Cell division cycle of cultured neural precursor cells from Drosophila

In Drosophila neuroblast cells, which give rise to the embryonic nervous system, undergo a limited number of asymmetric cell divisions. These cell lineages result in the formation of clusters of neurons when neuroblasts are isolated and cultured. A significant proportion of these neural cell clusters (NCC) arise from individual precursor cells. The formation of NCC containing more than two neurons is repressed when DNA synthesis is inhibited. Cell division during NCC development was examined by [3H]thymidine autoradiography. The pattern of DNA synthesis by neural cells was that expected based on observations in situ. The pattern in individual NCC was consistent with single precursor origins for more than 80% of NCC, under our conditions of culture. Based on this, we show that the largest neural precursors at gastrulation undergo the most cell divisions in culture. The neuroblast cell division cycle averages approximately 1.5 hr, and is similar to that of blastoderm cells.     

Glutamate Neurotoxicity and the Inhibition of Protein Synthesis in the Hippocampal Slice

In some animal models of ischemia, neuronal degeneration can be prevented by the selective antagonism of the N-methyl-D-aspartate (NMDA) glutamate receptor subtype, suggesting that glutamate released during ischemia causes injury by activating NMDA receptors. The rat hippocampal slice preparation was used as an in vitro model to study the pharmacology of glutamate toxicity and investigate why NMDA receptors are critical in ischemic injury. Acute toxicity was assessed by quantifying the inhibition of protein synthesis, which we confirmed by autoradiography to be primarily neuronal. The effect of NMDA was prevented by the specific antagonists MK-801 and ketamine, as well as by the less selective antagonist kynurenic acid. The less selective antagonists kynurenic acid and 6,7-dinitroquinoxaline-2,3-dione antagonized the effects of quisqualate and NMDA. In contrast to previous observations with dissociated neurons in tissue culture, the toxicity of glutamate was unaffected by antagonists, regardless of the glutamate concentration, the duration of exposure, or the presence of magnesium. The high concentration of glutamate required to inhibit protein synthesis and the inability of receptor antagonists to block the effect of glutamate suggest that either glutamate acts through a non-receptor-mediated mechanism, or that the receptor-mediated nature of glutamate effects are masked in the slice preparation, perhaps by the glial uptake of glutamate. The altered physiology induced by ischemia must potentiate the neurotoxicity of glutamate, because we observed with a brain slice preparation that only high concentrations of glutamate caused neurotoxicity in the presence of oxygen and glucose and that these effects were not reversed by glutamate receptor antagonists.     

Cerebellar granule cell neurogenesis is regulated by cell-cell interactions in vitro

When CNS precursor cells purified from the external germinal layer of the early postnatal mouse cerebellum are cultured in cellular reaggregates, DNA synthesis increased 10-fold above that of cells dispersed in a monolayer or embedded in a collagen matrix. Dividing precursor cells gave rise to neurons immunopositive for the neural antigens N-CAM, L1, and TAG-1, but not to astroglial cells immunopositive for glial filament protein. Moreover, proliferating precursor cells did not generate other types of cerebellar neurons, as judged by the lack of expression of glutamic acid decarboxylase, the synthetic enzyme for gamma-amino-n-butyric acid. By contrast, the addition of astroglial cells, or astroglial cell membranes, to cellular reaggregates of granule cell neuroblasts arrested precursor cell DNA synthesis in a dose-dependent manner. These results suggest that homotypic contact interactions among CNS neural progenitors control precursor cell proliferation and fate in generative zones of developing brain.     

