The hormone of love attracts a partner for life

Neurovascular integration during embryonic development is essential for adult physiology. In this issue of Developmental Cell, Gutnick et?al. (2011) report that hypothalamic neurons secrete oxytocin as a guidance cue for endothelial cells to establish their vascular supply-a prerequisite for neuroendocrine secretion from the neurohyophysis in adult life.     

Human stem cell-derived neurons: a system to study human tau function and dysfunction

Background

Intracellular filamentous deposits containing microtubule-associated protein tau constitute a defining characteristic of many neurodegenerative disorders. Current experimental models to study tau pathology in vitro do not usually recapitulate the tau expression pattern characteristic of adult human brain. In this study, we have investigated whether human embryonic stem cell-derived neurons could be a good model to study human tau distribution, function and dysfunction.

Methodology/Principal Findings

Using RT-PCR, immunohistochemistry, western blotting and cell transfections we have investigated whether all 6 adult human brain tau isoforms are expressed in neurons derived from human embryonic and fetal stem cells and whether 4 repeat tau over-expression alone, or with the F3 tau repeat fragment, (amino acid 258–380 of the 2N4R tau isoform with the ΔK280 mutation) affects tau distribution. We found that the shortest 3 repeat tau isoform, similarly to human brain, is the first to be expressed during neuronal differentiation while the other 5 tau isoforms are expressed later. Over expression of tau with 4 repeats affects tau cellular distribution and the short tau F3 fragment appears to increase tau phosphorylation but this effect does not appear to be toxic for the cell.

Conclusions

Our results indicate that human embryonic stem cell-derived neurons express all 6 tau isoforms and are a good model in which to study tau physiology and pathology.     

Impaired cerebral cortex development and blood pressure regulation in FGF-2-deficient mice.

Fibroblast growth factor-2 (FGF-2) has been implicated in various signaling processes which control embryonic growth and differentiation, adult physiology and pathology. To analyze the in vivo functions of this signaling molecule, the FGF-2 gene was inactivated by homologous recombination in mouse embryonic stem cells. FGF-2-deficient mice are viable, but display cerebral cortex defects at birth. Bromodeoxyuridine pulse labeling of embryos showed that proliferation of neuronal progenitors is normal, whereas a fraction of them fail to colonize their target layers in the cerebral cortex. A corresponding reduction in parvalbumin-positive neurons is observed in adult cortical layers. Neuronal defects are not limited to the cerebral cortex, as ectopic parvalbumin-positive neurons are present in the hippocampal commissure and neuronal deficiencies are observed in the cervical spinal cord. Physiological studies showed that FGF-2-deficient adult mice are hypotensive. They respond normally to angiotensin II-induced hypertension, whereas neural regulation of blood pressure by the baroreceptor reflex is impaired. The present genetic study establishes that FGF-2 participates in controlling fates, migration and differentiation of neuronal cells, whereas it is not essential for their proliferation. The observed autonomic dysfunction in FGF-2-deficient adult mice uncovers more general roles in neural development and function.     

Whole cell recordings from brain of adult Drosophila

In this video, we demonstrate the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons. We begin by describing the dissecting solution and capture of the adult females used in our studies. The procedure for removing the whole brain intact, including both optic lobes, is illustrated. Dissection of the overlying trachea is also shown. The isolated brain is not only small but needs special care in handling at this stage to prevent damage to the neurons, many of which are close to the outer surface of the tissue. We show how a special holder we developed is used to stabilize the brain in the recording chamber. A standard electrophysiology set up is used for recording from single neurons or pairs of neurons. A fluorescent image, viewed through the recording microscope, from a GAL4 line driving GFP expression (GH146) illustrates how projection neurons (PNs) are identified in the live brain. A high power Nomarski image shows a view of a single neuron that is being targeted for whole cell recording. When the brain is successfully removed without damage, the majority of the neurons are spontaneously active, firing action potentials and/or exhibiting spontaneous synaptic input. This in situ preparation, in which whole cell recording of identified neurons in the whole brain can be combined with genetic and pharmacological manipulations, is a useful model for exploring cellular physiology and plasticity in the adult CNS.     

