Target-derived neurotrophic factors regulate the death of developing forebrain neurons after a change in their trophic requirements.

Many neurons die as the normal brain develops. How this is regulated and whether the mechanism involves neurotrophic molecules from target cells are unknown. We found that cultured neurons from a key forebrain structure, the dorsal thalamus, develop a need for survival factors including brain-derived neurotrophic factor (BDNF) from their major target, the cerebral cortex, at the age at which they innervate it. Experiments in vivo have shown that rates of dorsal thalamic cell death are reduced by increasing cortical levels of BDNF and are increased in mutant mice lacking functional BDNF receptors or thalamocortical projections; these experiments have also shown that an increase in the rates of dorsal thalamic cell death can be achieved by blocking BDNF in the cortex. We suggest that the onset of a requirement for cortex-derived neurotrophic factors initiates a competitive mechanism regulating programmed cell death among dorsal thalamic neurons.

Effects of the thalamus on the development of cerebral cortical efferents in vitro.

The cerebral cortex is a multilayered tissue, with each layer differing in its cellular composition and connections. Axons from deep layer neurons project subcortically, many to the thalamus, whereas superficial layer neurons target other cortical areas. The mechanisms that regulate the development of this pattern of connections are not fully understood. Our experiments examined the potential of the thalamus to attract and/or select neurites from appropriate cortical layers. First, we cocultured murine cortical slices in close proximity to thalamic explants in collagen gels. The amount of neurite outgrowth from deep layer cells was enhanced by, but not attracted to, the thalamic explants. Second, we cocultured cortical slices in contact with thalamic or cortical explants to test for laminar specificity of connections. Specificity was apparent after culture for about a week, in that deep cortical layers contained the highest proportions of corticothalamic cells and superficial cortical layers contained the highest proportions of corticocortical cells. After shorter culture of only a few days, however, specificity was not apparent and there were larger numbers of corticothalamic projections from the superficial layers than after a week. To study how the early nonspecific pattern of corticothalamic connections was transformed into the more specific pattern, we labeled corticothalamic cells early, after 2 days, but let the cultures survive for 8 days. On day 8, the nonspecific pattern of early-labeled cells was still seen. We conclude that although the thalamus does not block the initial entry of inappropriate axons from the superficial layers, many of these axons are subsequently lost. This suggests that contact-mediated interactions between cortical axons and the thalamus allow cortical efferents from appropriate layers to be distinguished from those arising in inappropriate layers. This may contribute to the development of layer-specific cortical connections in vivo.

A role for neurotrophins in the survival of murine embryonic thalamic neurons.

The mechanisms that determine whether developing CNS neurons live or die are poorly understood. We studied the role of the neurotrophins and fibroblast growth factors in the survival of embryonic thalamic neurons in culture. Dissociated embryonic dorsal thalamic neurons cultured at high density in defined serum-free medium survived and grew neurites. As in vivo, they expressed all the neurotrophins, fibroblast growth factor-1 and their high-affinity tyrosine kinase receptors. The survival of these cells was reduced by the addition of the protein kinase inhibitor K252a at concentrations that block neurotrophin receptor activity but not the activity of other tyrosine kinase receptors. In low-density cultures, most dorsal thalamic neurons died, but their survival was increased by co-culture with thalamic explants or with most of the neurotrophins and fibroblast growth factor-1 added singly. These results indicate that thalamic neurons have remarkably promiscuous trophic responses to a battery of neurotrophins and fibroblast growth factors. They suggest that neurotrophins endogenous to the early embryonic thalamus may be required to promote the survival of its neurons.

Influences of the thalamus on the survival of subplate and cortical plate cells in cultured embryonic mouse brain.

The afferent and efferent connections of the cerebral neocortex develop simultaneously toward the end of embryogenesis. At this stage, the neocortex comprises two main cell-dense layers: the thicker and more superficial cortical plate (future layers 2-6) and the thinner underlying subplate. Many early thalamocortical projections temporarily innervate the subplate before leaving it to locate their ultimate targets in the overlying cortical plate. The subplate then disappears. In this study, we performed in vitro experiments on late embryonic murine brain to test whether the thalamus can influence the survival of cortical plate and subplate cells at this stage. In isolated organotypic cortical explants from embryonic day 19 mice, most of the cells that had formed the subplate died. Coculture with a thalamic explant prevented this loss; coculture with additional cortical or cerebellar explants did not. By contrast, many cells in or destined for the cortical plate survived even in isolated cortical explants; coculture with a thalamic explant did not alter the numbers of these cells that survived. Our results suggest that the thalamus provides trophic support for subplate cells but not for late embryonic cortical plate cells. In vivo, a loss of thalamic-derived trophic support for the subplate late in embryogenesis, consequent on the movement of thalamocortical axons into the cortical plate, may contribute to subplate death.

