Embryo Central Nervous System Research Articles

Repulsive guidance molecule A (RGMA) is widely expressed in the CNS of the sea lamprey Petromyzon marinus during early development.

RGM interactions with its receptor Neogenin play an important role in the regulation of axonal guidance or cell death in the developing central nervous system. The sea lamprey RGMA transcript has been recently identified. However, its expression has been only studied in the spinal cord of mature (premetamorphic) larval sea lampreys. Here, we report the expression of the sea lamprey RGMA transcript in developing embryos and prolarvae by means of in situ hybridization. Our data show that the RGMA transcript is broadly expressed in the central nervous system of embryos and prolarvae and with a rostro-caudal gradient of expression.

Cellular Imprinting Proteomics Assay: A Novel Method for Detection of Neural and Ocular Disorders Applied to Congenital Zika Virus Syndrome.

Congenital Zika syndrome was first described due to increased incidence of congenital abnormalities associated with Zika virus (ZIKV) infection. Since the eye develops as part of the embryo central nervous system (CNS) structure, it becomes a specialized compartment able to display symptoms of neurodegenerative diseases and has been proposed as a noninvasive approach to the early diagnosis of neurological diseases. Ocular lesions result from defects that occurred during embryogenesis and can become apparent in newborns exposed to ZIKV. Furthermore, the absence of microcephaly cannot exclude the occurrence of ocular lesions and other CNS manifestations. Considering the need for surveillance of newborns and infants with possible congenital exposure, we developed a method termed cellular imprinting proteomic assay (CImPA) to evaluate the ocular surface proteome specific to infants exposed to ZIKV during gestation compared to nonexposure. CImPA combines surface cells and fluid capture using membrane disks and a large-scale quantitative proteomics approach, which allowed the first-time report of molecular alterations such as neutrophil degranulation, cell death signaling, ocular and neurological pathways, which are associated with ZIKV infection with and without the development of congenital Zika syndrome, CZS. Particularly, infants exposed to ZIKV during gestation and without early clinical symptoms could be detected using the CImPA method. Lastly, this methodology has broad applicability as it could be translated in the study of several neurological diseases to identify novel diagnostic biomarkers. Data are available via ProteomeXchange with identifier PXD014038.

A novel vector for transgenesis in the rat CNS

The larger brain of the rat enables a much greater repertoire of complex behaviors than mice, likely making rats preferential for investigating neurodegeneration. Because molecular tools for specific expression of transgenes in the rat brain are sparse, we chose Prnp encoding the prion protein (PrP) to develop a novel vector to drive transgene expression in the rat brain. We compared the rat Prnp sequence with mouse and Syrian hamster Prnp sequences, identifying conserved genetic elements and hypothesizing that these elements would be able to drive neuronal transgene expression. We investigated this by generating a vector termed RaPrnp that encompasses portions of the rat Prnp gene. Importantly, we replaced the rat Prnp open reading frame (ORF) with a cloning site for rapid and seamless In-Fusion cloning. To validate the in vivo neuronal specificity of the RaPrnp vector in rats, we generated stable RaPrnp-LacZ/enhanced green fluorescent protein (EGFP) transgenic (Tg) rat lines, which led to robust LacZ activity and high EGFP fluorescence in the central nervous system of embryos and adult animals. Next, we restored the rat Prnp ORF and generated multiple Tg(RaPrnp-PrP) lines, demonstrating that overexpression of Prnp accelerates the onset of scrapie. While the incubation time in wild-type (WT) rats was 175 ± 3 days post inoculation (dpi), one line, Tg2919, overexpressed RaPrPC at 4.4-fold and exhibited a reduced incubation time of 149 ± 2 dpi. The second line, Tg2922, overexpressed RaPrPC at 9.7-fold compared with WT animals and had an incubation time of 112 ± 0 dpi. Tg2922 rats inoculated with rat RML showed extensive vacuolation of the brainstem in contrast to WT and Tg2919 animals in which vacuolation was most prominent in the hippocampus and striatum as well as the motor and sensory cortices. It is possible that construction of Tg rats with modified phenotypes will prove more advantageous than mice for neurodegeneration studies.

