accessing CPG interneurons by asking how motor neuron activation might induce locomotor-like activity. Through intracellular recording and morphological assays, I discovered that ventral spinocerebellar tract (VSCT) neurons are activated monosynaptically following motor neuron axon stimulation through chemical and electrical synapses. A subset of VSCT neurons were located close to or within the motor neuron nucleus. VSCT neurons were found to be excitatory, have descending spinal axon collaterals, and influence motor neuron output, suggesting that VSCT neurons are positioned advantageously to initiate and maintain locomotor-like rhythmogenesis. Intracellular recording from VSCT neurons revealed that they exhibit rhythmic activity during locomotor-like activity. VSCT neurons were found to contain the rhythmogenic pacemaker Ih current and to be connected to other VSCT neurons, at least through gap junctions. Optogenetic and chemogenetic manipulation of VSCT neuron activity provided evidence that VSCT neurons are both necessary and sufficient for the production of locomotor-like activity. Silencing VSCT neurons prevented the induction of such activity, whereas activation of VSCT neurons was capable of inducing locomotor-like activity. The production of locomotor-like activity by VSCT neuron photoactivation was dependent upon both electrical communication through gap junctions as well as the pacemaker Ih current. The evidence presented in this thesis suggests that VSCT neurons are critical components for rhythm generation in the mammalian CPG and are key mediators of locomotor activity.

The provision of proprioceptive feedback from limb muscle to spinal motor neuron is essential for the generation of coordinated movement. Proprioceptive sensory neurons form a precise matrix of connections with motor neurons and do so in the absence of patterned activity, implying the existence of proprioceptor subtype identities that mediate selective connectivity. The developing limb has been shown to influence the pattern of connections made by proprioceptors with motor neurons, suggesting that the patterning cues distributed along its cardinal axes are capable of influencing the molecular identities of proprioceptors. In this thesis, I describe efforts to characterize the molecular diversity of proprioceptors supplying distinct muscles located at different dorsoventral and proximodistal positions within the mouse hindlimb. I demonstrate the selective expression of several genes – cdh13, vstm2b, sema5a, and crtac1 – by proprioceptors supplying defined positional domains of the limb. I proceed to determine the limb tissue source of proprioceptor patterning information by examining the expression of these genes in mice in which one of three tissues encountered by proprioceptors – the motor axon, limb mesenchyme, and target muscle – has been genetically manipulated, revealing that both mesenchyme and muscle supply cues capable of directing proprioceptor gene expression. Finally, I show that one marker of proprioceptor muscle-type identity, cdh13, mediates the formation of selective connections between proprioceptors and motor neurons, thereby establishing a molecular link between proprioceptor subtype identity and patterned central connectivity.

During neural development, axons rely on extracellular cues to reach their target regions. Although extracellular signaling is one of the principal determinants for the growth of developing axons, only a small handful of known signaling cues has been identified. The existence of some 86 billion neurons of different subtypes, which ultimately form numerous functional circuits in the human nervous system, means an enormous number of extracellular cues would be required during development. Current views hold that even if more extracellular cues were to be discovered, they would never number large enough to account for the complexity of the human nervous system. Rather, intracellular signaling pathways and other cell-intrinsic mechanisms expand the ways in which a neuron can respond to extracellular cues by tuning the degree of responsiveness to them. Cell-intrinsic signaling pathways also give axons the ability to actively control their own development. These pathways can operate independently of the extracellular environment or even independently of the cell body, where the majority of protein synthesis takes place. For example, the local translation of proteins in the axon gives it autonomous control to immediately respond to changing demands in the environment. Local translation also occurs in other cell types, but the compartmentalized control over growth is especially important for neurons since the axon can extend up to a meter away from the cell body. In addition to local translation, axonally derived transcription factors, which can be locally synthesized in or localized to the axon, provide another means to control axon development. Axonally derived transcription factors act as physiological sensors and relay information about events happening in the periphery back to the cell body in order to effectuate a global response. It has recently been shown that transcription factors belonging to the OASIS family are activated by proteolysis in axons. Following their activation by proteolytic cleavage, the transcriptionally active N-terminus of these factors is transported to the cell body to activate global transcriptional pathways. For at least one OASIS family member, CREB3L2, this cleavage event simultaneously produces the C-terminus, which is capable of undergoing secretion. The secreted C-terminus of CREB3L2 acts as an accessory ligand for the activation of Hh pathways in chondrocytes. The generation of two bioactive proteins from one transcription factor, a transcriptionally active portion and a secreted portion, raised the question of whether there was a local function for OASIS transcription factors in axons. Through my research, I identified a mechanism in which DRG axons secrete the C-terminus of CREB3L2, which promotes axon growth in a paracrine manner. CREB3L2 is a transcription factor whose translation is induced by physiological ER stress. For CREB3L2 to be active, it must be cleaved by S2P, which I found is expressed in developing axons. Following proteolysis of CREB3L2 by S2P, the secreted C-terminus of CREB3L2 promotes the formation of Shh and Ptch1 complexes along axons. I found that upon depletion of the secreted CREB3L2 C-terminus, binding of Shh to the Ptch1 receptor is diminished. Returning the CREB3L2 C-terminus to the cultures exogenously was sufficient to rescue the formation of these complexes. These results highlight an intrinsic role for Shh signaling in developing DRG axons. Moreover, these results demonstrate how ER stress machinery is recruited to axons and promotes axon outgrowth. Finally, these results illustrate a novel, neuron-intrinsic mechanism by which developing axons actively regulate their own growth.

