Effects of coculture on the morphology of identified raphe and substantia nigra neurons from the embryonic rat brain

Abstract

The substantia nigra (SN) is one of the earliest targets of the 5‐HT neurons of the raphe nuclei (RN). To test the hypothesis that embryonic 5‐HT and catechol‐amine neurons may influence the differentiation of their target cells or source neurons, we have produced dissociated cell cultures from embryonic day 14 (E14) rat rhombencephalon (containing the serotonergic RN) and mesencephalon (containing the dopaminergic substantia nigra, SN). These cells were grown for 6 days in vitro, either as single cultures (RN or SN) or cocultures (RN + SN). Effects of coculture on the morphological development of neurons immunoreactive (IR) for 5‐HT or tyrosine hydroxylase (TH) were studied by measuring a series of morphological parameters related to size of the cell body and dendritic field, as well as to the complexity of neurites within this field, using computer‐assisted morphometry. Increases in a number of these parameters were found in cocultures compared to single cultures for both types of monoamine neurons, but a greater number of parameters were increased for TH‐IR cells, including size of the cell body. Although this might suggest that there was a greater effect of coculture on the TH‐IR (dopaminergic) cells of the SN than on the 5‐HT‐IR cells of the RN, we must consider the fact that a significant population of TH‐IR cells were present in single RN cultures, which contributed to the total population of TH‐IR cells in cocultures. Indeed, when morphometric parameters for TH‐IR cells in RN and SN single cultures were compared, it was found that TH‐IR cells from the RN were generally larger and more complex than those from the SN. Therefore, an analysis was made of which parameters were significantly increased for TH‐IR cells in cocultures compared to single cultures from both SN and RN. This was the case for two parameters: cell body size and absolute field area, indicating that these increases were probably due to the effects of coculture itself rather than to contamination by the larger and more complex TH‐IR cells from the RN. It is impossible to ascertain, however, whether this effect was on cells from the RN, SN, or both. Coculture effects on 5‐HT‐IR cells were easier to analyze, since no such cells were found in single cultures of SN. Three parameters were consistently increased for 5‐HT‐IR cells in coculture: cumulative length of all segments, segment length density, and absolute field area. This indicates that 5‐HT‐IR cells in coculture had larger dendritic fields that contained a more complex array of neurites than their counterparts in single cultures. In sum, coculture seems to promote the growth of both 5‐HT and TH‐IR neurons, but in different ways and to different degrees. These results suggest that some factor or factors related to growth of these monoamine neurons are altered when the rhombencephalon and mesencephalon are cultured together. The exact nature of this developmental signal remains to be determined.