Zika Virus Infection in Pregnant Women and Microcephaly.

From the discovery of the Zika virus (ZIKV) in 1947 in Uganda (Africa), until its arrival in South America, it was not known that it would affect human reproductive life so severely. Today, damage to the central nervous system is known to be multiple, and microcephaly is considered the tip of the iceberg. Microcephaly actually represents the epilogue of this infection's devastating process on the central nervous system of embryos and fetuses. As a result of central nervous system aggression by the ZIKV, this infection brings the possibility of arthrogryposis, dysphagia, deafness and visual impairment. All of these changes of varying severity directly or indirectly compromise the future life of these children, and are already considered a congenital syndrome linked to the ZIKV. Diagnosis is one of the main difficulties in the approach of this infection. Considering the clinical part, it has manifestations common to infections by the dengue virus and the chikungunya fever, varying only in subjective intensities. The most frequent clinical variables are rash, febrile state, non-purulent conjunctivitis and arthralgia, among others. In terms of laboratory resources, there are also limitations to the subsidiary diagnosis. Molecular biology tests are based on polymerase chain reaction (PCR) with reverse transcriptase (RT) action, since the ZIKV is a ribonucleic acid (RNA) virus. The RT-PCR shows serum or plasma positivity for a short period of time, no more than five days after the onset of the signs and symptoms. The ZIKV urine test is positive for a longer period, up to 14 days. There are still no reliable techniques for the serological diagnosis of this infection. If there are no complications (meningoencephalitis or Guillain-Barré syndrome), further examination is unnecessary to assess systemic impairment. However, evidence is needed to rule out other infections that also cause rashes, such as dengue, chikungunya, syphilis, toxoplasmosis, cytomegalovirus, rubella, and herpes. There is no specific antiviral therapy against ZIKV, and the therapeutic approach to infected pregnant women is limited to the use of antipyretics and analgesics. Anti-inflammatory drugs should be avoided until the diagnosis of dengue is discarded. There is no need to modify the schedule of prenatal visits for pregnant women infected by ZIKV, but it is necessary to guarantee three ultrasound examinations during pregnancy for low-risk pregnancies, and monthly for pregnant women with confirmed ZIKV infection. Vaginal delivery and natural breastfeeding are advised.

Spatial distribution and characterization of non-apical progenitors in the zebrafish embryo central nervous system.

Studies of non-apical progenitors (NAPs) have been largely limited to the developing mammalian cortex. They are postulated to generate the increase in neuron numbers that underlie mammalian brain expansion. Recently, NAPs have also been reported in the retina and central nervous system of non-mammalian species; in the latter, however, they remain poorly characterized. Here, we characterize NAP location along the zebrafish central nervous system during embryonic development, and determine their cellular and molecular characteristics and renewal capacity. We identified a small population of NAPs in the spinal cord, hindbrain and telencephalon of zebrafish embryos. Live-imaging analysis revealed at least two types of mitotic behaviour in the telencephalon: one NAP subtype retains the apical attachment during division, while another divides in a subapical position disconnected from the apical surface. All NAPs observed in spinal cord lost apical contact prior to mitoses. These NAPs express HuC and produce two neurons from a single division. Manipulation of Notch activity reveals that neurons and NAPs in the spinal cord use similar regulatory mechanisms. This work suggests that the majority of spinal NAPs in zebrafish share characteristics with basal progenitors in mammalian brains.

