Anatomy and Neuroscience - Theses
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ItemEnteric serotonin interneurons: connections and role in intestinal movement( 2008)5-HT powerfully affects gastrointestinal function. However, the study of these effects is complicated because 5-HT from both mucosa and a subset of enteric neurons acts on multiple receptor subtypes in enteric tissues. The role of neural 5-HT has been difficult to isolate with current techniques. This thesis aimed to elucidate the role of 5-HT neurons in motility using anatomical and functional methods. In Chapter 2, confocal microscopy was used to examine over 95% of myenteric neurons in guinea pig jejunum, categorized neurochemically, to identify neurons that received anatomically-defined input from 5-HT interneurons. The data showed that cholinergic secretomotor neurons were strongly targeted by 5-HT interneurons. In another key finding, excitatory motor neurons were surrounded by 5-HT terminals; this could provide an anatomical substrate for the descending excitation reflex. Subgroups of ascending interneurons and neurons with immunoreactivity for NOS, were also targeted by 5-HT interneurons. Thus, subtypes of these neurons might act in separate reflex pathways. Despite strong physiological evidence for 5-HT inputs to AH/Dogiel type II neurons, few contacts were identified. In Chapter 3, the confocal microscopy survey was extended to the three other interneuron classes (VIP/NOS and SOM descending interneurons; calretinin ascending interneurons) of guinea pig small intestine. A high degree of convergence between the otherwise polarized ascending and descending interneuron pathways was identified.
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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.
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ItemMorphological and functional consequences of ischemia/reperfusion injury in the enteric nervous system( 2011)Of all organs, the intestine is possibly the most susceptible to the damaging effects of ischemia and reperfusion (I/R) injury. I/R injury to the intestine occurs following shock, sepsis, vascular surgery, strangulation ileus, embolism, intestinal obstruction, necrotising enterocolitis and small intestine transplantation. I/R injury that occurs during intestinal transplantation is particularly relevant because small intestinal transplantation is being increasingly applied; it has become a treatment of choice for patients with short bowel syndrome who are on total parenteral nutrition and who have developed life-threatening complications. Studies of the consequences and mechanisms of I/R in the intestine have concentrated on damage to the epithelium and the breakdown of the mucosal barrier. However, I/R also affects enteric neurons, changing their properties and even causing their death. At the time I began my project, almost all studies of effects of I/R on enteric neurons have used only morphological methods, or markers of apoptosis, to document changes. My work has identified a selectivity of effects on nitric oxide synthase (NOS) neurons and on intrinsic primary afferent neurons (IPANs) following I/R. Following I/R, NOS neurons became swollen, and their dendrites were distorted, while IPANs shrank and showed signs of distortion of the surface membrane. In addition to the changes in cell morphology, my work has also developed further measures of effects of I/R on enteric neurons, including detection of neuronal apoptosis, the nitrosylation of proteins in enteric neurons and smooth muscle cells, and the translocation to the nucleus of the mRNA-binding protein, Hu. I have also made the first studies of the physiological consequences of intestinal I/R properties in enteric neurons by recording from enteric neurons with intracellular microelectrodes. Furthermore, I have shown that nNOS plays a protective role following intestinal I/R injury since the deletion of nNOS resulted in markedly more I/R-induced damage. Finally, this study showed that damage to the mucosa and muscle recovers quickly after I/R, but that changes in neurons persist. This suggests that neuronal damage may be involved in long-term functional changes after intestinal I/R. During my project, I had the opportunity to study the involvement of enteric neurons in intestinal dysfunction following I/R injury. By investigating the specificity of effects on different classes of neurons and the ways in which properties of the neurons are altered, strategies for coping with deleterious effects of the neuronal changes can possibly be identified. My work provides quantitative methods to assess neuronal responses to I/R injury, and thus a basis for assessing the effectiveness of protective strategies.