No description available.

Abstract not available

Medications that treat diseases such as Parkinson???s disease work by regulating dopamine transmission at synapses. Surprisingly, little is known about the mechanisms regulating dopamine release at synapses. In this thesis, we study mechanisms that regulate vesicle recycling in axons and dendrites of dopamine neurons. Key questions we addressed were: (1) Are vesicles in axons and dendrites associated with the same regulatory proteins, and thus by implication the same regulatory mechanisms, as in excitatory neurons; (2) Do vesicles undergo recycling, and (3) if so, are they characterised by a distinct pool size and rate of recycling. To study this, we cultured dopamine neurons and used immunocytochemistry to detect vesicular monoamine transporter 2 (VMAT2) and identify axons, dendrites and synaptic proteins, combined with labelling of recycling vesicles using FM 1-43. Vesicles in axons, but not in dendrites, were associated with presynaptic proteins such as Synaptophysin and Bassoon. We identified two kinds of presynaptic sites in axons: ???synaptic??? (located close to soma and dendrites??? and ???orphan???. The recycling vesicle pool size was smaller at orphan sites than at synaptic sites, and the initial rate of vesicle pool release was also lower at orphan sites. Both synaptic and orphan sites exhibited lower rates of vesicle pool release compared to hippocampal synapses, suggesting functional differences in presynaptic physiology between dopamine neurons and hippocampal neurons. In somatodendritic regions, VMAT2 was localised to the endoplasmic reticulum, Golgi, endosome, and large dense-core vesicles, suggesting that these vesicles might function as a part of the regulated secretory pathway in mediating dopamine release. None of the synaptic vesicle proteins we studied were detected in these regions, although some preliminary evidence of vesicle turnover was detected using FM 1-43 labelling. This thesis provides a detailed analysis of neurotransmitter release mechanisms in dopamine neurons. Our data suggests that presynaptic release of dopamine is mediated by mechanisms similar to those observed in excitatory neurons. In somatodendritic regions, our data suggests that VMAT2 is localised to organelles in secretory pathways, and that distinct mechanisms of release might be present at somatodendritic sites to those present in presynaptic sites. This thesis provides novel methods for analysing vesicle recycling in dopamine neurons, which provides the basis for further studies examining presynaptic function of dopamine neurons in normal brain function, disease, and therapeutic approaches.

The aim of this thesis was to investigate neuronal cellular mechanisms that may underlie the behavioural characteristics of the spontaneously hypertensive rat strain (SHR). The SHR was developed by selective breeding for elevated blood pressure and is also described as having increased levels of locomotor behaviour compared to its normotensive control strain, the Wistar-Kyoto. This hyperactivity and other behaviours, including altered sensitivity to reinforcement, have been used to model aspects of behaviour displayed in attention deficit hyperactivity disorder. In vivo intracellular recording of striatal spiny projection neuron activity in urethaneanaesthetised animals from three genetically related strains: the SHR, Wistar-Kyoto and standard Wistar, was employed to measure basic cellular properties and cellular mechanisms of reward-related learning. This population of neurons was chosen because alterations in their activity can influence behaviour and they are known to show cellular changes (synaptic plasticity) that are associated with learning. Cellular properties were measured in 71 neurons. Comparison between strains revealed a significant difference in action potential amplitude and duration between the SHR and Wistar-Kyoto strains. Interestingly, when measured at a later time, in a different sample of rats, the SUR action potential amplitude and duration were significantly different from the earlier sample. A change in the membrane potential repolarisation rate following action potential firing also occurred over this time. Twenty-nine of these neurons were also used in a study investigating the neuronal responses to a low dose of amphetamine (0.5 mg/kg). Changes were observed in some cellular properties following intraperitoneal administration of amphetamine. Synaptic plasticity at the corticostriatal synapses is sensitive to the timing of dopamine release in relation to cortical input. In anaesthetised preparations the spiny projection neuron membrane potential fluctuates between hyperpolarised (DOWN) and depolarised (UP) states, which reflect the level of cortical input. During the present study the responses of nine neurons to the induction of cortical spreading depression were observed to investigate the suitability of this method for use during synaptic plasticity experiments. Spiny projection neurons showed unpredictable responses to cortical spreading depression, therefore this method was not used further. Corticostriatal synaptic plasticity was induced in sixteen spiny projection neurons from two strains: SHR and Wistar. High frequency stimulation of the dopamine neurons in the substantia nigra, during the DOWN-state, did not induce any significant changes in corticostriatal synaptic efficacy. This was also true when high frequency stimulation of dopamine neurons was applied during the UP-state in neurons from the SHR strain. This thesis represents the first in vivo intracellular study of neuronal physiology in the SHR and Wistar-Kyoto rat strains. Results revealed action potential differences between these two behaviourally distinct rat strains. Synaptic mechanisms thought to underlie reward-related learning were not different between the SHR and Wistar strains, although the observed levels of plasticity were inconsistent with previous literature.