An Intronic Polymorphism in couch potato Is Not Distributed Clinally in European Drosophila melanogaster Populations nor Does It Affect Diapause Inducibility.

couch potato (cpo) encodes an RNA binding protein that has been reported to be expressed in the peripheral and central nervous system of embryos, larvae and adults, including the major endocrine organ, the ring gland. A polymorphism in the D. melanogaster cpo gene coding region displays a latitudinal cline in frequency in North American populations, but as cpo lies within the inversion In(3R)Payne, which is at high frequencies and itself shows a strong cline on this continent, interpretation of the cpo cline is not straightforward. A second downstream SNP in strong linkage disequilibrium with the first has been claimed to be primarily responsible for the latitudinal cline in diapause incidence in USA populations.Here, we investigate the frequencies of these two cpo SNPs in populations of Drosophila throughout continental Europe. The advantage of studying cpo variation in Europe is the very low frequency of In(3R)Payne, which we reveal here, does not appear to be clinally distributed. We observe a very different geographical scenario for cpo variation from the one in North America, suggesting that the downstream SNP does not play a role in diapause. In an attempt to verify whether the SNPs influence diapause we subsequently generated lines with different combinations of the two cpo SNPs on known timeless (tim) genetic backgrounds, because polymorphism in the clock gene tim plays a significant role in diapause inducibility. Our results reveal that the downstream cpo SNP does not seem to play any role in diapause induction in European populations in contrast to the upstream coding cpo SNP. Consequently, all future diapause studies on strains of D. melanogaster should initially determine their tim and cpo status.

MO‐D‐220‐08: Quantitative Embryonic Central Nervous System Imaging Using High‐ Frequency Annular Arrays

Purpose: The central nervous system (CNS) is the first organ to develop during mammalian embryogenesis and the CNS dynamically evolves well into the postnatal stages. Abnormal CNS phenotypes are normally visualized in vivo or with fixed samples using magnetic resonance imaging (MRI). Current MRI coil technology does not permit early‐stage in vivo imaging of the mouse embryo CNS. Here, a custom‐made, high‐frequency‐ultrasound annular‐array system operating at 38 MHz was utilized to perform in vivo imaging of early‐stage mouse embryo CNS. Methods: A custom‐made ultrasound scanning system using a piezopolymer‐based annular array was utilized for the imaging studies. The array had five equal‐area elements, a 6 mm total aperture, and a 12 mm geometric focus. The scanning system was designed to perform five scans across an object in order to acquire all transmit‐to‐receive data (25 data sets). This data was then synthetically focused to achieve enhanced depth of field and lateral resolution. In addition, the system utilized a coded‐excitation scheme to enhance SNR and penetration depth, and a respiratory gate to reduce motion artifacts. A full 3D volumetric data set could be acquired in approximately 2 minutes. Results: In vivo, in utero volumetric data were acquired from 78 mouse embryos spanning embryonic days 10 to 13. The data sets were processed with a semi‐automated image segmentation algorithm and brain volumes were calculated. The results indicated that during these mid‐gestational stages, there was an increase in CNS volume of 38% while volumetric renderings revealed the complex remodeling of the neuroarchitecture showing more cell proliferation than increase in brain mass. Conclusions: High‐frequency annular arrays with coded excitation offer an effective means of acquiring in vivo, in utero ultrasound data of the embryonic mouse and can be used to rapidly analyze the normal or abnormal development of the CNS during longitudinal studies.This research was supported by the National Institutes of Health (EB008606).

Bendiocarbamate Toxicity in the Chick Embryo

Carbamate pesticides generally possess low toxicity for warm-blooded vertebrates, but developmental data are scarce. We have therefore evaluated embryotoxicity of choline esterase inhibitor bendiocarbamate in the chick embryo. The pesticide was dissolved in 5% acetone in distilled water and a volume of 200 μl was administered over the embryo through membrana papyracea on embryonic days 2, 3, 4, 5, and 10. Sampling was performed on embryonic day 10, while the embryos treated on embryonic day 10 were sampled on embryonic day 17. The toxicity of bendiocarbamate was fairly low, and LD50 decreased with advancing development from 1 mg/ embryo on embryonic day 2 to 29 mg on embryonic day 5. Malformations in surviving embryos were observed rarely (< 3 %) and occurred in both control and experimental groups. There was a mild but statistically significant dose-dependent reduction in body weight, most pronounced in the treatment on embryonic days 5 and 10, but the maximum difference from controls was below 15 %. A small but not significant increase in the number of positive cells was observed in the eye, limb buds, and the central nervous system of embryos treated on embryonic days 3 and 4 and examined after supravital wholemount staining with Lysotracker Red for apoptosis. In agreement with previously published studies in other vertebrate animals, we conclude that bendiocarbamate does not possess significant toxicity in the avian embryo.