The stimulation of thalamic neurite outgrowth by cortex-derived growth factors in vitro: the influence of cortical age and activity.

Recent in vitro experiments have provided useful insights into the development of connections between the thalamus and the cortex. While most of these previous studies focused on neurite guidance and target recognition, our experiments used a serum-free culture system to examine the possible roles of unidentified diffusible cortex-derived growth factors. We demonstrated that occipital cortical explants release diffusible growth factors that enhance neurite outgrowth from explants of the posterior thalamus (the region around the developing lateral geniculate nucleus). The amount of thalamic outgrowth was dependent on the age of the cocultured cortical slices. Our results suggest that there is an overall increase in the release of cortex-derived growth factors during the first three postnatal weeks in mice; this parallels known postnatal increases in the production of several identified growth factors. We found evidence for two peaks in the release of cortex-derived growth factors during the general upward trend, the first at around postnatal day 6 (shortly after thalamocortical innervation of layer 4) and a second between postnatal days 14 and 18 (just after eye-opening). The increased release of cortex-derived growth factors was not found when cortical slices were from mice that had been dark-reared from birth, suggesting that neural activity may be important for enhancing release. Other regions of the central nervous system, including the cerebellum and medulla, were also capable of stimulating some thalamic outgrowth; neither additional explants of the thalamus nor hepatic explants enhanced outgrowth. Fibroblast growth factor is one substance that is distributed preferentially among those tissues that were stimulatory in our experiments. Its level of transcription is known to increase in the brain during the first three postnatal weeks and to be influenced by neural activity. At low doses, fibroblast growth factor greatly increased outgrowth from isolated posterior thalamic explants. Nerve growth factor, another candidate molecule, was less effective. Overall, our results complement the in vivo observations of others on the synthesis of identified growth factors in the cortex and the factors that influence their production. They suggest that growth factors may influence thalamic neurons, and indicate that fibroblast growth factor, and possibly nerve growth factor, are two candidates for molecules mediating the in vitro effects.

Growth-promoting interactions between the murine neocortex and thalamus in organotypic co-cultures.

The aim of this study was to assess whether developing cerebral cortex produces diffusible factors that can affect the growth of thalamic cells and, if so, what the role of these factors might be during the formation of thalamocortical connections. We studied interactions between cultured organotypic explants from mice maintained in defined serum-free medium. First, we cultured explants of embryonic dorsolateral thalamus in isolation from any other tissue; after culture, these explants were viewed intact and then sectioned. We estimated the numbers of healthy and pyknotic cells before and after culture, and the rates of mitosis in the explants during culture (using bromodeoxyuridine). Based on these data, we concluded that the majority of cells in the thalamic explants survived, although significant numbers of pyknotic cells did accumulate. Thalamic explants extended either very few or no neurites when cultured alone. We then cultured explants of embryonic thalamus near to explants from other tissues. A gap was always maintained between the explants, and we measured the length and density of neurite outgrowth from each thalamic explant. Slices of embryonic cortex promoted a small but significant increase in the amount of growth from thalamic explants. Postnatal cortex stimulated much more profuse neurite outgrowth; postnatal cerebellum had less of an effect, and postnatal medulla or liver had none. We showed that there was significantly more outgrowth from thalamic explants cultured in medium that had been preconditioned with cortical slices than from thalamic explants cultured in control medium, confirming that diffusible factors were produced by the cortex. The survival and mitotic rates of thalamic cells were unaffected by co-culture with the cortex. We conclude that the developing cortex releases diffusible factors that stimulate the growth of thalamic neurites and that other regions of the brain may also release the same substance(s). The lack of a specific source of thalamic growth promoting factor(s) argues against a role for these factors in guiding thalamic axons to specific targets; indeed, we were unable to demonstrate any chemotropic guidance of thalamic axons towards cortical explants in collagen gels. Since postnatal cortex has a more potent stimulatory effect than prenatal cortex, it seems possible that, in vivo, the cortical-derived factors act mainly on thalamocortical axons that have located their targets and are in the process of arborizing and refining their connections.