Wnt/��-catenin signaling in cerebral cortical development

The cerebral cortex is the multilayered sheet of neurons that underlies our highest cognitive abilities. Canonical Wnt/β-catenin signaling has well-known activities in tissue patterning in regulating rostral-caudal and medial-lateral patterning in the developing cortex. In addition, recent studies suggest that Wnt/β-catenin signaling also plays important roles in establishing the radial inside to outside organization of the cerebral cortex. Different Wnts, Wnt receptors and inhibitors are expressed in overlapping radial compartments of the cerebral cortex, and in vivo functional studies have provided evidence for Wnt/β-catenin regulation of neural precursor self-renewal, laminar fate determination and establishing or stabilizing the patterns of neuronal communication of cortical neurons. Wnt/β-catenin alterations have been observed in human brain tumors, and understanding its many diverse functions during normal neural development may provide greater insight into the mechanisms underlying the development and progression of neural tumors.Key words: cerebral cortex, neural stem cell, neural precursor, ventricular zone, laminar fate, regional specification, radial patterning     

Mitogen and substrate differentially affect the lineage restriction of adult rat subventricular zone neural precursor cell populations.

The effects of specific mitogens and substrates on the proliferative capacity and the differentiated phenotypic plasticity of neural precursor cell populations isolated from the adult rat subventricular zone (SVZ) were examined. SVZ cells were grown on uncoated tissue culture plastic, extracellular matrix, or poly-D-ornithine with either laminin or fibronectin. SVZ neural precursor cells could not be generated with platelet-derived growth factor (PDGF), granulocyte macrophage colony stimulating factor, stem cell factor, heparin-binding epidermal growth factor (HB-EGF), granulocyte colony stimulating factor, or ciliary neurotrophic factor (CNTF), but could be with EGF, fibroblast growth factor 2 (FGF2), and FGF2 plus heparin. Varying combinations of substrate and mitogen resulted in very different expansion rates and/or lineage potential. Neurons, oligodendrocytes, and astrocytes differentiated from all cultures, but EGF-generated neural precursor cells were more restricted to an astrocytic lineage and FGF2-generated neural precursor cells had a greater capacity for neuronal differentiation. In both EGF- and FGF2-generated cell populations, CNTF increased the number of differentiated astrocytes, triiodothyronine oligodendrocytes, PDGF neurons, and brain-derived neurotrophic factor neurons only from EGF cells. Electrophysiological analysis of differentiated cells showed three distinct phenotypes, glial, neuronal, and presumed precursor cells, although the neuronal properties were immature. Collectively, these data indicate that CNS neural precursor cell populations isolated with different mitogens and substrates are intrinsically different and their characteristics cannot be directly compared.     

Delta/Notch signaling promotes formation of zebrafish neural crest by repressing Neurogenin 1 function

In zebrafish, cells at the lateral edge of the neural plate become Rohon-Beard primary sensory neurons or neural crest. Delta/Notch signaling is required for neural crest formation. ngn1 is expressed in primary neurons; inhibiting Ngn1 activity prevents Rohon-Beard cell formation but not formation of other primary neurons. Reducing Ngn1 activity in embryos lacking Delta/Notch signaling restores neural crest formation, indicating Delta/Notch signaling inhibits neurogenesis without actively promoting neural crest. Ngn1 activity is also required for later development of dorsal root ganglion sensory neurons; however, Rohon-Beard neurons and dorsal root ganglion neurons are not necessarily derived from the same precursor cell. We propose that temporally distinct episodes of Ngn1 activity in the same precursor population specify these two different types of sensory neurons.     

Cellular and network contributions to excitability of layer 5 neocortical pyramidal neurons in the rat

There is a considerable gap between investigating the dynamics of single neurons and the computational aspects of neural networks. A growing number of studies have attempted to overcome this gap using the excitation in brain slices elicited by various chemical manipulations of the bath solution. However, there has been no quantitative study on the effects of these manipulations on the cellular and network factors controlling excitability. Using the whole-cell configuration of the patch-clamp technique we recorded the membrane potential from the soma of layer 5 pyramidal neurons in acute brain slices from the somatosensory cortex of young rats at 22 degrees C and 35 degrees C. Using blockers of synaptic transmission, we show distinct changes in cellular properties following modification of the ionic composition of the artificial cerebrospinal fluid (ACSF). Thus both cellular and network changes may contribute to the observed effects of slice excitation solutions on the physiology of single neurons. Furthermore, our data suggest that the difference in the ionic composition of current standard ACSF from that of CSF measured in vivo cause ACSF to depress network activity in acute brain slices. This may affect outcomes of experiments investigating biophysical and physiological properties of neurons in such preparations. Our results strongly advocate the necessity of redesigning experiments routinely carried out in the quiescent acute brain slice preparation.     