Molecular cloning, characterization, and expression in brain and gonad of Dmrt5 of zebrafish

The DM domain gene family has at least eight members with conserved DNA-binding DM domain, which encodes putative transcription factors related to the sexual regulator Dsx of Drosophila and Mab-3 of Caenorhabditis elegans. Although some of the DM genes are involved in sexual development, the function of most of these genes remains unclear. There is also few structural and functional analysis concerning DM domain genes of the model fish, zebrafish. We report here molecular cloning, sequence, and expression of zebrafish Dmrt5, which consists of two exons, and encodes a 440-amino acid protein with conserved DMA and DMB domains in addition to DM domain. Phylogenetic analysis shows that zebrafish Dmrt5 fits within the Dmrt5 clade of fish and mammals. Zebrafish Dmrt5 was expressed in early gastrula period, subsequently increased to a high level in late stage of gastrula period (bud stage) and lower until the hatch period. In situ hybridization analysis showed its expression in developing central nervous system of embryos, especially in mid-brain and mid-hind brain boundary. In adult, its expression was restricted in brain and developing germ cells, especially in spermatogonia, spermatocytes, spermatids, and sperm cells, and in developing oocytes, including early perinucleolus stage oocyte, late yolk vesicle stage oocyte, and oil drop stage oocyte. These data suggest that zebrafish Dmrt5 have potentially important roles in gonadal development and may have contributed to the functional endocrine axis.

Corticosteroids are unable to protect against pseudorabies virus-induced tissue damage in the developing brain.

After intraocular injection of the virulent pseudorabies virus (PRV) strain Becker into late-stage chicken embryos, the virus spreads and replicates in the brain, where severe edema and hemorrhaging follow. By contrast, the attenuated Bartha strain does not cause severe brain pathology despite viral replication and spread throughout the brain (B. W. Banfield, G. S. Yap, A. C. Knapp, and L. W. Enquist, J. Virol. 72:4580-4588, 1998). These observations prompted us to explore the mechanism by which the virulent Becker strain mediates pathology in the chicken embryo central nervous system (CNS). To test the hypothesis that Becker infection induced an inflammatory response in the developing CNS, we examined the ability of the anti-inflammatory corticosteroid dexamethasone (Dex) to protect chicken embryos from PRV-induced brain damage. We found that Dex is not sufficient to protect the chicken embryo CNS from damage due to Becker infection. Surprisingly, systemic Dex delivery appeared to potentiate CNS damage, which was preceded by petechial hemorrhaging in the optic lobes. Taken together, these data suggest that the severe pathology elicited during the Becker infection is due not to immunopathology but to damage by the virus itself, possibly through the damage to or destruction of endothelial cells.

Genetic analyses of essential genes in cytological region 61D1-2 to 61F1-2 of Drosophila melanogaster.

We performed a systematic mutagenesis screen for lethals in the genomic region 61D1-2 to 61F1-2 on chromosomal arm 3L of Drosophila melanogaster. Our genetic analyses revealed that this region contains eight essential complementation groups including trio, Glut1 and extra macrochaetae (emc). For the trio locus, 22 mutant alleles were identified, and all of the alleles analyzed resulted in defects in the central nervous system of embryos, indicating that trio functions in the control of axon extension or guidance. Western analysis showed that at least three proteins are derived from trio and also suggested that a polypeptide of over 200 kDa plays a crucial role in embryonic or larval development. In addition, a newly identified emc allele was associated with several defects in embryonic morphogenesis, including abnormalities in head involution, gut formation and dorsal closure, thus revealing multiple roles for emc in embryonic development. We also performed preliminary phenotypic analyses on stocks bearing mutations belonging to the other lethal complementation groups. These genes function in essential biological events, but the mutations do not result in gross morphological changes during embryonic stages. The present study extends our knowledge of the Drosophila gene set, by identifying most of the essential genes in the chromosomal region 61D1-2 to 61F1-2.