Novel effects of serotonin on neurite outgrowth in neurons cultured from embryos of Helisoma trivolvis

The neurotransmitter serotonin has been shown to inhibit neurite outgrowth in specific identified neurons isolated from adult Helisoma. While in vivo experiments on Helisoma embryos have supported the hypothesis that endogenous serotonin regulates neurite outgrowth during embryonic development, direct effects of serotonin on embryonic neurons have not been measured. In the present study, cultures of dissociated embryonic neurons were used to test the direct actions of serotonin on developing embryonic neurons. Serotonin arrested neurite outgrowth in a significant percentage of elongating neurites in a dose-dependent manner. Furthermore, analysis of neurons with stable, nonelongating neurites revealed a novel response. Serotonin caused the reinitiation of neurite outgrowth in a significant percentage of nonelongating neurites. The arrestment of outgrowth and reinitiation of outgrowth occurred in similar percentages of elongating and nonelongating neurites, respectively. Parallel experiments on cultures of dissociated adult neurons were carried out to determine whether serotonin could also induce both inhibitory and stimulatory responses in adult cells. Serotonin arrested neurite outgrowth in a similar percentage of neurites to that observed in cultures of embryonic neurons. In contrast, serotonin did not reinitiate neurite outgrowth in a significant percentage of adult neurites. These data support the hypothesis that serotonin regulates neurite outgrowth in developing embryonic neurons. Furthermore, only some of these regulatory effects appear to be conserved from embryonic to adult neurons.     

Is the Outgrowth of Transplant-Derived Axons Guided by Host Astrocytes and Myelin Loss?

Loss of cortical neurons may lead to sever and sometimes irreversible deficits in motor function in a number of neuropathological conditions. Absence of spontaneous axonal regeneration following trauma in the adult central nervous system (CNS) is attributed to inhibitory factors associated to the CNS white matter and to the non-permissive environment provided by reactive astrocytes that form a physical and biochemical barrier scar. Neural transplantation of embryonic neurons has been widely assessed as a potential approach to overcome the generally limited capacity of the mature CNS to regenerate axons or to generate new neurons in response to cell loss. We have recently shown that embryonic (E14) mouse motor cortical tissue transplanted into the damaged motor cortex of adult mice developed efferent projections to appropriate cortical and subcortical host targets including distant areas such as the spinal cord, with a topographical organization similar to that of intact motor cortex. Several parameters might account for the outgrowth of axonal projections from embryonic neurons within a presumably non-permissive adult brain, among which are astroglial reactions and myelin formation. In the present study, we have examined the role of astrocytes and myelin in the axonal outgrowth of transplanted neurons.     

Transplanted neurons form both normal and ectopic projections in the adult brain

Transplantation of embryonic or stem cell derived neurons has been proposed as a potential therapy for several neurological diseases. Previous studies reported that transplanted embryonic neurons extended long-distance projections through the adult brain exclusively to appropriate targets. We transplanted E14 lateral ganglionic eminence (LGE) and E15 cortical precursors from embryonic mice into the intact adult brain and analyzed the projections formed by transplanted neurons. In contrast to previous studies, we found that transplanted embryonic neurons formed distinct long-distance projections to both appropriate and ectopic targets. LGE neurons transplanted into the adult striatum formed projections not only to the substantia nigra, a normal target, but also to the claustrum and through all layers of fronto-orbital cortex, regions that do not normally receive striatal input. In some cases, inappropriate projections outnumbered appropriate projections. To examine the relationship between the donor cells and host brain in establishing the pattern of projections, we transplanted cortical precursors into the adult striatum. Despite their heterotopic location, cortical precursors not only predominantly formed projections appropriate for cortical neurons, but they also formed projections to inappropriate targets. Transplantation of GFP-expressing cells into beta-galactosidase-expressing mice confirmed that the axonal projections were not created by the fusion of donor and host cells. These results suggest that repairing the brain using transplantation may be more complicated than previously expected, because exuberant ectopic projections could result in brain dysfunction. Understanding the signals regulating axonal extension in the adult brain will be necessary to harness stem cells or embryonic neurons for effective neuronal-replacement therapies.     

Is the Outgrowth of Transplant-Derived Axons Guided by Host Astrocytes and Myelin Loss?

