Organization of the neuronal circuits in the central nervous system during development.

UNLABELLED: The human brain is a product of genetic instructions, cellular interactions and influences of innate activity and external stimulation. The formation of the neural tube and the patterning of the brain are determined by homeotic genes. After a prosencephalic phase with the formation of the hemispheres, the neurons prolipherate to number about 100 billion halfway through gestation. They also migrate to their final positions in an inside-outside fashion with the newly formed neurons at the outer layer of the cortex, followed by synaptogenesis, programmed cell death and organization of the neuronal circuits. This phase is probably determined not only by genes but also by innate activity, which for example has been detected in the foetal retina: "Cells that fire together wire together while those which don't won't". CONCLUSION: Development of the neuronal circuits in the CNS can be viewed as epigenetic. i.e. many different components must come together at the right time and place.

Brain derived neurotrophic factor is an endothelial cell survival factor required for intramyocardial vessel stabilization.

Brain derived neurotrophic factor, BDNF, is a neurotrophin best characterized for its survival and differentiative effects on neurons expressing the trk B receptor tyrosine kinase. Although many of these neurons are lost in the BDNF(-)(/)(- )mouse, the early postnatal lethality of these animals suggests a wider function for this growth factor. Here, we demonstrate that deficient expression of BDNF impairs the survival of endothelial cells in intramyocardial arteries and capillaries in the early postnatal period, although the embryonic vasculature can remodel into arteries, capillaries and veins. BDNF deficiency results in a reduction in endothelial cell-cell contacts and in endothelial cell apoptosis, leading to intraventricular wall hemorrhage, depressed cardiac contractility and early postnatal death. Vascular hemorrhage is restricted to cardiac vessels, reflecting the localized expression of BDNF and trk B by capillaries and arterioles in this vascular bed. Conversely, ectopic BDNF overexpression in midgestational mouse hearts results in an increase in capillary density. Moreover, BDNF activation of endogenous trk B receptors supports the survival of cardiac microvascular endothelial cells cultured from neonatal mice. These results establish an essential role for BDNF in maintaining vessel stability in the heart through direct angiogenic actions on endothelial cells.

Slit inhibition of retinal axon growth and its role in retinal axon pathfinding and innervation patterns in the diencephalon.

We have analyzed the role of the Slit family of repellent axon guidance molecules in the patterning of the axonal projections of retinal ganglion cells (RGCs) within the embryonic rat diencephalon and whether the slits can account for a repellent activity for retinal axons released by hypothalamus and epithalamus. At the time RGC axons extend over the diencephalon, slit1 and slit2 are expressed in hypothalamus and epithalamus but not in the lateral part of dorsal thalamus, a retinal target. slit3 expression is low or undetectable. The Slit receptors robo2, and to a limited extent robo1, are expressed in the RGC layer, as are slit1 and slit2. In collagen gels, axon outgrowth from rat retinal explants is biased away from slit2-transfected 293T cells, and the number and length of axons are decreased on the explant side facing the cells. In addition, in the presence of Slit2, overall axon outgrowth is decreased, and bundles of retinal axons are more tightly fasciculated. This action of Slit2 as a growth inhibitor of retinal axons and the expression patterns of slit1 and slit2 correlate with the fasciculation and innervation patterns of RGC axons within the diencephalon and implicate the Slits as components of the axon repellent activity associated with the hypothalamus and epithalamus. Our findings suggest that in vivo the Slits control RGC axon pathfinding and targeting within the diencephalon by regulating their fasciculation, preventing them or their branches from invading nontarget tissues, and steering them toward their most distal target, the superior colliculus.

C-fos gene expression in rat brain around birth: effect of asphyxia and catecholamines.

At birth, the mammalian nervous system must adapt rapidly to the new conditions it encounters in the extra-uterine environment. One aspect of this adaptation, known as arousal, is mediated by catecholamines, the levels of which in the brain increase rapidly after birth. The pattern of gene expression also changes. Shortly after birth, expression of the immediate early gene c-fos, known to reflect general neural activity, is up-regulated. Furthermore, asphyxia often occurs in connection with birth. In order to examine the effects of this phenomenon on the expression of c-fos, as well as on the rate of noradrenaline (NA) turnover, asphyxia was induced in rat pups delivered by caesarean section. Northern blot analysis and in situ hybridization revealed that the increase in expression of c-fos in certain areas of the brain was greatly enhanced by asphyxia of moderate duration; whereas more prolonged asphyxia lowered the level of c-fos mRNA. Asphyxia had a similar effect on the rate of NA turnover. Adrenergic receptor antagonists administered prior to birth attenuated the birth-related induction of c-fos mRNA. However, the potentiation of c-fos expression by asphyxia was not altered by these antagonists. Therefore, we propose that while catecholamines play an important role in the induction of c-fos in the brain at birth, the effects of asphyxia involve a different mechanism.

