Dendritic morphology and spine density is not altered in motor cortex and dentate granular cells in mice lacking the ganglioside biosynthetic gene B4galnt1 - A quantitative Golgi cox study

Gangliosides are characteristic plasma membrane constituents of vertebrate brain used as milestones of neuronal development. As neuronal morphology is a good indicator of neuronal differentiation, we analyzed how lack of the ganglioside biosynthetic gene Galgt1 whose product is critical for production of four major adult mammalian brain complex gangliosides (GM1, GD1a, GD1b and GT1b) affects neuronal maturation in vivo. To define maturation of cortical neurons in mice lacking B4galnt1 we performed a morphological analysis of Golgi-Cox impregnated pyramidal neurons in primary motor cortex and granular cells of dentate gyrus in 3, 21 and 150 days old B4galnt1-null and wild type mice. Quantitative analysis of basal dendritic tree on layer III pyramidal neurons in the motor cortex showed very immature dendritic picture in both mice at postnatal day 3. At postnatal day 21 both mice reached adult values in dendritic length, complexity and spine density. No quantitative differences were found between B4galnt1-null and wild type mice in pyramidal cells of motor cortex or granular cells of dentate gyrus at any examined age. In addition, the general structural and neuronal organization of all brain structures, qualitatively observed on Nissl and Golgi-Cox, were similar Our results demonstrate that neurons can develop normal dendritic complexity and length without presence of complex gangliosides in vivo. Therefore, behavioral differences observed in B4galnt1-null mice may be attributed to functional rather than morphological level of dendrites and spines of cortical pyramidal neurons.     

Central nervous system inflammation and neurological disease in transgenic mice expressing the CC chemokine CCL21 in oligodendrocytes.

To study the biological role of the chemokine ligands CCL19 and CCL21, we generated transgenic mice expressing either gene in oligodendrocytes of the CNS. While all transgenic mice expressing CCL19 in the CNS developed normally, most (18 of 26) of the CCL21 founder mice developed a neurological disease that was characterized by loss of landing reflex, tremor, and ataxia. These neurological signs were observed as early as postnatal day 9 and were associated with weight loss and death during the first 4 wk of life. Microscopic examination of the brain and spinal cord of CCL21 transgenic mice revealed scattered leukocytic infiltrates that consisted primarily of neutrophils and eosinophils. Additional findings included hypomyelination, spongiform myelinopathy with evidence of myelin breakdown, and reactive gliosis. Thus, ectopic expression of the CC chemokine CCL21, but not CCL19, induced a significant inflammatory response in the CNS. However, neither chemokine was sufficient to recruit lymphocytes into the CNS. These observations are in striking contrast to the reported activities of these molecules in vitro and may indicate specific requirements for their biological activity in vivo.     

Specific neuronal expression of human NGF receptors in the basal forebrain and cerebellum of transgenic mice

Transgenic mice carrying multiple copies of the human NGF receptor gene have been generated. Using a monoclonal antibody specific for the human receptor, we have detected specific expression in cholinergic neurons in the basal forebrain and Purkinje cells in the cerebellum during the postnatal period. Expression in the PNS was exemplified by immunostaining of sympathetic and sensory neurons during an early embryonic age. Transection of the sciatic nerve in transgenic animals resulted in induction of human NGF receptors, indicating that the inserted gene can be appropriately regulated. These transgenic mice will provide an opportunity to study the elements regulating the NGF receptor. Furthermore, the ability to obtain specific expression in transgenic mice will permit directed expression of heterologous genes in discrete cells important in the cholinergic septal-hippocampal pathway and the PNS.     

Enriched Population of PNS Neurons Derived from Human Embryonic Stem Cells as a Platform for Studying Peripheral Neuropathies

Background

The absence of a suitable cellular model is a major obstacle for the study of peripheral neuropathies. Human embryonic stem cells hold the potential to be differentiated into peripheral neurons which makes them a suitable candidate for this purpose. However, so far the potential of hESC to differentiate into derivatives of the peripheral nervous system (PNS) was not investigated enough and in particular, the few trials conducted resulted in low yields of PNS neurons. Here we describe a novel hESC differentiation method to produce enriched populations of PNS mature neurons. By plating 8 weeks hESC derived neural progenitors (hESC-NPs) on laminin for two weeks in a defined medium, we demonstrate that over 70% of the resulting neurons express PNS markers and 30% of these cells are sensory neurons.

