Serotonin promotes the differentiation of glutamate neurons in organotypic slice cultures of the developing cerebral cortex

Abstract

The monoamines serotonin (5-HT), noradrenaline (NA), and dopamine (DA), which are present in the developing brain apparently before they assume their neurotransmitter functions, are regarded as strong candidates for a role in the maturation of the cerebral cortex. Here we sought to investigate their effects on the generation and differentiation of cortical cell types. Slice cultures, prepared from the cortices of embryonic day (E) 14, E16, and E19 rat fetuses, were kept in defined medium or in defined medium plus 5-HT for 7 d. E16 cortices were also exposed to NA or DA for the same period. At the end of this period, the proportions of the neuronal [glutamate (Glu)-, GABA-, calbindin-, calretinin-labeled], glial (GFAP), and neuroepithelial (nestin) cell types were estimated for all conditions. We found that in E16 cultures, application of 5-HT, but not of NA or DA, significantly increased the proportion of Glu-containing neurons without affecting the overall neuronal population or the proportions of any other cell types. A similar effect was observed in co-cultures of E16 cortex with slices through the midbrain raphe nuclei of E19 rats. The total amount of cortical Glu, as measured with HPLC, was also increased in these co-cultures. To investigate whether the effect of 5-HT was the result of changes in cell proliferation, we exposed slices to bromodeoxyuridine (BrdU) and found that the proportion of BrdU-labeled cells was similar in the 5-HT-treated and control slices. These results indicate that 5-HT promotes the differentiation of cortical Glu-containing neurons without affecting neuroepithelial cell proliferation.

Fig. 1.

Immunolabeled cells from E16 cortices kept for 7 DIV, as they appeared in whole-mount preparations (A, B) and after dissociation and embedding in agarose (C, D). In the whole-mount preparations, Glu- (A) and GABA-containing (B) cells showed darkly stained somata with short and sparsely branched processes. In agarose films, cells labeled for Glu (C) and GABA (D) appeared round and darkly stained.Long arrows point to a number of labeled neurons, andshort arrows point to some unlabeled cells. Scale bars:A, B, 20 μm; C, D, 50 μm.

Fig. 2.

The proportions of cells immunoreactive for cell-specific markers (Glu, GABA, CB, CR, nestin, GFAP) in cortical slices prepared at E14 (A), E16 (B), and E19 (C) and cultured both in the presence of a monoamine and under control conditions. The presence of 5-HT in the medium significantly increased the proportion of Glu-containing cells in cultures prepared at E16 (B, asterisk indicates statistical significance).

Fig. 3.

Co-cultures of cortical slices with slices through the midbrain containing the raphe nuclei. A, Numerous serotonergic neurons were visualized in the raphe slice. 5-HT-containing fibers innervated the entire cortical slice as shown in a camera lucida drawing (C); pia is at thetop. The 5-HT fibers in the cortex were thick and varicose, often having growth cones at their tips (B). Scale bars: A, 20 μm;C, 100 μm.

Fig. 4.

The effects of co-culturing cortical slices with slices containing the raphe nuclei. Both the proportion of Glu-containing neurons in the culture (A) and the Glu content of the culture as measured by HPLC (B) were significantly increased as compared with cortices kept in control conditions.

Fig. 5.

BrdU incorporation in cortical slices. In cryostat sections of cortical slices, BrdU-immunolabeled cell nuclei appeared darkly stained (A). Scale bar, 50 μm. The incorporation of BrdU did not differ between cultures exposed to 5-HT and cultures kept in control conditions; in both cases, the proportion of immunolabeled cells ranged from ∼50% (on the first day) to ∼10% (on the seventh day); note the very rapid drop in BrdU incorporation after 3 DIV (B).