Association of polymorphisms in complement component C3 gene with susceptibility to systemic lupus erythematosus.

OBJECTIVE: Identification of the genes responsible for systemic lupus erythematosus (SLE). METHODS: All the exons and putative promoter regions of 53 candidate genes (TNFRSF6/Fas, TNFSF6/FasL, Fli1, TNFSF10/TRAIL, TNFSF12/TWEAK, Bcl-2, PTEN, FADD, TRADD, CDKN1A, TNFRSF1A/TNFR1, TNFRSF4/OX40, TNFSF4/OX40L, TNFSF5/CD40L, TNFSF13B/BAFF, ICOS, CTLA4, CD28, FYN, G2A, CR2, PTPRC/CD45, CD22, CD19, Lyn, PDCD1, PTPN6, TGFB1, TGFB2, TGFB3, TGFBR1, TGFBR2, TGFBR3, CD3Z, DNASE1, APCS, MERTK, C3, C1QA, C1QB, C1QG, C2, MBL2, IGHM, IL-2, IL-4, IL-10, IFNG, TNFA, MAN2A1, TNFRSF11A/RANK, TNFRSF11B/OPG, TNFSF11/OPGL) were screened for single nucleotide polymorphisms (SNPs) and their association with SLE was assessed by case-control studies. A total of 509 cases and 964 controls of Japanese descent were enrolled. RESULTS: A total of 316 SNPs was identified. When analysed in the Japanese population, the allele frequencies of T at rs7951 and G at rs2230201 of the C3 gene were 0.110 and 0.626, respectively, in SLE patients; significantly higher than the frequencies of 0.081 and 0.584, respectively, in controls [odds ratio (OR) = 1.40, 95% confidence interval (CI) = 1.05-1.86, P = 0.016 and OR=1.19, 95% CI = 1.01-1.41, P = 0.038, respectively]. The mean serum C3 level of carriers of the rs7951 T allele was significantly lower than that of non-carriers of the T allele in 87 SLE patients whose medical records were available (P = 0.0018). CONCLUSION: rs7951 T allele of the C3 gene was significantly associated with SLE, and decreased serum level of C3 seems to be correlated with this allele.

Growth and differentiation of astrocytes and neural progenitor cells on micropatterned polymer films.

This paper investigates the influence of micropatterned polymers and chemical modification on neural progenitor cell growth and differentiation in co-culture systems with astrocytes. We sought to develop strategies to facilitate nerve regeneration using a synergistic combination of guidance cues, investigating the cellular mechanisms of nerve repair using adult rat hippocampal progenitor cells (AHPCs). Our studies have shown that this synergistic combination of physical, chemical, and biological cues can lead to oriented growth of astrocytes and progenitor cells, can control and accelerate neurite outgrowth and alignment in vitro, and may influence differentiation of progenitor cells.

Neural progenitor cell transplants into the developing and mature central nervous system.

When developing cell transplant strategies to repair the diseased or injured central nervous system (CNS), it is essential to consider host-graft interactions and how they may influence the outcome of the transplants. Recent studies have demonstrated that transplanted neural progenitor cells (NPCs) can differentiate and integrate morphologically into developing mammalian retinas. Is the ability to differentiate and to undergo structural integration into the CNS unique to specific progenitor cells, or is this plasticity a function of host environment, or both? To address these issues we have used the developing retina of the Brazilian opossum and have compared the structural integration of brain and retinal progenitor cells transplanted into the eyes at different developmental stages. The Brazilian opossum, Monodelphis domestica, is a small pouchless marsupial native to South America. This animal's lack of a pouch and fetal-like nature at birth circumvents the need for in utero surgical procedures, and thus provides an ideal environment in which to study the interactions between developing host tissues and transplanted NPCs. To test whether NPCs affect visual function we transplanted adult hippocampal progenitor cells (AHPCs) into normal, healthy adult rat eyes and performed noninvasive functional recordings. Monitoring of the retina and optic nerve over time by electroretinography and pupillometry revealed no severe perturbation in visual function in the transplant recipient eyes. Taken together, our findings suggest that the age of the host environment can strongly influence NPC differentiation and that transplantation of neural progenitor cells may be a useful strategy aimed at treating neurodegeneration and pathology of the CNS.

Oriented astroglial cell growth on micropatterned polystyrene substrates.

