Abstract
The overall aims of my PhD research project were to identify which particular gamma-amino butyric acid A receptor (GABAAR) subunits are expressed by neurochemically defined cell-types of the enteric nervous system (ENS) of the mouse colon, and what effect the pharmacological activation of specific receptor subtypes had on colonic contractility, as well as assessing whether two key determinants of gastrointestinal (GI) plasticity, namely stress and ageing, altered such GABAAR expression and functional phenotypes in the colon. My main findings were:1) Using RT-PCR and homogenates of whole mouse colon, I demonstrated that the majority of mRNA transcripts for GABAAR subunits are expressed within the mouse colon, apart from the GABAAR α6 and ε subunits (Chapter 2).
2) Using immunohistochemistry and confocal microscopy on fixed whole-mount preparations of the mouse colon, I discovered that immunoreactivity for the GABAAR alphas (α) 1-5 and gamma (γ) 2 subunits was divergently localised to ENS neurons in a cell-type (based on neurochemical signature), cell-domain (somata; dendrite; axon), subunit and ENS plexus (myenteric or submucosal)-specific manner (Chapter 2).
3) Using organ bath preparations of the isolated, intact mouse colon and subunit-preferring GABAAR ligands, I demonstrated that the activation of specific GABAAR subtypes had contrasting effects on the force and frequency of spontaneous contractions of colonic longitudinal smooth muscles as follows: 1) the activation of α1-γ2-GABAARs and α4-GABAARs increased the force of spontaneous contractions, but not their frequency; 2) the activation of α2-γ2-GABAARs increased their frequency, but not their force; 3) the activation of α3-γ2-GABAARs decreased their force and; 4) an inverse agonist at α5-γ2-GABAARs decreased their force (Chapter 3). This suggests that the engagement of various GABAAR subtypes within the cellular networks of the ENS cooperate to modulate the distinct physiological processes, which underlie coordinated GI contractility and thus motility.
4) Using various animal models of stress, either in early life, or in adulthood, I discovered that different forms of stress, which generally induce changes in CNS function as well as in behaviour, potently engage the ENS and the GABAAR system of the mouse colon in a stress- and GABAAR subunit-specific manner, by demonstrating the stress-induced changes in native colonic contractility, colonic GABAAR subunit expression and GABAAR-mediated colonic contractions (Chapter 4).
5) Using mouse subjects of varying ages (postnatal days 10; 15; 60 and 18 months), quantitative RT-PCR (qRT-PCR), organ bath preparations of isolated mouse colon and subunit-preferring GABAAR ligands, I discovered that: 1) GABAAR subunit expression varies significantly with
age, with the highest relative mRNA expression levels occurring at early
postnatal stages and then decreasing dramatically during adulthood, in a
subunit-specific manner; 2) the effects of the pharmacologically mediated
activation of GABAAR subtypes on the force and frequency of longitudinal
colonic smooth muscles also varies significantly across ages (Chapter 4).
Collectively, these data demonstrate the central role that the GABAAR
system plays in at least one aspect of GI function, namely motility.
Furthermore, the profound changes in the expression and functional
phenotypes of the GABAAR system across the life-time of an individual,
either in response to emotional challenges, or healthy ageing, appears to
parallel the functional plasticity of the GI system itself under such diverse
conditions. This leads me to conclude that the colonic GABAAR system plays
an integral role in native GI physiology and represents a potential therapeutic
target for GI disorders arising either due to emotional challenges or the
ageing process. The data arising from this thesis thus provide the scientific
rationale for future translational studies targeting selectively the GI GABAAR
system in various GI disorders using GABAAR subtype-specific ligands which
do not cross the blood brain barrier and are thus devoid of any unwanted
CNS effects.
| Date of Award | May 2015 |
|---|---|
| Original language | English |
| Awarding Institution |
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| Supervisor | Jerome Swinny (Supervisor), James Brown (Supervisor) & Jeremy Mills (Supervisor) |