Anatomy and Neuroscience - Theses
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ItemA morphological characterisation of central neural pathways to the kidney( 2005-04)This study was undertaken to locate and characterise the neurons in the central nervous system that project to the kidney. In particular, the aim was to illustrate and characterise the neural link between regions in the hypothalamus known to influence renal function and fluid balance, and nerves known to innervate the kidney.
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ItemEarly emergence of neural activity in the developing mouse enteric nervous system( 2012)The enteric nervous system (ENS) is a crucial part of the autonomic nervous system that controls or regulates various aspects of gastrointestinal (GI) function, including motility and secretion (Furness, 2006). The development of a functioning ENS is thus vital for correct control of GI activity. All the neurons and glia of the ENS arise from neural crest-derived cells that migrate into the foregut during embryonic development, and colonise the GI tract in a rostral-to-caudal wave. A series of co-ordinated events is required to produce the mature ENS from the enteric neural crest-derived cells (ENCCs), including proliferation, migration, differentiation into neurons of various neurochemical phenotypes, formation of ganglia, the development of neuronal activity, as well as axon extension and synaptogenesis. Although the early events in ENS development such as ENCC proliferation and migration have been extensively studied, little is known about the later events in ENS development, in particular, the development of neural activity. It is well established that a subpopulation of ENCCs express pan-neuronal markers at early stages of ENS development. At embryonic day (E) 10.5 in the mouse, 15-20% of ENCCs in the developing small intestine express pan-neuronal markers (Baetge and Gershon, 1989; Young et al., 1999). Some studies have suggested these cells cannot be labelled as “neurons” as they have not yet exited the cell cycle (Teitelman, 1981; Baetge and Gershon, 1989). Prior to commencing this thesis, it was not known whether the early ENCCs that express pan-neuronal markers were electrically active. However, there has been indirect evidence for neural activity during embryonic murine ENS development, as recent studies have shown that inhibition of various forms of activity, or genetic deletion of some neurotransmitter synthetic enzymes or transporters, impact on developmental events such as ENCC migration and differentiation (Vohra et al., 2006; Li et al., 2010; Li et al., 2011). Hence, it is important to investigate how neural activity develops in the ENS, and how it contributes to the formation of a mature functioning ENS. The aims of this thesis are to examine: (i) At what age do enteric “neurons” become electrically active and what are the early forms of neural activity? (ii) How does electrical activity develop over time? (iii) What is the role of neural activity on early vs. later events in ENS development? In Chapter 1, I provide an overview of the literature on the mature ENS, the development of the ENS, and investigations of neural activity during the development of other parts of the nervous system. Chapters 2-4 are the three main experimental studies comprising this thesis. In the first study described in Chapter 2, I used Ca2+ imaging to examine the response of embryonic enteric neurons to electrical and chemical stimulation. Sharp increases in intracellular Ca2+ concentration ([Ca2+]i) were observed in response to electrical stimulation at E11.5, indicating that neurons are electrically active at early stages of ENS development. [Ca2+]i transients were also observed in response to several neurotransmitter receptor agonists, indicating that the machinery to respond to neurotransmitters is also present during embryonic development. In addition, spontaneous [Ca2+]i transients were also observed. In the second study described in Chapter 3, I examined the electrical activity of enteric neurons in further detail using whole-cell patch-clamp electrophysiology. Based on their firing properties, neurons at E11.5 and E12.5 could be classified into 3 main groups: (i) action potential (AP)-firing neurons; (ii) neurons that fired immature electrical responses; and (iii) inactive neurons. Spontaneous synaptic activity was also recorded in cells at E12.5. In the last study described in Chapter 4, I examined the differentiation of subtypes of enteric neurons at E11.5. Nitrergic and calbindin-immunoreactive neurons were identified at E11.5, as well as expression of the intermediate conductance K+ channel in neuronal fibres. Neural activity was then inhibited in developing explants of embryonic gut. Inhibition of the voltage-dependent Na+ channels (VGSCs) resulted in a decrease in the differentiation of nitrergic subtype of enteric neurons. These studies have directly examined the presence of neural activity at early stages of ENS development, and identified that this electrical activity is important for guiding differentiation.