In an effort to develop a permissive environment for neural stem cell differentiation, directional growth of astrocytes has been achieved on polymer substrates in vitro. Manipulating a combination of physical and chemical cues, astrocyte adhesion and alignment in vitro were examined. To provide physical guidance, micropatterned polymer substrates of polystyrene (PS) were fabricated. Laminin was selectively adsorbed onto the grooves of the patterned surface. Rat type-1 astrocytes were seeded onto the micropatterned PS substrates, and the effects of substrate topography and the adsorption of laminin to the PS substrates on the behavior and morphology of the astrocytes were explored. The astrocytes were found to align parallel to the micropatterned grooves at initial seeding densities of approximately 7500, 13,000, and 20,000 cells/cm(2) due to the effects of the physical and chemical guidance mechanisms. Adsorbing laminin in the microgrooves of the micropatterned PS substrates improved cell adhesion and spreading of cytoskeletal filaments significantly. At these initial seeding densities, over 85% astrocyte alignment in the direction of the grooves was achieved on the micropatterned PS substrates with laminin adsorbed in the grooves. This combination of guidance cues has the potential to provide a permissive substrate for in vivo regeneration within the central nervous system.

Transplantation of neural progenitor cells into the developing retina of the Brazilian opossum: an in vivo system for studying stem/progenitor cell plasticity.

In developing cell transplant strategies to repair the diseased or injured retina is essential to consider host-graft interactions and how they may influence the outcome of the transplants. In the present study we evaluated the influence of the host microenvironment upon neural progenitor cells (NPCs) transplanted into the developing and mature retina of the Brazilian opossum, Monodelphis domestica. Monodelphis pups are born in an extremely immature state and the neonatal pups provide a fetal-like environment in which to study the interactions between host tissues and transplanted NPCs. Three different populations of GFP-expressing NPCs were transplanted by intraocular injection in hosts ranging in age from 5 days postnatal to adult. Extensive survival, differentiation and morphological integration of NPCs were observed within the developing retina. These results suggest that the age of the host environment can strongly influence NPC differentiation and integration.

Differentiation and morphological integration of neural progenitor cells transplanted into the developing mammalian eye.

Transplantation of neural stem/progenitor cells has been proposed as a novel approach for the replacement and repair of damaged CNS tissues. We have evaluated the influence of the host cellular microenvironment upon the survival, differentiation, and integration of neural progenitor cells transplanted into the CNS. Using this approach, we have investigated the fate of neural progenitor cells in vivo following transplantation into the developing mammalian eye. Murine brain progenitor cells (mBPCs) isolated from neonatal mice expressing the green fluorescent protein (GFP) transgene were transplanted into the eyes of Brazilian opossums (Monodelphis domestica). Monodelphis pups are born in an extremely immature, fetal-like state. The eyes of neonatal pups provide a fetal-like environment in which to study cellular interactions between host tissues and transplanted neural progenitor cells. mBPCs were transplanted by intraocular injection in hosts ranging in age from 5 days postnatal to adult. The transplanted cells were easily identified because of their GFP fluorescence. Extensive survival, differentiation, and morphological integration of mBPCs within the host tissue was observed. We found that the younger retinas provided a more supportive environment for the morphological integration of the transplanted mBPCs. Cells with morphologies characteristic of specific retinal cell types were observed. Moreover, some transplanted mBPCs were labeled with antibodies characteristic of specific neural/retinal phenotypes. These results suggest that the host environment strongly influences progenitor cell differentiation and that transplantation of neural progenitor cells may be a useful approach aimed at treating degeneration and pathology of the CNS.

Differential localization of SNARE complex proteins SNAP-25, syntaxin, and VAMP during development of the mammalian retina.

SNARE complex proteins have critical functions during regulated vesicular release of neurotransmitter. In addition, they play critical roles during neurite outgrowth and synaptogenesis. Although it is clear that the function of any one SNARE complex protein during release of neurotransmitter is dependent on its association with other members of the complex, it is less certain whether their function during development and differentiation is dependent on interaction with one another. Previously, we have observed transient high levels of SNARE complex protein SNAP-25 in developing cholinergic amacrine cells (West Greenlee et al. [1998] J Comp Neurol 394:374-385). In addition, we detected, high levels of SNAP-25 in developing and mature photoreceptors. To better understand the functional significance of these high levels of SNAP-25 expression, we used immunocytochemistry to examine the developmental expression of the three members of the SNARE complex, SNAP-25, Syntaxin, and vesicle associated membrane protein (VAMP/also Synaptobrevin). Our results demonstrate that the high levels of SNAP-25 in cholinergic amacrine cells and photoreceptors are not accompanied by the same relatively high levels of other SNARE complex proteins. These results suggest that high levels of SNAP-25 in specific cell types may function independently of association with Syntaxin and VAMP. In this analysis, we characterized the changing patterns of immunoreactivity for the three SNARE complex proteins during the development and differentiation of the mammalian retina. We have compared the pattern of expression of the core SNARE complex proteins in the Brazilian opossum, Monodelphis domestica, and in the rat and found common patterns of expression between these diverse mammalian species. We observed temporal differences in the onset of immunoreactivity between these three proteins, and differences in their localization within synaptic layers in the developing and mature mammalian retina. This study is the first to characterize the changing expression patterns of the three SNARE complex proteins in the developing central nervous system. The differential distribution of SNAP-25, Syntaxin, and VAMP may indicate additional roles for these proteins during vesicle trafficking events, which are independent of their association with one another.