Extensive Proliferation of Oligodendrocyte Precursors in the Parenchyma of the Embryonic Chick Central Nervous System

The proliferation of oligodendrocyte lineage cells in the chick embryo central nervous system (CNS) was examined by double-immunolabeling with a lineage marker monoclonal antibody (mAb) O4 or mAb O1 and 5-bromo-3′-deoxyuridine (BrdU). In all regions examined, the first O4-positive (O4+) cells appeared in restricted regions of the ventricular zone (VZ), regarded as a site of oligodendrocyte origin. Within the O4+ focus, less than 20% of the O4+ cells incorporated BrdU. In contrast, O4+ cells in the parenchyma were mitotically active; for example, 40–50% of early O4+ cells were labeled with BrdU. Some of these were unipolar in shape, indicative of migratory precursor cells. The frequency of O4+/BrdU+ cell appearance decreased to less than 20% with further development. O1+ oligodendrocytes were largely mitotically inactive, with only approximately 5% of O1+ cells incorporating BrdU. These results clearly demonstrated that the VZ generates relatively few precursor cells and that these oligodendrocyte precursors actively generate their cohort in the parenchyma of the CNS.

Protein kinase C‐ε is a developmentally regulated, neuronal isoform in the chick embryo central nervous system

Protein kinase C (PKC) is expressed as many isoforms and in high quantities in the central nervous system (CNS), which suggests an important role for this enzyme in neuronal development and function. We used specific antibodies to investigate the expression of the known PKC isoforms in extracts from chick major CNS areas during embryogenesis, from day 3 (E3) of incubation to day 1 post-hatching (P1). PKC-epsilon was the predominant isoform and was expressed from E6 onward in all brain regions, except retina (E12 and on). PKC-alpha/beta and -zeta isoforms were expressed at lower levels prior to PKC-epsilon expression and throughout embryogenesis. No other isoforms were detected in neural tissue preparations. We then used neural culture systems derived from the chick CNS to study the expression of PKC isoforms in neuroblasts, cortical neurons, and cortical glial cells. Western blotting and immunostaining of neuroblast-enriched cultures, derived from E3 CNS, showed only the Ca(2+)-dependent PKC-alpha/beta to be present. Studies on neuronal cultures derived from E6 cerebral hemispheres revealed only the Ca(2+)-independent PKC-epsilon to be expressed in neurons, as predicted by the developmental studies on tissue homogenates. PKC-epsilon immunoreactivity was seen intracellularly in differentiating neurons, regardless of their neurotransmitter phenotypes, and it correlated well with the level of neuronal activity. Furthermore, PKC-alpha/beta immunoreactivity was verified on glia cells, as the glial lineage emerges in E15 cortical cultures. These data suggest that PKC-epsilon expression is associated with the final neuroblast division in neurons, and the correlation of PKC isoform expression and neural cell lineage is discussed.

Immunohistochemical and biochemical analysis of N-cadherin expression during CNS development.

The expression of the calcium-dependent adhesion molecule N-cadherin during chick embryo central nervous system (CNS) development was examined by immunohistochemistry and electrophoresis and immunoblotting. During histogenesis, N-cadherin is expressed at high levels in a uniform fashion in many regions of the CNS. However, during later stages of development, expression becomes restricted to the ependymal cells lining the ventricular system and in the choroid plexus. This down-regulation was confirmed by both immunohistochemical and biochemical techniques. The program of expression lags behind in the cerebellum in concert with the delayed development of this region of the brain. A high level of N-cadherin was found to be expressed in the brainstem and spinal cord floorplate, while a low level was detected at the optic nerve head. The results indicate that while, in general, the program of N-cadherin expression is similar in the retina and the brain, certain structures unique to the eye and brain express locally high or low levels of this adhesion protein.

Expression of developmentally relevant proteins by rodent embryo CNS cells in vivo and in vitro: proto-oncogene pp60c-src and high molecular weight neurofilament protein.