Loss of cortical neurons may lead to sever and sometimes irreversible deficits in motor function in a number of neuropathological conditions. Absence of spontaneous axonal regeneration following trauma in the adult central nervous system (CNS) is attributed to inhibitory factors associated to the CNS white matter and to the non-permissive environment provided by reactive astrocytes that form a physical and biochemical barrier scar. Neural transplantation of embryonic neurons has been widely assessed as a potential approach to overcome the generally limited capacity of the mature CNS to regenerate axons or to generate new neurons in response to cell loss. We have recently shown that embryonic (E14) mouse motor cortical tissue transplanted into the damaged motor cortex of adult mice developed efferent projections to appropriate cortical and subcortical host targets including distant areas such as the spinal cord, with a topographical organization similar to that of intact motor cortex. Several parameters might account for the outgrowth of axonal projections from embryonic neurons within a presumably non-permissive adult brain, among which are astroglial reactions and myelin formation. In the present study, we have examined the role of astrocytes and myelin in the axonal outgrowth of transplanted neurons.Key Words: motor cortex, neuronal transplantation, embryonic cells, GFP, GFAP, PLP     

Immunocytochemical study of catecholaminergic neurons in grafted mesencephalon transplanted into the hypothalamus of the rat

Mesencephalic fragments from 14 day old embryonic rat brain were transplanted into the third ventricle of adult rats neonatally treated with monosodium glutamate. From two to twelve months after grafting, the implanted tissue was still present in the ventricle and contained TH immunoreactive neurons which displayed a normal appearance at ultrastructural level. While endogenous TH containing neurons were still present in dopaminergic regions of the recipient hypothalamus, grafted mesencephalic fragments could survive and develop. They contained TH immunopositive most probably dopaminergic neurons which are able, in some cases, to innervate the host brain. This model should be of interest in the study of neuroendocrine functions of dopaminergic neurons.     

Two members of the Fxr gene family, Fmr1 and Fxr1, are differentially expressed in Xenopus tropicalis

The Fxr gene family is composed of three members, FMR1, FXR1 and FXR2. The FMR1 gene is involved in the fragile X syndrome, whereas for the other two members, no human disorder has been identified yet. An appropriate animal model to study in vivo gene function is essential to unravel the cellular function of the gene products FMRP, FXR1P and FXR2P, respectively. In Xenopus tropicalis both Fmr1 and Fxr1 were identified; however, unexpectedly Fxr2 was not. Here we describe the characterization of both Fmrp and Fxr1p in Xenopus tropicalis. Fmrp is expressed ubiquitously throughout the embryo during embryonic development, whereas Fxr1p shows a more tissue-specific expression particularly during late embryonic development. In adult frogs both proteins are highly expressed in most neurons of the central nervous system and in all spermatogenic cells in the testis. In addition, Fxr1p is also highly expressed in striated muscle tissue. Western blotting experiments revealed only one prominent isoform for both proteins using different tissue homogenates from adult frogs. Thus, for in vivo gene function studies, this relative simple animal model may serve as a highly advantageous and complementary model.     

Embryonic development of the histaminergic system in the ventral nerve cord of the Marbled Crayfish (Marmorkrebs)

The embryonic development of neurotransmitter systems in crustaceans so far is poorly understood. Therefore, in the current study we monitored the ontogeny of histamine-immunoreactive neurons in the ventral nerve cord of the Marbled Crayfish, an emerging crustacean model system for developmental studies. The first histaminergic neurons arise around 60% of embryonic development, well after the primordial axonal scaffold of the ventral nerve cord has been established. This suggests that histaminergic neurons do not serve as pioneer neurons but that their axons follow well established axonal tracts. The developmental sequence of the different types of histaminergic neurons is charted in this study. The analysis of the histaminergic structures is also extended into adult specimens, showing a persistence of embryonic histaminergic neurons into adulthood. Our data are compared to the pattern of histaminergic neurons in other crustaceans and discussed with regard to our knowledge on other aspects of neurogenesis in Crustacea. Furthermore, the possible role of histaminergic neurons as characters in evolutionary considerations is evaluated.     