The steroid hormone of sunlight soltriol (vitamin D) as a seasonal regulator of biological activities and photoperiodic rhythms

Neural and systemic somatotrophic effects of the ultraviolet component of sunlight through the skin-vitamin D endocrine system are considered as alternate or additional to the neuroendocrine effects of the visual component of light through the retino-diencephalic input. The extensive distribution of soltriol nuclear receptor cells, revealed by autoradiography with tritium-labeled 1,25 dihydroxycholecalciferol (vitamin D, soltriol) and related effects, indicate an involvement of vitamin D-soltriol in the actinic induction of seasonal biorhythms. This is considered to be independent of the traditionally assigned effects of vitamin D on systemic calcium regulation. Skin-soltriol mediated seasonal, and to a degree daily, genomic activation involves many target regions in the brain. These include neurons in the central nucleus of the amygdala, in the linked part of the bed nucleus of the stria terminalis, in periventricular hypothalamic neurons, dorsal raphe nucleus, reticular thalamic nucleus and autonomic, endocrine as well as sensory and motor components of the brainstem and spinal cord. Additional to the eye-regulated "suprachiasmatic clock", existence of a soltriol-vitamin D regulated neural "timing circuit(s)" is proposed. Both, activational and organizational effects of soltriol on mature and developing brain regions, respectively are likely to play a role in the regulation of neuronal functions that include the modulation and entrainment of biorhythms. Soltriol's central effects correlate with peripheral effects on elements in skin, bone, teeth, kidney, intestine, heart and blood vessels, endocrine organs, and tissues of the immune and reproductive system.     

Biochemical and Autoradiographical Determination of Protein Synthesis in Experimental Brain Tumors of Rats

The rate of leucine incorporation into brain proteins was studied in rats with experimental brain tumors produced by intracerebral transplantation of the glioma clone F98. Incorporation was measured with [14C]leucine using a controlled infusion technique for maintaining constant specific activity of [14C]leucine in plasma, followed by quantitative autoradiography and biochemical tissue analysis. After 45 min the specific activity of free [14C]leucine in plasma was 2.5-3 times higher than in brain and brain tumor, indicating that the precursor pool for protein synthesis was fueled both by exogenous (plasma-derived) and endogenous (proteolysis-derived) amino acids. Endogenous recycling of amino acids amounted to 73% of total free leucine pool in brain tumors and to 60-70% in normal brain. Taking endogenous amino acid recycling into account, leucine incorporation was 78.7 +/- 16.0 nmol/g of tissue/min in brain tumor, and 17.2 +/- 4.2 and 9.7 +/- 3.3 nmol/g/min in normal frontal cortex and striatum, respectively. Leucine incorporation within tumor tissue was markedly heterogeneous, depending on the local pattern of tumor proliferation and necrosis. Our results demonstrate that quantitative measurement of leucine incorporation into brain proteins requires estimation of recycling of amino acids derived from proteolysis and, in consequence, biochemical determination of the free amino acid precursor pool in tissue samples. With the present approach such measurements are possible and provide the quantitative basis for the evaluation of therapeutic interventions.     