The expression of proteins that play a role in neuronal differentiation was examined in central nervous system (CNS) micromass embryo cell cultures and compared to expression at comparable developmental stages in vivo. The protein product of the src proto-oncogene (pp60c-src) has been postulated to have a specific role in development because, although it is expressed in many tissues, marked increases in amount and activity of pp60c-src occur in neurons at the time of differentiation. Another protein of interest, high molecular weight neurofilament (NF) protein, is found in differentiated neurons. In the present study, changes over time in the expression of these two proteins in vitro and in vivo were examined. In the micromass cell cultures, primary cells from day 12 rat embryo CNS are plated at high density and differentiate into neurons during five days in culture. Tissues from embryos grown in vivo were assessed at 12 and 17 days post-coitum. Proteins were quantified by PAGE separation of equal amounts of total protein followed by transfer to membranes, immunoblotting, and densitometric scanning of blots. Increases in the amount of both proteins with neuronal differentiation was shown. Protein kinase activity of immunoprecipitated pp60c-src also increased in cell cultures and in embryos. Similarity in patterns of expression between in vitro and in vivo tissue samples provides further evidence that the cultures closely simulate in vivo differentiation and are a useful system for examining expression of developmental genes in vitro.

Insulin and Neuroparsin Promote Neurite Outgrowth in Cultured Locust CNS.

Organotypic cultures of locust embryo central nervous system (CNS) were used to study the influence of hormonal factors on neurite outgrowth. Explants from the third thoracic ganglion (embryonic day 9) were grown in a serum-free medium and exposed to insulin, neuroparsin, a recently characterized insect neurohormone, somatostatin and two insect hormones: 20-hydroxyecdysone and juvenile hormone. These hormonal factors were tested either alone or in several combinations. In our culture system both insulin and neuroparsin promoted extensive neurite outgrowth. Their effects were quantitatively similar. Somatostatin had no effect on neurite formation, but was a growth factor for glial cells. 20-hydroxyecdysone was a required factor for glial cell and neurite survival. Juvenile hormone did not have any observable effects in this culture system. When insulin was added in combination with 20-hydroxyecdysone the stimulation of neurite outgrowth exceeded that seen when insulin was added alone. A similar enhancement was observed with the combination of neuroparsin and 20-hydroxyecdysone. We conclude that in locusts: (i) insulin can be implicated as a neurotrophic factor as in vertebrates; and (ii) neuroparsin, a locust neurohormone, can also be characterized as a neurotrophic factor for the locust. Moreover, both neurohormones interact synergistically with the steroid hormone 20-hydroxyecdysone to enhance neurite growth.

Roles of Glycinergic Inhibition and N-Methyl-D-Aspartate Receptor Mediated Excitation in the Locomotor Rhythmicity of One Half of the Xenopus Embryo Central Nervous System.

Recent investigations into the neural basis for swimming in Xenopus embryos have pointed to central roles played by N-methyl-D-aspartate (NMDA) receptor-mediated excitation acting within and glycinergic reciprocal inhibition acting between motor systems for the muscular antagonists on the two sides of the CNS. A 'reduced' preparation consisting of only one half of the CNS divided sagittally along its midline is used here to examine the basis for rhythmicity within each side in the absence of reciprocal connections. Divided preparations transected rostrally at levels between the otic capsule and the obex can all generate a rhythmic pattern of motor discharge similar to that which underlies swimming. All rhythm generation is blocked by the NMDA antagonist (+/-)-2-amino-5-phosphonovaleric acid (AP5) at 20 microM. However, neither glycinergic nor GABAergic inhibition is required for a basic rhythmicity since some rhythm persists in the presence of 10 microM strychnine and 50 microM bicuculline, though it is no longer sustained. In the divided spinal cord alone, rhythm generation requires extracellular Mg2+. If the most caudal segment of the divided hindbrain is left attached, extracellular Mg2+ is required only if strychnine is present. If more of the hindbrain is included, extracellular Mg2+ is no longer necessary for rhythm generation even in the presence of strychnine. It seems that rhythm generation by a single side of the spinal cord requires NMDA receptor-mediated excitation together with the voltage dependency conferred on it by extracellular Mg2+, but not inhibition in order to occur. As more of the hindbrain is left attached, the requirement for extracellular Mg2+ becomes progressively less strong. For sustained rhythm generation, one side of the CNS requires both excitation and glycinergic inhibition.