High-affinity sodium-vitamin C co-transporters (SVCT) expression in embryonic mouse neurons

The sodium-vitamin C co-transporters SVCT1 and SVCT2 transport the reduced form of vitamin C, ascorbic acid. High expression of the SVCT2 has been demonstrated in adult neurons and choroid plexus cells by in situ hybridization. Additionally, embryonic mesencephalic dopaminergic neurons express the SVCT2 transporter. However, there have not been molecular and kinetic analyses addressing the expression of SVCTs in cortical embryonic neurons. In this work, we confirmed the expression of a SVCT2-like transporter in different regions of the fetal mouse brain and in primary cultures of neurons by RT-PCR. Kinetic analysis of the ascorbic acid uptake demonstrated the presence of two affinity constants, 103 microM and 8 microM. A K(m) of 103 microM corresponds to a similar affinity constant reported for SVCT2, while the K(m) of 8 microM might suggest the expression of a very high affinity transporter for ascorbic acid. Our uptake analyses also suggest that neurons take up dehydroascorbic acid, the oxidized form of vitamin C, through the glucose transporters. We consider that the early expression of SVCTs transporters in neurons is important in the uptake of vitamin C, an essential molecule for the fetal brain physiology. Vitamin C that is found at high concentration in fetal brain may function in preventing oxidative free radical damage, because antioxidant radical enzymes mature only late in the developing brain.     

In vitro studies on the control of nerve fiber growth by the extracellular matrix of the nervous system

1. Cultured neurons from embryonic chick sympathetic ganglia or dorsal root ganglia grow nerve fibers extensively on simple substrata containing fibronectin, collagens (types I, III, IV), and especially laminin. 2. The same neurons cultured on substrata containing glycosaminoglycans grow poorly. Glycosaminoglycans (heparin) inhibit nerve fiber growth on fibronectin substrata. 3. Proteolytic fragments of fibronectin support nerve fiber growth only when the cell attachment region is intact. For example, a 105 kD fragment, encompassing the cell attachment region, supports growth when immobilized in a substratum, but a 93 kD subfragment, lacking the cell attachment region, is unable to support fiber growth. When it is added to the culture medium, the 105 kD fragment inhibits fiber growth on substrata containing native fibronectin. 4. In culture medium lacking NGF, DRG neurons extend nerve fibers only on laminin and not on fibronectin, collagen or polylysine. Studies with radioiodinated laminin indicate that laminin binds with a relatively high affinity (kd approximately equal to 10(-9) M) to DRG neurons, and to a variety of other neural cells (NG108 cells, PC12 cells, rat astrocytes, chick optic lobe cells). We have isolated a membrane protein (67 kD) by affinity chromatography on laminin columns and are characterizing this putative laminin receptor. 5. Dissociated DRG neurons or ganglionic explants cultured on complex substrata consisting of tissue sections of CNS or PNS tissues extend nerve fibers onto the PNS (adult rat sciatic nerve) but not CNS (adult rat optic nerve) substrata. Other tissue substrata which support fiber growth in vivo (embryonic rat spinal cord, goldfish optic nerve) support growth in culture. While substrata from adult CNS, which support meager regeneration in vivo (adult rat spinal cord) support little fiber growth in culture. 6. Ganglionic explants cultured in a narrow space between a section of rat sciatic nerve and optic nerve grow preferentially onto the sciatic nerve suggesting that diffusible growth factors are not responsible for the differential growth on the two types of tissues. 7. Dissociated neurons adhere better to sections of sciatic nerve than optic nerve. Laminin, rather than fibronectin or heparan sulfate proteoglycan, is most consistently identifiable by immunocytochemistry in tissues (sciatic nerve, embryonic spinal cord, goldfish optic nerve) which support nerve fiber growth. Taken together, these data suggest that ECM adhesive proteins are important determinants of nerve regeneration.     

Distribution of neuropeptide Y and vasoactive intestinal polypeptide immunoreactive nerves in normal and transplanted pancreatic tissue

Neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) immunoreactive nerves were demonstrated in 21-day-old embryonic pancreatic tissue fragments transplanted into the anterior eye chamber of rats for 22, 45 and 109 days and in 60-day-old normal adult pancreas using immunohistochemical technique. In normal adult tissue, NPY-positive neurons lie close to the basal and lateral walls of the acinar cells. NPY-containing nerve fiber plexuses were found around blood vessels. VIP-immunopositive nerves were also discernible in the outer parts of the islets of Langerhans and on pancreatic ducts. In the transplants, it is not only the neural elements that survived but also the pancreatic ducts and the endocrine cells. VIP- and NPY-positive neurons were found in the stroma of the surviving pancreatic tissue. The distribution of these neural elements is similar to that of normal tissue in the surviving pancreatic ducts but different with regards to the acinar tissue. This study confirms that intrinsic nerves can survive and synthesize polypeptides even after 109 days of transplantation into the anterior eye chamber.     