Role of brain-derived neurotrophic factor in target invasion in the gustatory system.

Brain-derived neurotrophic factor (BDNF) is a survival factor for different classes of neurons, including gustatory neurons. We have studied innervation and development of the gustatory system in transgenic mice overexpressing BDNF under the control of regulatory sequences from the nestin gene, an intermediate filament gene expressed in precursor cells of the developing nervous system and muscle. In transgenic mice, the number and size of gustatory papillae were decreased, circumvallate papillae had a deranged morphology, and there was also a severe loss of lingual taste buds. Paradoxically, similar deficits have been found in BDNF knock-out mice, which lack gustatory neurons. However, the number of neurons in gustatory ganglia was increased in BDNF-overproducing mice. Although gustatory fibers reached the tongue in normal numbers, the amount and density of nerve fibers in gustatory papillae were reduced in transgenic mice compared with wild-type littermates. Gustatory fibers appeared stalled at the base of the tongue, a site of ectopic BDNF expression, where they formed abnormal branches and sprouts. Interestingly, palatal taste buds, which are innervated by gustatory neurons whose afferents do not traverse sites of ectopic BDNF expression, appeared unaffected. We suggest that lingual gustatory deficits in BDNF overexpressing mice are a consequence of the failure of their BDNF-dependent afferents to reach their targets because of the effects of ectopically expressed BDNF on fiber growth. Our findings suggest that mammalian taste buds and gustatory papillae require proper BDNF-dependent gustatory innervation for development and that the correct spatial expression of BDNF in the tongue epithelium is crucial for appropriate target invasion and innervation.

Development of fusimotor innervation correlates with group Ia afferents but is independent of neurotrophin-3.

Fusimotor neurons, group Ia afferents and muscle spindles are absent in mutant mice lacking the gene for neurotrophin-3 (NT3). To partition the effect of Ia afferent or spindle absence from that of NT3 deprivation on fusimotor neuron development, we examined the fusimotor system in a mutant mouse (NesPIXpNT3) that lacks Ia afferents and spindles, but has normal or elevated tissue levels of NT3 during embryogenesis. Fusimotor fibers were absent in lumbar ventral spinal roots, and limb muscles were devoid of Ia afferents and spindles in adult NesPIXpNT3 mice. In contrast, no deficiency in motoneuron numbers was observed in the trigeminal nucleus which contains cell bodies of motor axons innervating muscles of mastication. Spindles and Ia afferents were also present in the masticatory muscles. Thus, the development and/or survival of fusimotor neurons correlates with the presence of Ia afferents and/or spindles, and not with the amount of NT3 in the spinal cord or muscle.

BDNF regulates reelin expression and Cajal-Retzius cell development in the cerebral cortex.

Cajal-Retzius (CR) cells of the cerebral cortex express receptors for the neurotrophin brain-derived neurotrophic factor (BDNF) and downregulate expression of the extracellular matrix protein Reelin during early postnatal development, coincident with the onset of cortical BDNF expression. During this period, mice lacking BDNF have elevated levels of Reelin in CR cells. Acute BDNF stimulation of cortical neuron cultures and overexpression of BDNF in the developing brain of transgenic mice prior to the onset of endogenous production causes a profound, dose-dependent reduction of Reelin expression in CR cells. In addition, overexpression of BDNF produces gaps and heterotopias in the marginal zone and disorganization and aggregation of cortical CR cells and induces several other malformations, including aberrant cortical lamination, similar to the phenotype of reeler mutant mice, which lack Reelin. These results demonstrate a role for BDNF on cortical CR cells and identify Reelin as a direct effector of this neurotrophin during brain development.

Limb proprioceptive deficits without neuronal loss in transgenic mice overexpressing neurotrophin-3 in the developing nervous system.

The role of neurotrophin-3 (NT3) during sensory neuron development was investigated in transgenic mice overexpressing NT3 under the control of the promoter and enhancer regions of the nestin gene, an intermediate filament gene widely expressed in the developing nervous system. Most of these mice died during the first postnatal day, and all showed severe limb ataxia suggestive of limb proprioceptive dysfunction. Tracing and histological analyses revealed a complete loss of spindles in limb muscles, absence of peripheral and central Ia projections, and lack of cells immunoreactive to parvalbumin in the dorsal root ganglion (DRG). Despite these deficits, there was no neuronal loss in the DRG of these mice. At birth, transgenic DRG showed increased neuron numbers, and displayed a normal proportion of neurons expressing substance P, calcitonin gene-related peptide and the NT3 receptor trkC. Transgenic dorsal roots exhibited an increased number of axons at birth, indicating that all sensory neurons in transgenic mice projected to the dorsal spinal cord. Despite the absence of central Ia afferents reaching motorneurons, several sensory fibers were seen projecting towards ectopic high levels of NT3 in the midline of transgenic spinal cords. These findings suggest novel roles for NT3 in differentiation of proprioceptive neurons, target invasion and formation of Ia projections which are independent from its effects on neuronal survival.