"April 2003" / Bibliography: leaves 124-144. / xvii, 144 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, School of Molecular and Biomedical Sciences, Discipline of Physiology, 2003

All motor behaviours are expressed via the activation of alpha motoneurones, the final common path of the central nervous system. The corticospinal tract conveys neural information from the motor cortex to motoneurones. This thesis focuses on the corticospinal control of human motoneurones during voluntary contraction and fatigue. First, output of motoneurones to corticospinal inputs is described for a wide range of contraction strengths. Results show that motoneurones become less responsive during strong contractions whereas motor cortical output cells are not limited in the same way. Comparison of motoneurone output to different strength corticospinal inputs and of different motoneurone pools demonstrates an important role for motor unit firing rates in determining the excitability of motoneurones during strong contractions. Next, the reflex actions of group III and IV muscle afferents on motoneurones are investigated. These studies address a long and ongoing debate about the role of these afferents to the slowing of motor unit firing rates during sustained contractions. It was believed that these afferents inhibit motoneurones and contribute to fatigue. However, findings demonstrate that human motoneurones innervating extensor and flexor muscles are not uniformly affected by fatigue-sensitive afferents. Thus afferent inputs from homonymous and antagonist muscles depress extensor motoneurones but facilitate flexor motoneurones. When group III and IV muscle afferents are activated by hypertonic saline, motoneurones of both extensors and flexors are facilitated. This demonstrates parallel excitatory and inhibitory pathways from group III and IV muscle afferents to extensor motoneurones, which are activated under different conditions. Furthermore, the excitation is more pronounced for high-threshold motoneurones. In addition to the effects mediated at motoneurones, activity in group III and IV afferents inhibits motor cortical cells. The final studies investigate changes in the cervical propriospinal pathway with fatigue. This pathway transmits part of the voluntary drive to motoneurones, in parallel with the direct corticospinal pathway. The studies demonstrate that during fatigue, there are coordinated changes in the excitation mediated via this pathway to motoneurones of both fatigued and non-fatigued muscles of the upper limb. In summary, this thesis demonstrates novel aspects of the corticospinal control of motoneurones during voluntary contraction and fatigue.

Amyotrophic Lateral Sclerosis (ALS) is a fatal disease characterized by the progressive degeneration of motor neurons. Although there has been some progress in the identification of genes linked to inherited cases of ALS, the etiology of this disease remains largely unknown. Clinical progression of motor neuron diseases is associated with the degeneration of the axon preceding cell death. Elucidating novel mechanisms important for motor axon maintenance will help gain greater insight into disease pathogenesis. Here, I report that mutations in ras-opposite (rop), which encodes the Drosophila homologue of mammalian Sec1/Munc18, cause progressive degeneration of motor axons while sensory axons are largely unaffected. While mutations in mammalian munc18-1 have been linked to degeneration of the spinal cord, the mechanisms by which this occurs are unknown. Using Drosophila, I found that RNAi-induced knockdown of rop leads to severe motor deficits in adult flies. In addition, I discovered that motor axon degeneration in rop mutants could be delayed by overexpression of the neuronal maintenance factor Nmnat. Interestingly, I found that Rop is localized with Nmnat at the neuromuscular junction and that Rop physically interacts with Nmnat in vivo. These data indicate a novel role for Rop in motor axon maintenance and provide insight into the pathogenesis of neurodegenerative diseases targeting motor neurons, such as ALS.