Adult murine neurons: their chromatin and chromosome changes and failure to support embryonic development as revealed by nuclear transfer

Fully differentiated neurons in adult mammalian brains do not divide; consequently, their metaphase chromosomes have never been examined. Here we report metaphase chromosome constitutions of cortical neurons in adult mice visualized by a nuclear transfer technique. We found that although some reconstructed oocytes cloned from neuronal nuclei have an apparently normal karyotype, the majority do not. Regardless of chromosome morphology, nuclei of adult neurons totally lack the ability to support embryonic development. These findings support the hypothesis that fully differentiated neurons in adult mammalian brains are genomically altered.     

A motor-driven mechanism for cell-length sensing

Size homeostasis is fundamental in cell biology, but it is not clear how large cells such as neurons can assess their own size or length. We examined a role for molecular motors in intracellular length sensing.Computational simulations suggest that spatial information can be encoded by the frequency of an oscillating retrograde signal arising from a composite negative feedback loop between bidirectional motor-dependent signals. The model predicts that decreasing either or both anterograde or retrograde signals should increase cell length, and this prediction was confirmed upon application of siRNAs for specific kinesin and/or dynein heavy chains in adult sensory neurons. Heterozygous dynein heavy chain 1 mutant sensory neurons also exhibited increased lengths both in vitro and during embryonic development.Moreover, similar length increases were observed in mouse embryonic fibroblasts upon partial downregulation of dynein heavy chain 1.Thus, molecular motors critically influence cell length sensing and growth control.     

The Insulin-Like Growth Factor System

The insulin-like growth factor (IGF) system in ubiquitous and plays a role in every tissue of the body. It is comprised of ligands, receptors and binding proteins, each with specific functions. While it plays an essential role in embryonic and post-natal development, the IGF system is also important in normal adult physiology. There are now numerous examples of diseases such as diabetes, cancer, and malnutrition in which the IGF system is a major player and, not surprisingly, there are attempts to affect these disorders by manipulating the system.     

Immunocytochemical demonstration of vimentin in astrocytes and ependymal cells of developing and adult mouse nervous system

The occurrence of vimentin, a specific intermediate filament protein, has been studied by immunoflourescence microscopy in tissue of adult and embryonic brain as well as in cell cultures from nervous tissue. By double imminofluorescence labeling, the distribution of vimentin has been compared with that of subunit proteins of other types of intermediate filaments (glial fibrillary acidic [GFA] protein, neurofilament protein, prekeratin) and other cell-type specific markers (fibronectin, tetanus toxin receptor, 04 antigen). In adult brain tissue, vimentin is found not only in fibroblasts and cells of larger blood vessels but also in ependymal cells and astrocytes. In embryonic brain tissue, vimentin is detectable as early as embryonic day 11, the earliest stage tested, and is located in radial fibers spanning the neural tube, in ventricular cells, and in blood vessels. At all stages tested, oligodendrocytes and neurons do not express detectable amounts of vimentin. In primary cultures of early postnatal mouse cerebellum, a coincident location of vimentin and GFA protein is seen in astrocytes, and both types of filament proteins are included in the perinuclear aggregates formed upon exposure of the cells to colcemid. In cerebellar cell cultures of embryonic-day-13 mice, vimentin is seen in various cell types of epithelioid or fibroblastlike morphology but is absent from cells expressing tetanus toxin receptors. Among these embryonic, vimentin-positive cells, a certain cell type reacting neither with tetanus toxin nor with antibodies to fibronectin or GFA protein has been tentatively identified as precursor to more mature astrocytes. The results show that, in the neuroectoderm, vimentin is a specific marker for astrocytes and ependymal cells. It is expressed in the mouse in astrocytes and glial precursors well before the onset of GFA protein expression and might therefore serve as an early marker of glial differentiation. Our results show that vimentin and GFA protein coexist in one cell type not only in primary cultures in vitro but also in the intact tissue in situ.