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.

Radial migration in the cerebral cortex is enhanced by signals from thalamus.

The six layered cerebral cortex derives from cells that divide in the ventricular zone and migrate to their final destination in the cortical plate (future cortex). In the mouse, cortical layer III and IV neurons undergo their final mitotic division at around E16, at which time thalamic axons are beginning to enter the cortex. We used bromodeoxyuridine-birth dating of cells in cortical slice cultures to show that the thalamus enhances the migration out of the ventricular zone of future layer III/IV cells. When cortical slices were cultured alone, less than 35% of cells born in vitro on E16 were present in the pial half of the slice after 48 h in culture. In contrast, when cortical slices were cocultured with thalamus, 69% of these cells were found in the pial half of the slice. Explants of other developing tissues did not mimic the effect of the thalamus. The thalamus had no obvious effect on cortical radial glial cells, cortical cell viability or maintenance of cortical slice structure. We found that most precursors born at a similar age but in vivo, shortly before cortical slices were isolated, migrated to the pial half of the cultured slices in the absence of a cocultured thalamic explant. Thus, E16 cortical slices cultured without thalamus permit migration of cells born in vivo and therefore already exposed to the thalamus. Our results indicate that the thalamus provides factors to E16-born cortical precursors that enhance their directed migration out of the ventricular zone to the cortical plate.

A role for Pax6 in the normal development of dorsal thalamus and its cortical connections.

The transcription factor Pax6 is widely expressed throughout the developing nervous system, including most alar regions of the newly formed murine diencephalon. Later in embryogenesis its diencephalic expression becomes more restricted. It persists in the developing anterior thalamus (conventionally termed "ventral" thalamus) and pretectum but is downregulated in the body of the posterior (dorsal) thalamus. At the time of this downregulation, the dorsal thalamus forms its major axonal efferent pathway via the ventral telencephalon to the cerebral cortex. This pathway is absent in mice lacking functional Pax6 (small eye homozygotes: Sey/Sey). We tested whether the mechanism underlying this defect includes abnormalities of the dorsal thalamus itself. We exploited a new transgenic mouse ubiquitously expressing green fluorescent protein tagged with tau, in which axonal tracts are clearly visible, and co-cultured dorsal thalamic explants from Pax6(+/+ )or Pax6(Sey/Sey )embryos carrying the transgene with wild-type tissues from other regions of the forebrain. Whereas Pax6(+/+ )thalamic explants produced strong innervation of wild-type ventral telencephalic explants in a pattern that mimicked the thalamocortical tract in vivo, Pax6(Sey)(/Sey) explants did not, indicating a defect in the ability of mutant dorsal thalamic cells to respond to signals normally present in ventral telencephalon. Pax6(Sey)(/Sey) embryos also showed early alterations in the expression of regulatory genes in the region destined to become dorsal thalamus. Whereas in normal mice Nkx2.2 and Lim1/Lhx1 are expressed ventral to this region, in the mutants their expression domains are throughout it, suggesting that a primary action of Pax6 is to generate correct dorsoventral patterning in the diencephalon. Our results suggest that normal thalamocortical development requires the actions of Pax6 within the dorsal thalamus itself.

Defect of tyrosine hydroxylase-immunoreactive neurons in the brains of mice lacking the transcription factor Pax6.

In the CNS, the lack of the transcription factor Pax6 has been associated with early defects in cell proliferation, cell specification, and axonal pathfinding of discrete neuronal populations. In this study, we show that Pax6 is expressed in discrete catecholaminergic neuronal populations of the developing ventral thalamus, hypothalamus, and telencephalon. In mice lacking Pax6, these catecholaminergic populations develop abnormally: those in the telencephalon are reduced in cell number or absent, whereas those in the ventral thalamus and hypothalamus are greatly displaced and densely packed. Catecholaminergic neurons of the substantia nigra (SN) and the ventral tegmental area (VTA) do not express Pax6 protein. Nevertheless, mice lacking Pax6 display an altered pathfinding of SN-VTA projections: instead of following the route of the medial forebrain bundle ventrally, most of the SN-VTA projections are deflected dorsorostrally at the pretectal-dorsal thalamic transition zone and in the dorsal thalamic alar plate. Moreover, some catecholaminergic neurons are displaced dorsally to an ectopic location at the pretectal-dorsal thalamic transition zone. Interestingly, from the pretectal-dorsal thalamic to the dorsal thalamic-ventral thalamic transition zones, mice lacking Pax6 display an ectopic ventral to dorsal expansion of the chemorepellant/chemoattractive molecule, Netrin-1. This may be responsible for both the altered pathway of catecholaminergic fibers and the ectopic location of catecholaminergic neurons in this region.