Methods/Findings

Our method shows that the hNPs express neuronal crest lineage markers in a temporal manner, and by plating 8 weeks hESC-NPs into laminin coated dishes these hNPs were promoted to differentiate and give rise to homogeneous PNS neuronal populations, expressing several PNS lineage-specific markers. Importantly, these cultures produced functional neurons with electrophysiological activities typical of mature neurons. Moreover, supporting this physiological capacity implantation of 8 weeks old hESC-NPs into the neural tube of chick embryos also produced human neurons expressing specific PNS markers in vivo in just a few days. Having the enriched PNS differentiation system in hand, we show for the first time in human PNS neurons the expression of IKAP/hELP1 protein, where a splicing mutation on the gene encoding this protein causes the peripheral neuropathy Familial Dysautonomia.

Conclusions/Significance

We conclude that this differentiation system to produce high numbers of human PNS neurons will be useful for studying PNS related neuropathies and for developing future drug screening applications for these diseases.     

Schwann cell-derived factors support serotoninergic neuron survival and promote neurite outgrowth

During embryogenesis and the postnatal period, neurons and glia interact in the development and differentiation of specific populations of nerve cells. Both in the peripheral (PNS) and in the central nervous system (CNS), glial cells have been shown in various experimental conditions to constitute a favorable substrate for neural adhesion, neural polarity, shape and axonal extension, while numerous soluble molecules secreted by neurons influence the survival and differentiation of the glial cells themselves. The aim of the present work was to investigate the influence of postnatal Schwann cells (SC) on embryonic serotoninergic (5-HT) neurons of the raphe, in order to study the possible influence of the peripheral glia on the CNS neurons. Cultures of SC from sciatic nerve of postnatal rats and neurons from rat embryonic rhombencephalon were successfully established and cells were immunocytochemically characterized. The number of 5-HT neurons, and the number and length of their branches were quantified in the cultures of 5-HT neurons, in cultures added with Nerve Growth Factor (NGF) and Insulin-like Growth Factor I (IGF-I), in co-cultures with SC and in cultures added with conditioned medium obtained from SC cultures. The results indicated that SC have the capacity to promote the survival and growth of 5-HT neurons in culture, and that this activity is mediated by soluble factors. Although the precise nature and mechanism of action of the growth factor or factors produced by SC in the presence of 5-HT neurons was not identified, our results add more data on the possible activity of the peripheral glia in promoting and enhancing the survival and outgrowth of the CNS neurons.     

Comparative electron microscopic study of the visual cortex neurons of the cat in postnatal ontogeny

The maturation of layers II-VI of neurons and perineuronal neuropil of the cat visual cortex (field 17) was studied from postnatal day 1 to day 21. The differentiation of large, small (associate) pyramid and stellate neurons was described. During the first postnatal week, the somata of layers II-VI of neurons undergo significant changes, the perikaryal cytoplasm increases in volume. Cell bodies of large pyramidal neurons mature by day 15. During the second postnatal week and almost till day 15, the rough endoplasmic reticulum of small pyramidal and stellate neurons undergoes proliferation; dendritic processes are branching. In stellate neurons the amount of cytoplasmic organelles increases dramatically only after the second postnatal week, and this is presumably induced by the opening of eyes on day 12. The second postnatal week is the period of greatest growth of dendritic, axonal and glial processes in perineural neuropil of layers V-VI. In the perineuronal neuropil of large pyramidal neurons (layers V-VI) there appear symmetric synapses with pyramidal cells, dendritic processes and dendritic spines. This occurs just at the time when kittens first open the eyes. From this time and during postnatal days 15-21, asymmetric synapses appear in the perineuronal neuropil of large pyramidal neurons. In the perineuronal neuropil of small pyramidal and stellate neurons. (layers II-IV), synapses reveal the mature appearance by day 15. After the opening of the eyes and up to postnatal day 21, dendritic growth and spine production occur in the perineuronal neuropil of small pyramidal and stellate neurons.     

