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.