Serotonin receptor activation enhances neurite outgrowth of thalamic neurones in rodents.

Serotonin (5-HT) has been shown to influence the development of the rodent barrel field by affecting the patterning of thalamic axons in the somatic sensory cortex. To determine whether this is a direct effect on thalamocortical neurones, we analyzed primary thalamic cultures taken from E15 mouse embryos. We show that 5-HT enhances neurite outgrowth of thalamic neurones. The sodium channel blocker, TTX, blocks these effects, whereas the selective 5-HT1B agonist CGS-12066A maleate reproduced 5-HT's effect. Using PCR and immunocytochemistry, we found that 5-HT1B receptors are already expressed by thalamic neurones at E15, and that this expression is maintained in vitro. These results suggest that 5-HT-1B receptor activation directly affects the growth of thalamocortical axons.

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.

Effects of monoamine oxidase A inhibition on barrel formation in the mouse somatosensory cortex: determination of a sensitive developmental period.

Genetic inactivation of monoamine oxidase A (MAOA) in C3H/HeJ mice causes a complete absence of barrels in the somatosensory cortex, and similar alterations are caused by pharmacological inhibition of MAOA in wild type mice. To determine when and how MAOA inhibition affects the development of the barrel field, the MAOA inhibitor clorgyline was administered to mice of the outbred strain OF1 for various time periods between embryonic day 15 (E15) and postnatal day 7 (P7), and the barrel fields were analyzed with cytochrome oxidase and Nissl stains in P10 and adult mice. High-pressure liquid chromatography measures of brain serotonin (5-HT) showed three- to eightfold increases during the periods of clorgyline administration. Perinatal mortality was increased and weight gain was slowed between P3 and P6. Clorgyline treatments from E15 to P7 or from P0 to P7 disrupted the formation of barrels in the anterior snout representation and in parts of the posteromedial barrel subfield (PMBSF). Treatments from P0 to P4 caused similar although less severe barrel field alterations. Clorgyline treatments only during embryonic life or starting on P4 caused no detectable abnormalities. In cases with barrel field alterations, a rostral-to-caudal gradient of changes was noted: Rostral barrels of the PMBSF were most frequently fused and displayed an increased size tangentially. Thus, MAOA inhibition resulting in increased brain levels of 5-HT affects barrel development during the entire first postnatal week, with a sensitive period between P0 and P4. The rostral-to-caudal gradient of changes in the barrel field parallels known developmental gradients in the sensory periphery and in the maturation thalamocortical afferents. The observed barrel fusions could correspond to a default in the initial segregation of thalamic fibers or to a continued, exuberant growth of these fibers that overrides the tangential domain that is normally devoted to individual whiskers.

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.

Trophic and outgrowth-promoting effects of K(+)-induced depolarization on developing thalamic cells in organotypic culture.

The aim of this study was to investigate how different levels of K(+)-induced depolarization affect the survival and growth of isolated, cultured thalamic explants from mice aged embryonic day 13 to postnatal day 2. K+ was added to explants in serum-free culture medium. After culture for three days, explants were sectioned and Nissl-stained or photographed under phase contrast for quantification of neurite outgrowth. Viable and pyknotic cells were counted in sectioned material. The results revealed that, with no added K+, both viability and neurite outgrowth decreased as the age of the thalamic explant increased: most cells survived in embryonic day 13 explants, most died in postnatal day 2 explants. Adding K+ had an age- and dose-dependent effect on viability and neurite outgrowth. The greatest viability-promoting effect of adding K+ was at embryonic day 19: adding 5 mM K+ rescued the majority of these cells, although there was no effect on neurite outgrowth at this age (i.e., enhanced viability did not necessarily produce increased outgrowth). This same dose of K+ had its greatest effect on neurite outgrowth at embryonic day 17. No dose of added K+ had a stimulatory effect on viability and neurite outgrowth after embryonic day 19. The highest dose of K+ used here (50 mM) inhibited thalamic cell survival. We suggest that the survival and growth of the prenatal thalamus can occur without external influences. This intrinsic control may use an autocrine mechanism that becomes increasingly reliant on neural activity for its maintenance as it ages. After birth, when thalamic cells may switch their dependence to cortex-derived growth factors, this intrinsic control may become ineffective.

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.