The original Bio-Rad gene gun was unable to transfect acute or organotypic brain slices, as the amount of helium gas used, the distance for the gold-coated microcarriers to travel to target area were not optimised for fragile tissues, such as the brain. Typically, tissues were severely damaged by a helium shock wave and only a few cells were transfected. It was essential to improve gene gun accuracy by restricting the gold particles from being propelled superficially over a wide area. It was also necessary to increase the amount of DNA or dye delivery into intact tissues. Furthermore, for the gene gun to perform successfully on brain slices the helium gas pressure had to be lowered thereby reducing the degree of cell damage incurred during a biolistic delivery. Without knowing it at the time, the modified gene gun had worked particularly well on a variety of other fragile tissues, and not just the brain. However, the modified gun was not optimised for cultured cells as other transfection methods were available. A particularly notable point of this work was the successful labelling of individual Purkinje dendritic spines from live nerve cells in the cerebellum region of the brain. Biolistic images of Purkinje cells show that the distribution of dendritic spines are not random (O’Brien and Unwin, 2006). Spines were shown to grow in elaborate regular linear arrays, that trace short-pitch helical paths around the dendrites. It was apparent that the spines are arranged to maximize the probability that the dendritic arbour would interact with any afferent axon. This was an important discovery as there has been much debate as to how spines develop on a dendritic shaft. There are three general views to this question, each proposing a theory describing a model for spinogenesis. Classification of the three models in relation to our findings is described in chapter six of this thesis. The Investigation of spine morphology by biolistics was further optimized; gold particles were reduced from a micrometre to forty nanometres (O’Brien and Lummis, 2011), demonstrating that it is possible to use gold-coated DNA nanoparticles of this size to transfect tissue revealing exquisite structural detail. It was possible to observe boutons making synaptic contacts with the pyramidal nerve spines in the hippocampal region of the brain. The findings so far have shown spines from the pyramidal shaft are similar to the spines in the cerebellum, forming regular linear arrays. Recent studies had linked defects in the function of presynaptic boutons to the etiology of several neurodevelopment and neurodegenerative diseases, including autism and Alzheimer’s disease. Our discovery could help to understand why there are abnormalities in dendritic spines which are associated with pathological conditions characterized by cognitive decline, such as mental retardation, Alzheimer’s, stroke and schizophrenia (Yuste and Bonhoeffer, 2001). This thesis provides a synthesis of knowledge about biolistic technology. It is presented as a narrative from improving the gene gun transfection efficiency in brain slices to the development of nano-biolistics. The delivery of DNA and fluorescent dyes into living cells by biolistic delivery should enable a detailed map of the anatomical connections between individual cells and groups of cells to be constructed, providing a “wiring diagram” of connections. The implications of this are discussed in Chapter twelve. The original Bio-Rad gene gun was unable to transfect acute or organotypic brain slices, as the amount of helium gas used, the distance for the gold-coated microcarriers to travel to target area were not optimised for fragile tissues, such as the brain. Typically, tissues were severely damaged by a helium shock wave and only a few cells were transfected. It was essential to improve gene gun accuracy by restricting the gold particles from being propelled superficially over a wide area. It was also necessary to increase the amount of DNA or dye delivery into intact tissues. Furthermore, for the gene gun to perform successfully on brain slices the helium gas pressure had to be lowered thereby reducing the degree of cell damage incurred during a biolistic delivery. Without knowing it at the time, the modified gene gun had worked particularly well on a variety of other fragile tissues, and not just the brain. However, the modified gun was not optimised for cultured cells as other transfection methods were available. A particularly notable point of this work was the successful labelling of individual Purkinje dendritic spines from live nerve cells in the cerebellum region of the brain. Biolistic images of Purkinje cells show that the distribution of dendritic spines are not random (O’Brien and Unwin, 2006). Spines were shown to grow in elaborate regular linear arrays, that trace short-pitch helical paths around the dendrites. It was apparent that the spines are arranged to maximize the probability that the dendritic arbour would interact with any afferent axon. This was an important discovery as there has been much debate as to how spines develop on a dendritic shaft. There are three general views to this question, each proposing a theory describing a model for spinogenesis. Classification of the three models in relation to our findings is described in chapter six of this thesis. The Investigation of spine morphology by biolistics was further optimized; gold particles were reduced from a micrometre to forty nanometres (O’Brien and Lummis, 2011), demonstrating that it is possible to use gold-coated DNA nanoparticles of this size to transfect tissue revealing exquisite structural detail. It was possible to observe boutons making synaptic contacts with the pyramidal nerve spines in the hippocampal region of the brain. The findings so far have shown spines from the pyramidal shaft are similar to the spines in the cerebellum, forming regular linear arrays. Recent studies had linked defects in the function of presynaptic boutons to the etiology of several neurodevelopment and neurodegenerative diseases, including autism and Alzheimer’s disease. Our discovery could help to understand why there are abnormalities in dendritic spines which are associated with pathological conditions characterized by cognitive decline, such as mental retardation, Alzheimer’s, stroke and schizophrenia (Yuste and Bonhoeffer, 2001). This thesis provides a synthesis of knowledge about biolistic technology. It is presented as a narrative from improving the gene gun transfection efficiency in brain slices to the development of nano-biolistics. The delivery of DNA and fluorescent dyes into living cells by biolistic delivery should enable a detailed map of the anatomical connections between individual cells and groups of cells to be constructed, providing a “wiring diagram” of connections. The implications of this are discussed in Chapter twelve.