Influence of monensin on ganglioside anabolism and neurite stability in cultured chick neurons

We have examined the effects of monensin, a monovalent cationophore that disrupts exo- and endocytosis of membrane vesicles and diminishes Golgi anabolic function, on the incorporation of [3H]-galactose into glycosphingolipids in neurited primary cultures of chick embryo central nervous system neurons. A linear rate of incorporation into all ganglioside species from extracellular-labeled galactose was observed. Specific activity of anabolic labeling was markedly lower in GT1b and GQ1b than in the other major gangliosides of the embryonic neuron (GM3, GD3; GM2, GD2; GM1, GD1a, GD1b). With 100 nM monensin in the extracellular medium, the rate of labeling of GT1b diminished markedly to 20% of control; GD1a, GD1b, and GD2, to 35%; GQ1B to 48%; GD3 to 60%. Vigorous incorporation of label into GM3 was entirely undiminished by monensin. From these findings, it is suggested that ganglioside biosynthesis is compartmentalized in the cytodifferentiating embryonic neuron, with GM3 entirely, and GD3 and GQ1b partially, an extra-Golgi product. Extensive loss of neurites that occurred after several hours of exposure of the neurons to monensin could not be correlated directly with decreased ganglioside anabolism.

Differentiation of glial cells and neurite outgrowth obtained from embryonic locust central nervous system explants

Up to the present it has not been possible to obtain viable glial cells from dissociated insect nervous system cultures. We report here that the use of explant culture of locust embryo central nervous system (CNS) has been successful in allowing the proliferation of glial cells derived from glial precursors located at the periphery of the embryonic CNS. In such cultures, maintained for 3 months under specific conditions, 4 cell types at intermediate stages of differentiation can be distinguished around the explants after 2 weeks in vitro. They have been identified by scanning and transmission electron microscopy. The first type (stage 1) consists of flat epithelioid cells with an abundant and lucent cytoplasm containing little granular endoplasmic reticulum. These earliest cells subsequently develop flat ameboid prolongations forming an intermediate cell type (stage 2) which then differentiates into protuberant bipolar cells (stage 3) in which appear well-organized cisterns of granular endoplasmic reticulum. The last stage of differentiation (stage 4) is composed of multipolar cells with an electron-dense cytoplasm and well-defined processes characterized by the presence of ribosomes and granular endoplasmic reticulum. These (stage 4) differentiated cells resemble mature glial cells In the same in vitro system, neurites, growing from neurons originating in the explants, form a network lying upon the glial cells and in close relationship with them. Neurites present growth cones and lack ribosomes and granular endoplasmic reticulum. We conclude that this model is a potentially useful system for use in in vitro studies of insect glia and neurite-glia interactions.

Inhibitory neurones of a motor pattern generator in Xenopus revealed by antibodies to glycine.

Glycine and gamma-aminobutyric acid (GABA) are inhibitory transmitters of major importance. Whereas neurones using GABA as the transmitter can be visualized by immunocytochemical methods for glutamate decarboxylase (GAD) or GABA, no comparable techniques have been available for the selective visualization of glycinergic neurones. We have now produced polyclonal antibodies which specifically recognize glycine in glutaraldehyde-fixed tissue. We used these antibodies to investigate the distribution of glycine in the simple central nervous system (CNS) of the Xenopus embryo, which contains an anatomically and physiologically defined class of reciprocal inhibitory interneurones, the commissural interneurones. These interneurones have an important role in the generation of the swimming motor pattern and are thought to be glycinergic. The glycine antibodies specifically stain these interneurones, revealing their distribution and number in the embryo CNS. This is the first demonstration of the selective localization of glycine-like immunoreactivity in a putative glycinergic class of neurone that has been characterized physiologically, pharmacologically and anatomically.