Cortical migration defects in mice expressing A-RAF from the B-RAF locus

We have previously shown that mice lacking the protein kinase B-RAF have defects in both neural and endothelial cell lineages and die around embryonic day 12 (E12). To delineate the function of B-RAF in the brain, B-RAF KIN/KIN mice lacking B-RAF and expressing A-RAF under the control of the B-RAF locus were created. B-RAF KIN/KIN embryos displayed no vascular defects, no endothelial and neuronal apoptosis, or gross developmental abnormalities, and a significant proportion of these animals survived for up to 8 weeks. Cell proliferation in the neocortex was reduced from E14.5 onwards. Newborn cortical neurons were impaired in their migration toward the cortical plate, causing a depletion of Brn-2-expressing pyramidal neurons in layers II, III, and V of the postnatal cortex. Our data reveal that B-RAF is an important mediator of neuronal survival, migration, and dendrite formation and that A-RAF cannot fully compensate for these functions.     

Immunoreactive myelin basic proteins are not detected when shiverer mutant Schwann cells and fibroblasts are co-cultured with normal neurons

Shiverer (shi) is an autosomal recessive mutation in mice that results in hypomyelination in the central nervous system (CNS) but normal myelination in the peripheral nervous system (PNS). Myelin basic proteins (MBPs) are virtually absent in both PNS and CNS. It is not known whether the cellular target in the PNS is the myelin-forming Schwann cell or another cell type which secondarily affects the Schwann cell. To determine the cellular target of the shi gene, we have adapted tissue culture techniques that allow co-culture of pure populations of mouse sensory neurons of one genotype with Schwann cells and fibroblasts of another genotype under conditions that permit myelin formation. These cultures were stained immunocytochemically as whole mounts to determine whether MBPs were expressed under various in vitro conditions. In single-genotype cultures, presence or absence of MBPs was consistent with earlier in vivo results: +/+ cultures were MBP-positive and shi/shi cultures were MBP-negative. In mixed-genotype cultures, visualization of MBPs in myelin accorded with the genotype of the non-neuronal Schwann cells and fibroblasts and not with the neurons--those cultures that contained +/+ non-neuronal cells were MBP-positive and those with shi/shi non-neuronal cells were MBP-negative, independent of the neuronal genotype. These results rule out neurons or circulating substances as mediators of the influence of the shi genetic locus on MBP synthesis and deposition in peripheral myelin.     

Proteolipid protein gene: Pelizaeus-Merzbacher disease in humans and neurodegeneration in mice

The dosage of the myelin gene and mutant forms of the protein can affect the CNS and PNS. Pelizaeus-Merzbacher disease (PMD) is a myelin disorder of the CNS that arises from both mutational mechanisms. Investigating the molecular basis of PMD in patients and animal models is furthering our understanding of the disease, dosage sensitivity and proteolipid protein function during myelinogenesis.     

Neural expression of alpha-internexin promoter in vitro and in vivo

alpha-Internexin is a 66 kDa neuronal intermediate filament protein found most abundantly in the neurons of the nervous systems during early development. To characterize the function of mouse alpha-internexin promoter, we designed two different expression constructs driven by 0.7 kb or 1.3 kb of mouse alpha-internexin 5'-flanking sequences; one was the enhanced green fluorescent protein (EGFP) reporter for monitoring specific expression in vitro, and the other was the cre for studying the functional DNA recombinase in transgenic mice. After introducing DNA constructs into non-neuronal 3T3 fibroblasts and a neuronal Neuro2A cell line by lipofectamine transfection, we observed that the expression of EGFP with 1.3 kb mouse alpha-internexin promoter was in a neuron-dominant manner. To establish a tissue-specific pattern in the nervous system, we generated a transgenic mouse line expressing Cre DNA recombinase under the control of 1.3 kb alpha-Internexin promoter. The activity of the Cre recombinase at postnatal day 1 was examined by mating the cre transgenic mice to ROSA26 reporter (R26R) mice with knock-in Cre-mediated recombination. Analyses of postnatal day 1 (P1) newborns showed that beta-galactosidase activity was detected in the peripheral nervous system (PNS), such as cranial nerves innervating the tongue and the skin as well as spinal nerves to the body trunk. Furthermore, X-gal-labeled dorsal root ganglionic (DRG) neurons showed positive for alpha-Internexin in cell bodies but negative in their spinal nerves. The motor neurons in the spinal cord did not exhibit any beta-galactosidase activity. Therefore, the cre transgene driven by mouse alpha-internexin promoter, described here, provides a useful animal model to specifically manipulate genes in the developing nervous system.     