Activin receptor-like kinase 2 can mediate atrioventricular cushion transformation

The development of enteric and sympathetic neurons from neural crest precursor cells is regulated by signals produced by the embryonic environments to which the cells migrate. Bone morphogenetic proteins (BMPs) are present in the developing embryo and act to induce neuronal differentiation and noradrenergic properties of neural crest cells. We have investigated the role of BMP2 in regulating the appearance of distinct populations of autonomic neurons from postmigratory, HNK-1-positive neural crest precursor cells. BMP2 promotes neuronal differentiation of sympathetic and enteric precursor cells isolated from E14.5 rat. The effects of BMP2 change over time, resulting in a decrease in neuron number that can be attributed to apoptotic cell death. BMP2-dependent neuron death is rescued by gut-derived factors that provide trophic support to maturing neurons, indicating that BMP2 regulates the acquisition of trophic dependence of developing peripheral neurons. In addition to regulating neuron number, BMP2 promotes both panneuronal maturation and the acquisition of an enteric phenotype, as measured by lineage-specific changes in the expression of tyrosine hydroxylase and MASH-1. While BMP2 is sufficient to induce neuronal differentiation and panneuronal development, these results suggest that additional factors in the environment must collaborate with BMP2 to promote the final noradrenergic phenotype of sympathetic neurons.     

Adult human brain cell culture for neuroscience research

Studies of the brain have progressed enormously through the use of in vivo and in vitro non-human models. However, it is unlikely such studies alone will unravel the complexities of the human brain and so far no neuroprotective treatment developed in animals has worked in humans. In this review we discuss the use of adult human brain cell culture methods in brain research to unravel the biology of the normal and diseased human brain. The advantages of using adult human brain cells as tools to study human brain function from both historical and future perspectives are discussed. In particular, studies using dissociated cultures of adult human microglia, astrocytes, oligodendrocytes and neurons are described and the applications of these types of study are evaluated. Alternative sources of human brain cells such as adult neural stem cells, induced pluripotent stem cells and slice cultures of adult human brain tissue are also reviewed. These adult human brain cell culture methods could benefit basic research and more importantly, facilitate the translation of basic neuroscience research to the clinic for the treatment of brain disorders.     

Role of norepinephrine in regulating the activity of serotonin-containing dorsal raphe neurons

Previous studies have yielded conflicting results concerning the role of noradrenergic afferents to the dorsal raphe nucleus in regulating the activity of serotonergic neurons. In the present study, we recorded the activity of serotonin-containing dorsal raphe neurons in mouse brain slices $\text{in vitro}$ under the following conditions: (a) no treatment, (b) phenylephrine added to the incubation medium, (c) in tissue obtained from mice that were anesthetized with halothane, (d) same condition as c, with phenylephrine added to the incubation medium, and (e) same as condition c, with the addition of bicuculline to the incubation medium. The data revealed that the neurons recorded with no treatment exhibited a spontaneous discharge rate of 3.40 ± 0.29 spikes/sec and a cell/tract ratio of 1.15, while cells recorded from tissue slices obtained from halothane anesthetized mice exhibited a discharge rate of 2.01 ± 0.27 spikes/sec and a cell/track ratio of 0.58. Addition of phenylephrine to the incubation media in slices obtained from anesthetized mice increased both the discharge rate (4.23 ± 0.30 spikes/sec) and cell/tract ratio (1.28). Similarly, addition of bicuculline to the incubation media increased both the discharge rate (4.09 ± 0.46 spikes/sec) and cell/tract ratio (1.21) in mouse brain slices obtained from anesthetized animals. Thus, we conclude that a noradrenergic input (which is removed in the tissue slice preparation) is not necessary to maintain the spontaneous activity of serotonergic dorsal raphe units. Halothane anesthesia depressed the activity of these neurons, presumably by releasing GABA from interneurons. Finally, while dorsal raphe neurons are not dependent upon an excitatory noradrenergic input to maintain their spontaneous activity, these neurons can be excited by noradrenergic a fferents under certain conditions.     

Characterization of cortical neuronal and glial alterations during culture of organotypic whole brain slices from neonatal and mature mice

Background

Organotypic brain slice culturing techniques are extensively used in a wide range of experimental procedures and are particularly useful in providing mechanistic insights into neurological disorders or injury. The cellular and morphological alterations associated with hippocampal brain slice cultures has been well established, however, the neuronal response of mouse cortical neurons to culture is not well documented.

