Target-dependent synaptogenesis: Inductive effect of pituitary melanotrophs on their central innervation

Abstract

The glandular activity of the vertebrate pituitary intermediate lobe (IL) is regulated by direct cellular innervation, in contrast with the purely humoral regulation of adjacent pituitary anterior lobe (AL). Thus in the rat IL, melanotrophs receive a dopaminergic and GABAergic innervation from the basal hypothalamus, which tonically inhibit their glandular activity. We studied this model of neuron-target interactions in cocultures in defined medium of fetal hypothalamic neurons with neonate pituitary glandular cells. In the cocultures with IL cells, neuroglandular contacts occurred after 4 days in vitro (DIV) but required another 8 DIV to exhibit ultrastructural and immunocytochemical features of fully differentiated functional synapses; by contrast, neuroneuronal synapses developed much faster and could already be detected after 4 DIV. In the cocultures with AL cells, neuroglandular contacts never mature in differentiated synapses. Confocal microscope observation revealed that dopaminergic neurons, which represented less than 1% of total neurons in the cocultures, established 50% of the synapses detected on the melanotrophs. These cells are thus able, contrary to the AL cells, to promote the establishment of functional synapses and, to some extent, to select their specific innervation.

Differentiation of rat hypothalamic dopaminergic neurons is stimulated in vitro by target cells: the melanotrophs

“…Although POMC is also expressed in situ by a subpopulation of neurons in the arcuate nucleus (Mansour et al, 1988), in our cultures of hypothalamic neurons alone, neither MSH immunoreactivity nor secretion of the peptide in the culture medium was detectable. In co-cultures with nb NIL cells, the level of secreted αMSH (about 1 nM/day and well, René et al, 1997) is only attributable to melanotrophs, since neurons are not immunoreactive for αMSH. It is now well established that POMC-derived peptides can exert neurotrophic effects on several areas of the central nervous system (for reviews see Strand et al, 1993;Darlington et al, 1996).…”

“…In our conditions, the DA neurons contacting the melanotrophs are also immunoreactive for GAD, as those innervating the IL in situ . Moreover, in co‐cultures of foetal hypothalamic neurons with nb IL cells, we have recently reported the establishment of target‐dependent and, to some extent, neuron‐selective synapses (Egles et al 1997) between GABAergic/DA neurons and melanotrophs, and demonstrated their functionality (Poisbeau et al 1996). These observations clearly suggest neurotrophic effects of the target (melanotrophs) on their afferent neurons which may most probably correspond to the population of dopaminergic/GABAergic neurons originating from the A14 hypothalamic nucleus, which innervate the melanotrophs in situ .…”

“…Diencephalic areas corresponding to the basal hypothalamus were dissected from 15-day-old foetuses (day 0 of gestation corresponding to the day of fecondation). Hypothalami were dissociated according to René et al (1997); briefly, enzymatic dissociation with trypsin-EDTA (0.05%-0.02%, Gibco France) was followed by mechanical dissociation performed by aspiration into fire-polished Pasteur pipettes with decreasing tip diameter. After centrifugation, the cells were resuspended in a serum-free Dulbecco's modified Eagle medium (Sigma, France), supplemented as described by Bottenstein & Sato (1979).…”

“…To test whether these peptides are implicated during the development of the hypothalamo‐pituitary intermediate lobe axis, we examined the influence of melanotrophs and their secretory products, the melanocortins, on their afferent neurons, the DA/GABAergic neurons of the hypothalamus. This study was performed in vitro , using a co‐culture system of melanotrophs and foetal E15 hypothalamic neurons developed in our laboratory (René et al 1997), which reproduces faithfully the establishment of specific (Egles et al 1997) and functional (Poisbeau et al 1996) neuroglandular synapses, as in the IL in situ . In the present study, we compared the influence of melanotrophs on hypothalamic neurons taken at two developmental stages: E15 when neurons start to express TH, and immediately after birth when they invade the IL to innervate their target cells.…”