This series of experiments investigated dystrophin localization in the normal cerebellum and examined Purkinje neuron function in normal and dystrophin-deficient mice to better understand the physiological basis for cognitive deficits associated with Duchenne muscular dystrophy (DMD), a common genetic disorder among children. Cognitive impairments are consistently reported in DMD, yet precise mechanisms for their occurrence are unknown. Dystrophin protein, which is absent in DMD, is normally localized to muscles and specific neurons in the brain. Purkinje neurons are rich in dystrophin, specifically in somatic and dendritic membranes. Studies demonstrate perturbed cerebellar function in the absence of dystrophin, suggesting that DMD should be regarded as a cerebellar disorder in addition to being considered a neuromuscular disorder. However, theory and evidence are not generated from overlapping information: research investigating cerebellar involvement in DMD has focused on the vermal region, associated with motor function. The lateral region, implicated in cognition, has not been explicitly examined in DMD. The first experiment revisited the issue of dystrophin distribution in the mouse cerebellum using immunohistochemistry to investigate qualitative and quantitative differences between cerebellar regions. Both regions showed dystrophin localized to Purkinje neuron somatic and dendritic membranes, but dystrophin density was 30% greater in the lateral than the vermal region. The second experiment examined intrinsic electrophysiological properties of vermal and lateral Purkinje neurons from wild-type (WT) mice and from the mdx mouse model of DMD which lack dystrophin. Significant differences in action potential firing frequency, regularity, and shape were found between cerebellar regions. Purkinje neurons from mdx mouse cerebellum exhibited membrane hyperpolarization and irregular action potential firing, regardless of region. Spontaneous action potential firing frequency was reduced in Purkinje neurons from lateral cerebellum in mdx mice relative to controls, demonstrating that a loss of dystrophin causes a potent dysregulation of Purkinje neuron function in the region associated with cognition. This research extends our understanding of cerebellar pathology in DMD and its potential relevance to cognitive deficits in the disorder. Moreover, this research further supports the role of the cerebellum as a structure important for cognition and contributes to our understanding of dystrophin’s role in the brain.

This series of experiments investigated dystrophin localization in the normal cerebellum and examined Purkinje neuron function in normal and dystrophin-deficient mice to better understand the physiological basis for cognitive deficits associated with Duchenne muscular dystrophy (DMD), a common genetic disorder among children. Cognitive impairments are consistently reported in DMD, yet precise mechanisms for their occurrence are unknown. Dystrophin protein, which is absent in DMD, is normally localized to muscles and specific neurons in the brain. Purkinje neurons are rich in dystrophin, specifically in somatic and dendritic membranes. Studies demonstrate perturbed cerebellar function in the absence of dystrophin, suggesting that DMD should be regarded as a cerebellar disorder in addition to being considered a neuromuscular disorder. However, theory and evidence are not generated from overlapping information: research investigating cerebellar involvement in DMD has focused on the vermal region, associated with motor function. The lateral region, implicated in cognition, has not been explicitly examined in DMD. The first experiment revisited the issue of dystrophin distribution in the mouse cerebellum using immunohistochemistry to investigate qualitative and quantitative differences between cerebellar regions. Both regions showed dystrophin localized to Purkinje neuron somatic and dendritic membranes, but dystrophin density was 30% greater in the lateral than the vermal region. The second experiment examined intrinsic electrophysiological properties of vermal and lateral Purkinje neurons from wild-type (WT) mice and from the mdx mouse model of DMD which lack dystrophin. Significant differences in action potential firing frequency, regularity, and shape were found between cerebellar regions. Purkinje neurons from mdx mouse cerebellum exhibited membrane hyperpolarization and irregular action potential firing, regardless of region. Spontaneous action potential firing frequency was reduced in Purkinje neurons from lateral cerebellum in mdx mice relative to controls, demonstrating that a loss of dystrophin causes a potent dysregulation of Purkinje neuron function in the region associated with cognition. This research extends our understanding of cerebellar pathology in DMD and its potential relevance to cognitive deficits in the disorder. Moreover, this research further supports the role of the cerebellum as a structure important for cognition and contributes to our understanding of dystrophin’s role in the brain.