Methods

In the current study, we compared the cell viability, as well as phenotypic and protein expression changes in cortical neurons, in whole brain slice cultures from mouse neonates (P4–6), adolescent animals (P25–28) and mature adults (P50+). Cultures were prepared using the membrane interface method.

Results

Propidium iodide labeling of nuclei (due to compromised cell membrane) and AlamarBlue™ (cell respiration) analysis demonstrated that neonatal tissue was significantly less vulnerable to long-term culture in comparison to the more mature brain tissues. Cultures from P6 animals showed a significant increase in the expression of synaptic markers and a decrease in growth-associated proteins over the entire culture period. However, morphological analysis of organotypic brain slices cultured from neonatal tissue demonstrated that there were substantial changes to neuronal and glial organization within the neocortex, with a distinct loss of cytoarchitectural stratification and increased GFAP expression (p<0.05). Additionally, cultures from neonatal tissue had no glial limitans and, after 14 DIV, displayed substantial cellular protrusions from slice edges, including cells that expressed both glial and neuronal markers.

Conclusion

In summary, we present a substantial evaluation of the viability and morphological changes that occur in the neocortex of whole brain tissue cultures, from different ages, over an extended period of culture.     

Cellular localization of apolipoprotein D and lecithin:cholesterol acyltransferase mRNA in rhesus monkey tissues by in situ hybridization

Apolipoprotein D (apoD) and lecithin:cholesterol acyl transferase (LCAT) are found on high density lipoprotein particles (HDLs) and have been postulated to form part of a complex involved in the transport of cholesterol from peripheral tissues to the liver for excretion. We have examined the sites of synthesis of the mRNAs for these two proteins in the rhesus monkey by in situ hybridization. ApoD mRNA-containing cells were widely distributed throughout peripheral tissues in interstitial and connective tissue fibroblasts often associated with blood vessels or capillaries. ApoD mRNA was also found localized in cells associated with peripheral nerves, neuroglial cells, cells in the subarachnoid space on the surface of the brain including the pial cells, perivascular cells, and scattered neurons in the brain. LCAT demonstrated a much more restricted pattern of synthesis and was found to be synthesized by hepatocytes, the basal cell layer of the epidermis, and in brain cell populations distinct from those that synthesize apoD. In the brain LCAT was synthesized by scattered neurons, neuroglial cells, ependymal cells, as well as a discrete cell layer in the cerebellum. ApoD has been shown to possess extensive homology to retinol binding protein, which has a binding pocket for vitamin A. We propose that apoD may also function to bind cholesterol or its derivatives in compartments not in direct contact with the blood. The findings of both apoD and LCAT synthesis in the brain suggest that they play a significant role in lipid transport in the brain.     

Dynamic interplay between GABAergic networks and developing neurons in the adult hippocampus

Neurogenesis is a powerful mechanism for structural and functional remodeling that occurs in restricted areas of the adult brain. Although different neurotransmitters regulate various aspects of the progression from neural stem cell quiescence to neuronal maturation, GABA is the main player. The developmental switch from excitation to inhibition combined with a heterogeneous population of GABAergic interneurons that target different subcellular compartments provides multiple points for the regulation of development and function of new neurons. This complexity is enhanced by feedback and feedforward networks that act as sensors and controllers of circuit activity, impinging directly or indirectly onto developing granule cells and, subsequently, on mature neurons. Newly generated granule cells ultimately connect with input and output partners in a manner that is largely sculpted by the activity of local circuits.     

Distinct Neuronal Lineages of the Ascidian Embryo Revealed by Expression of a Sodium Channel Gene