Programmed cell death without DNA fragmentation in the jimpy mouse: secreted factors can enhance survival

Jimpy is one of many related mutations affecting the myelin proteolipid protein gene that causes severe hypomyelination in the central nervous system (CNS). Underlying the hypomyelination is a failure of oligodendrocytes (OLs) to differentiate, and the premature death of large numbers of OLs during the developmental period. Previous light and electron microscopic evidence suggested that jimpy OLs die in a manner consistent with programmed cell death. We have used TUNEL staining as a biochemical marker for apoptosis in conjunction with immunostaining for OL and myelin markers. At 13 - 14 days postnatal, a time when the number of dying OLs in jimpy CNS is increased more than five times normal, there are only modest increases (70% in spinal cord; 20% in cerebral cortex) in TUNEL labeled cells in mutant CNS tissues. The results in vitro are similar, and only a small per cent of TUNEL labeled cells have the antigenic phenotype of OLs. The discrepancy between numbers of dying and TUNEL labeled cells suggests either that most jimpy OLs do not undergo programmed cell death or that the biochemical pathways leading to their death do not involve DNA fragmentation which is detected by the TUNEL method. We also present evidence that jimpy OLs show increased survival and enhanced differentiation when they are grown in vitro in medium conditioned by cells lines which express products of the proteolipid protein gene. Cell lines expressing proteolipid protein and the alternatively spliced DM20 protein have differential effects on cell numbers and production of myelin-like membranes.     

HGF regulates the development of cortical pyramidal dendrites

Although hepatocyte growth factor (HGF) and its receptor tyrosine kinase MET are widely expressed in the developing and mature central nervous system, little is known about the role of MET signaling in the brain. We have used particle-mediated gene transfer in cortical organotypic slice cultures established from early postnatal mice to study the effects of HGF on the development of dendritic arbors of pyramidal neurons. Compared with untreated control cultures, exogenous HGF promoted a highly significant increase in dendritic growth and branching of layer 2 pyramidal neurons, whereas inactivation of endogenous HGF with function-blocking, anti-HGF antibody caused a marked reduction in size and complexity of the dendritic arbors of these neurons. Furthermore, pyramidal neurons transfected with an MET dominant-negative mutant receptor likewise had much smaller and less complex dendritic arbors than did control transfected neurons. Our results indicate that HGF plays a role in regulating dendritic morphology in the developing cerebral cortex.     

Satb1 ablation alters temporal expression of immediate early genes and reduces dendritic spine density during postnatal brain development

Complex behaviors, such as learning and memory, are associated with rapid changes in gene expression of neurons and subsequent formation of new synaptic connections. However, how external signals are processed to drive specific changes in gene expression is largely unknown. We found that the genome organizer protein Satb1 is highly expressed in mature neurons, primarily in the cerebral cortex, dentate hilus, and amygdala. In Satb1-null mice, cortical layer morphology was normal. However, in postnatal Satb1-null cortical pyramidal neurons, we found a substantial decrease in the density of dendritic spines, which play critical roles in synaptic transmission and plasticity. Further, we found that in the cerebral cortex, Satb1 binds to genomic loci of multiple immediate early genes (IEGs) (Fos, Fosb, Egr1, Egr2, Arc, and Bdnf) and other key neuronal genes, many of which have been implicated in synaptic plasticity. Loss of Satb1 resulted in greatly alters timing and expression levels of these IEGs during early postnatal cerebral cortical development and also upon stimulation in cortical organotypic cultures. These data indicate that Satb1 is required for proper temporal dynamics of IEG expression. Based on these findings, we propose that Satb1 plays a critical role in cortical neurons to facilitate neuronal plasticity.     

