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Title: An investigation of the morphological and functional properties of rat hypothalamic dopaminergic systems in vitro
Author: Murray, Hilary Elaine
Awarding Body: University of London
Current Institution: Imperial College London
Date of Award: 1994
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Hypothalamic neurotransmitter systems are likely to play an important role in bringing about the changes in neuroendocrine function which occur throughout development. In particular, the dopaminergic neurones intrinsic to the hypothalamus are widely implicated in the regulation of the reproductive and growth axes which exhibit clear ontogenetic changes in their endocrine profiles. Therefore, using foetal rat hypothalamic cells in primary culture, maintained in a serum-free defined medium, we have investigated the morphological and functional development of the intrahypothalamic dopaminergic systems. Immunocytochemical studies demonstrated the presence of three morphologically distinct sub-types of tyrosine hydroxylase-immunopositive (THIP) neurones. On day three in vitro unipolar, bipolar and multipolar cell types were apparent. The latter two sub-types persisted to later stages in culture and increased both in perikarya size and neurite length. All subtypes have been shown to have correlates in vivo. Biochemical studies employing tritiated dopamine ([3H]DA) demonstrated a time- and temperature- dependent uptake mechanism within the cultures which was significantly attenuated by uptake inhibitors such as benztropine and nomifensine in a dose - dependent manner. The degree of uptake into the hypothalamic cells in culture increased as a function with time reflecting a maturation of the uptake mechanism in play. Certain experiments suggest that the uptake may also be influenced by dopamine receptor modulation. [3H]DA was released under both basal and potassium (56mM) - stimulated conditions and the magnitude of the response was reduced by exclusion of calcium from the release medium. The ability of the cells to release pH]DA also increased with the age of the culture again indicating a functional maturation of the DA containing neurones within this preparation. Functional maturation of these systems was also reflected in studies involving the measurement of endogenous DA using high performance liquid chromatography coupled with electrochemical detection (HPLC - EC). The neurones displayed an inherent maturation as shown by an increase in the magnitude of release (basal and potassium - stimulated) of endogenous catecholamine with advancing cultivation time. Potassium - depolarization was seen to enhance the rate of development/improve the efficiency of the release mechanism. In addition, we found that cultures initiated from tissue collected at different gestational ages showed differential behaviour in terms of DA output. The role of 17 6 - oestradiol (E2) in regulating hypothalamic dopaminergic function was also investigated both indirectly with the use of pH]DA and by direct measurement of endogenous DA. Both uptake and release of [^HJDA and release of endogenous DA were significantly modulated by the concentration of the steroid in the defined medium. The optimal concentration for uptake ([^HJDA) and release (pH]DA and endogenous DA) were 10 and 10 respectively. These levels of oestrogen are likely to be within the physiological range. The parameters under investigation were also influenced by progesterone, but in a manner which was distinctly different from that of oestrogen. The effect of prolactin and gonadotrophin - hormone - releasing hormone on the release of endogenous DA was also studied to investigate whether intrahypothalamic dopaminergic systems might be involved in negative feedback regulation of hormone release. While prolactin was ineffective in modulating DA output GnRH was a potent secretagogue for the dopaminergic systems within the culture model. GABA-ergic neurones within the cultures were also studied in order to test a) the specificity of the results with the DA neurones and b) a functional interaction between hypothalamic GABA-ergic and dopaminergic neurones. Studies on the uptake and release of [^HJGABA in hypothalamic cells in culture highlighted marked differences in behaviour of GABA-ergic and dopaminergic systems both in terms of their pattern of maturation and their responsiveness to gonadal steroids. Bicuculline (GABAA receptor antagonist) significantly (p < 0.005) enhanced the release of endogenous DA at concentrations ranging from to 10"^M and both GABAA and GABAB agonists (muscimol and baclofen respectively) were also found to modulate the release of endogenous DA. Marked differences in the behaviour of [^H]DA release compared with that of the endogenous amine were noted and suggest caution when interpreting pharmacological studies using cells pre-loaded with [^H] - labelled neurotransmitters. In conclusion, these results demonstrate that hypothalamic dopaminergic neurones in primary culture undergo a pre-programmed morphological and functional maturation which have several correlates in vivo. At least one sub-population of these neurones is responsive to gonadal steroids and influenced by hypothalamic GABAergic systems. As hypothalamic dopaminergic pathways are essential components in the regulation of neuroendocrine activity we propose that this model may serve as a valuable tool for the investigation of the ontogeny of DA systems intrinsic to the hypothalamus and also their interaction with other hypothalamic neurotransmitters and neuropeptides.
Supervisor: Gillies, Glenda Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available