The ascidian larva contains tubular neural tissue, one of the prominent anatomical features of the chordates. The cell-cleavage pattern and cell maps of the nervous system have been described in the ascidian larva in great detail. Cell types in the neural tube, however, have not yet been defined due to the lack of a suitable molecular marker. In the present work, we identified neuronal cells in the caudal neural tube of theHalocynthiaembryo by utilizing a voltage-gated Na⁺channel gene, TuNa I, as a molecular marker. Microinjection of a lineage tracer revealed that TuNa I-positive neurons in the brain and in the trunk epidermis are derived from the a-line of the eight-cell embryo, which includes cell fates to epidermal and neural tissue. On the other hand, TuNa I-positive cells in the more caudal part of the neural tissue were not stained by microinjection into the a-line. These neurons are derived from the A-line, which contains fates of notochord and muscle, but not of epidermis. Electron microscopic observation confirmed that A-line-derived neurons consist of motor neurons innervating the dorsal and ventral muscle cells. Isolated A-line blastomeres have active membrane excitability distinct from those of the a-line-derived neuronal cells after culture under cleavage arrest, suggesting that the A-line gives rise to a neuronal cell distinct from that of the a-lineage. TuNa I expression in the a-line requires signals from another cell lineage, whereas that in the A-line occurs without tight cell contact. Thus, there are at least two distinct neuronal lineages with distinct cellular behaviors in the ascidian larva: the a-line gives rise to numerous neuronal cells, including sensory cells, controlled by a mechanism similar to vertebrate neural induction, whereas A-line cells give rise to motor neurons and ependymal cells in the caudal neural tube that develop in close association with the notochord or muscle lineage, but not with the epidermal lineage.     

Establishment and characterization of immortalized hippocampal neural precursor cell lines

In the mammalian central nervous system, a complexcircuit of neurons contributes to higher behaviors.Each region of the brain has a unique function derivedfrom various types of neurons. Several neuralprecursor cell lines have been established from basalganglia of fetal brain. In this study, hippocampalneural precursor cell lines were established from thehippocampus of p53^-/- embryos. By means ofintegration of a MycER regulatable oncoprotein intop53^-/- neural precursor cells, several immortallines were established from embryonic hippocampalprimordium, with bFGF and estrogen continuouslysupplied for activation of the MycER protein. A dualluciferase study demonstrated that the MycER proteinblocked the expression of a glial cell marker protein,GFAP, probably contributing to the persistent celldivision of the immortalized neural precursor cells.These cell lines differentiate into neuronal and glialcell types after withdrawal of bFGF. The phenotype ofthe hippocampal cell lines differed from that of thebasal ganglia cell lines as observed in a clonaldensity culture. This result implies that each regionof the brain has a unique developmental program, thatmay be imprinted in each of the neural precursor cells.     

Genetic selection of sox1GFP-expressing neural precursors removes residual tumorigenic pluripotent stem cells and attenuates tumor formation after transplantation

Because of their ability to proliferate and to differentiate into diverse cell types, embryonic stem (ES) cells are a potential source of cells for transplantation therapy of various diseases, including Parkinson's disease. A critical issue for this potential therapy is the elimination of undifferentiated cells that, even in low numbers, could result in teratoma formation in the host brain. We hypothesize that an efficient solution would consist of purifying the desired cell types, such as neural precursors, prior to transplantation. To test this hypothesis, we differentiated sox1-green fluorescent protein (GFP) knock-in ES cells in vitro, purified neural precursor cells by fluorescence-activated cell sorting (FACS), and characterized the purified cells in vitro as well as in vivo. Immunocytofluorescence and RT-PCR analyses showed that this genetic purification procedure efficiently removed undifferentiated pluripotent stem cells. Furthermore, when differentiated into mature neurons in vitro, the purified GFP+ cell population generated enriched neuronal populations, whereas the GFP- population generated much fewer neurons. When treated with dopaminergic inducing signals such as sonic hedgehog (SHH) and fibroblast growth factor-8 (FGF8), FACS-purified neural precursor cells responded to these molecules and generated dopaminergic neurons as well as other neural subtypes. When transplanted, the GFP+ cell population generated well contained grafts containing dopaminergic neurons, whereas the GFP- population generated significantly larger grafts (about 20-fold) and frequent tumor-related deaths in the transplanted animals. Taken together, our results demonstrate that genetic purification of neural precursor cells using FACS isolation can effectively remove unwanted proliferating cell types and avoid tumor formation after transplantation.