Some data on postnatal maturation of the cerebral cortex in cat

This paper describes the neurons in different cortical areas and traces their postnatal changes. Rapid Golgi and Golgi--Kopsch impregnation were carried out in 1-day-old and 9-day-old kittens. The maturation of the pyramidal neurons can be observed mainly on their basal dendritic orientation and on development of the dendritic spines. The differentiation of the interneurons (non-pyramidal) also proceeds on the first postnatal days. These, though slightly less mature than the associated pyramidal neurons, are identifiable already on the first postnatal day. It is concluded that there are significant differences in the maturation of the neurons in the various cortical areas.     

The expression of α-internexin and peripherin in the developing mouse pineal gland

Summary The mammalian pineal gland contains pinealocytes, interstitial glial cells, perivascular macrophages, neurons and neuron-like cells. The neuronal identity of neurons and neuron-like cells was an enigma. α-Internexin and peripherin are specific neuronal intermediate filament proteins and are expressed differentially in the CNS and PNS. We investigated the development of immunoreactivity and expression patterns of mRNAs for α-internexin and peripherin in the mouse pineal gland to determine the neuronal identity of these cells. Both α-internexin- and peripherin-immunoreactive cells were readily visualized only after birth. Both proteins were at the highest level on the postnatal day 7 (P7), rapidly declined at P14, and obtained their adult level at P21. Both protein and mRNA of α-internexin are expressed in some cells and nerve processes, but not all, of adult mouse pineal gland. Less number of peripherin immunoreactive or RNA-expressing cells and nerve processes were identified. Accumulations of α-internexin and peripherin proteins were also found in the cells from the aged pineal gland (P360). We concluded that some cells in the developing mouse pineal gland may differentiated into neurons and neuron-like cells expressing both α-internexin and/or peripherin only postnatally, and these cells possess dual properties of CNS and PNS neurons in nature. We suggested that they may act as interneurons between the pinealocyte and the distal neurons innervating the pinealocytes, or form a local circuitry with pinealocytes to play a role of paracrine regulatory function on the pinealocytes.     

Gliogenesis and myelination in the optic nerve of trisomy 19 mice. A quantitative electron-microscopic study

Trisomy 19 (ts19) of the mouse permits detailed studies on the influence of an extra autosome upon the postnatal development of the central nervous system. To examine gliogenesis and myelinogenesis, the optic nerves of 19 ts19 pugs aged 1-15 days have been examined by light and electron microscopy and compared to those of litter-mate controls. Differentiation of astrocytes and oligodendrocytes, myelinogenesis as well as the opening of the eyes are each delayed by about 2 days. Myelin sheaths are normally structured in ts19. There is a decrease in the percentage of myelinated fibres. The cross-sectional area of the ts19 optic nerve is reduced. The fibre density, which decreases with age both in ts19 and control mice, is higher in ts19 mice. Both with ts19 and control animals, the distribution of fibre diameters of myelinated axons overlaps with that of promyelinated and unmyelinated fibres, but myelinated axons cannot be observed below a diameter of 0.3 micron, and unmyelinated axons are always smaller than 1 micron. The mean diameter of promyelinated axons is identical in ts19 and control animals. Myelination is therefore not severely disturbed in the ts19 optic nerve. As retinal differentiation in ts19 is delayed by 2 days as well, reports on an asynchronous development of neurons and myelin sheaths cannot be confirmed for the visual system.     

Cerebral myelinogenesis in theSnell dwarf mouse: Stimulatory effects of GH and T4 restricted to the first 20 days of postnatal life

We attempted to define the critical time period during early postnatal life when GH and T₄ are essential for myelination. We administered bGH and T₄ toSnell dwarf mice during the first and second 20 days after birth. Positive results were obtained only when hormones were given during the first 20 days of postnatal life. We observed a distinct increase in brain weight, DNA content, CNPase activity and a remarkably increased level of spontaneous locomotion activity with a diurnal periodicity. Morphological observations of brain sections stained for myelin basic protein (MBP) correlated the biochemical findings. The later administration of hormones was ineffective. Our interpretation is that the administration of exogenous hormones led to increased myelinogenesis through their stimulatory effects on glial proliferation, as evidenced by the increase in cerebral DNA content.