Expression of c-fos, tyrosine hydroxylase, and neuropeptide mRNA in the rat brain around birth: effects of hypoxia and hypothermia.

Arousal at birth is likely to be accompanied by changes in gene expression patterns in the brain. We analyzed the expression levels of genes that may be involved in neonatal adaptation. We have also tried to dissect the effect of hypoxia and hypothermia, two components that may play a role in gene expression at birth. Therefore, we analyzed the expression patterns of the c-fos, tyrosine hydroxylase, enkephalin, preprotachykinin-A, and neuropeptide Y genes in various brain regions of rat pups at various time points after cesarean section under normal conditions and after exposure to hypoxia and hypothermia. We found that c-fos RNA was up-regulated transiently after birth in neocortex, midbrain, and pons-medulla with a maximum of 30 min after cesarean section, and that this transient increase was not further augmented by hypoxia and hypothermia. The expression patterns of the other genes were not significantly altered, with the exception of a very slight increase in tyrosine hydroxylase RNA levels. We discuss tentative mechanisms for the transient increase in c-fos expression and the possible involvement of catecholamines in this process.

Birth-related changes of expression and turnover of some neuroactive agents and respiratory control.

Respiratory movements are partially inhibited before birth and central and peripheral chemoreceptors are probably less sensitive. The transition to continuous breathing might be due to the switch-off of inhibitory neuromodulators and the switch-on of excitatory neuroactive agents. This hypothesis has been explored mainly in rats. One example is dopamine which was found to have a high turnover in carotid bodies in newborn pups prior to decreasing rapidly. It is suggested to inhibit the carotid bodies before birth. In the brain, the noradrenergic system has been found to be activated at birth, possibly promoting neural transmission and arousal. Increased expressions of mRNA for c-fos, tyrosine hydroxylase and some neuropeptides have also been found.

Rapid increase of BDNF mRNA levels in cortical neurons following spreading depression: regulation by glutamatergic mechanisms independent of seizure activity.

Levels of mRNA for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and the tyrosine kinase receptors trkB and trkC have been studied using in situ hybridization in the rat brain after topical application of KCl to the cortical surface (which induces spreading depression). Repeated episodes of spreading depression during 2 h caused a rapid and marked increase of BDNF mRNA levels in deep and, in particular, superficial cortical layers of the ipsilateral hemisphere (to 213 and 417% of control, respectively). Maximal levels were reached within 2 h after the cessation of spreading depression and at 24 h BDNF mRNA expression had returned to control values. Levels of BDNF mRNA were unaffected in the hippocampus, in areas outside the cerebral cortex and in the contralateral hemisphere. Furthermore, no change of the expression of mRNA for NGF, NT-3, trkC or the full length trkB receptor was detected at any time point. However, at 2 h after spreading depression there was an increased level (150% of control) in superficial cortical layers of mRNA hybridizing to an oligonucleotide probe detecting both truncated receptors lacking the tyrosine kinase domain and full length trkB receptors. Also one single episode of spreading depression gave rise to a significant increase of cortical BDNF mRNA levels (to 207% of control), which was attenuated (by 61%) after administration of the competitive NMDA receptor antagonist CGS 19755. The results provide evidence that mild brain insults associated with glutamate release and elevated intracellular calcium, such as spreading depression, also in the absence of seizure activity can lead to activation of the BDNF gene in cortical neurons.

Expression of members of the trk family in the developing postnatal rat brain.

Tyrosine protein kinases trk, trkB and trkC are essential components of the high affinity receptors necessary to mediate biological effects of the neurotrophins NGF, BDNF, NT-3 and NT-4. Here we report on the expression of these receptors during postnatal development in the rat brain. Cells expressing mRNAs encoding different members of the trk family were identified by in situ hybridization using oligonucleotides complementary to their respective mRNA. In septum, striatum and brainstem, higher levels of trk mRNA were detected at 2 and 4 weeks than at 1 weeks of age. In thalamic nuclei associated with the limbic system, trkB and trkC mRNA were highly expressed at P1 to P7, but the expression declined gradually in 2 and 4 week old animals. Other structures where a developmentally regulated expression was seen included the tenia tecta and piriform cortex where trkB mRNA was not detected until 2 weeks of age. A high labeling was found for trkC mRNA in the deeper parts of neocortex in P1 and P4 animals, while in 2 and 4 weeks old animals the highest labeling was seen over the outer neocortical layers. Several brainstem nuclei showed a higher labeling for trkC mRNA at P1 to P7 than in animals of older age. These data show that expression of members of the trk family is developmentally regulated during postnatal brain development and suggest that high affinity neurotrophin receptors mediate a transient response to neurotrophins in many regions during brain ontogeny.