Mutation of Ha-Ras C terminus changes effector pathway utilization.

In PC12 cells, Ha-Ras modulates multiple effector proteins that induce neuronal differentiation. To regulate these pathways Ha-Ras must be located at the plasma membrane, a process normally requiring attachment of farnesyl and palmitate lipids to the C terminus. Ext61L, a constitutively activated and palmitoylated Ha-Ras that lacks a farnesyl group, induced neurites with more actin cytoskeletal changes and lamellipodia than were induced by farnesylated Ha-Ras61L. Ext61L-triggered neurite outgrowth was prevented easily by co-expressing inhibitory Rho, Cdc42, or p21-activated kinase but required increased amounts of inhibitory Rac. Compared with Ha-Ras61L, Ext61L caused 2-fold greater Rac GTP binding and phosphatidylinositol 3-kinase activity in membranes, a hyperactivation that explained the numerous lamellipodia and ineffectiveness of Rac(N17). In contrast, Ext61L activated B-Raf kinase and ERK phosphorylation more poorly than Ha-Ras61L. Thus, accentuated differentiation by Ext61L apparently results from heightened activation of one Ras effector (phosphatidylinositol 3-kinase) and suboptimal activation of another (B-Raf). This surprising unbalanced effector activation, without changes in the designated Ras effector domain, indicates the Ext61L C-terminal alternations are a new way to influence Ha-Ras-effector utilization and suggest a broader role of the lipidated C terminus in Ha-Ras biological functions.

Disruption of actin-myosin interactions results in the inhibition of focal adhesion assembly in Xenopus XR1 glial cells.

In the present study we have investigated the role of actin-myosin interactions in regulating focal adhesion assembly in Xenopus XR1 glial cells. Actin-myosin interactions, stress fiber formation, and focal adhesion assembly are thought to allow cells to exert tension in the surrounding extracellular matrix, a process essential during morphogenesis and wound healing. Immunocytochemical analysis has revealed that myosin heavy chain-A (MHC-A), the predominant isoform in XR1 cells, was distributed in a filamentous pattern in the central region but was more diffuse towards the cell periphery. Myosin heavy chain-A-like immunoreactivity (IR) partially colocalized with phalloidin stained F-actin microfilaments in XR1 cells but not with microtubules. Furthermore, MHC-A-IR colocalized with immunoreactivity for beta1 integrin receptors and vinculin at focal adhesions located more centrally along the ventral surface of the cells. The partial colocalization of MHC-A with the F-actin cytoskeleton, as well as at focal adhesions, provides evidence that actin-myosin interactions may be involved in regulating focal adhesion assembly and stabilization. To examine this possibility, we have used drugs shown to inhibit cell contractility: the kinase inhibitors H7 and HA100, and 2,3-butanedione 2-monoxime (BDM), which inhibits muscle and nonmuscle ATPase activity. Compared to control cultures, those treated with the inhibitors exhibited a dose-dependent decrease in the percentage of cells that displayed focal adhesions. In addition, these cells also displayed disrupted actin cytoskeletons and a similar disruption in myosin filaments. Taken together, these results provide evidence for an important role of actin-myosin generated forces during focal adhesion assembly in glial cells.

Development of the facial and hypoglossal motor nuclei in the neonatal Brazilian opossum brain.

The development of the facial and hypoglossal motor nuclei were examined in the neonatal Brazilian opossum (Monodelphis domestica), a marsupial in which postnatal central nervous system development has been well characterized. In this study, we utilized postnatal injection of the retrograde tracer cholera toxin subunit B (CtB) to characterize the formation of the facial and hypoglossal motor nuclei in the developing neonatal opossum brainstem. Injections of CtB were made into the cheek/lip region or tongue of opossum pups to retrogradely label the facial or hypoglossal motor nuclei, respectively. Following a 2 h survival time, facial motoneurons in newborn opossum pups (1 PN) exhibited CtB labeling, with their cell bodies localized near the developing cranial abducens nucleus. At 3 and 5 PN, following a 48 h survival time, CtB-labeled facial motoneurons were observed in and migrating to the region of the adult facial motor nucleus in the rostral medulla. Between 7 and 10 PN, almost all facial motoneurons had migrated to their destination within the facial motor nucleus. Hypoglossal motoneurons also exhibited CtB labeling from 1 PN; however, their cell bodies were localized within the hypoglossal motor nucleus at the earliest age examined. Double label studies, to examine guidance of facial motoneurons during migration, demonstrated that CtB-labeled facial motoneurons are in close proximity to vimentin-like immunostained radial glial fibers during migration. These results suggest: (1) migration of facial motoneurons to the facial motor nucleus is a postnatal event, (2) efferent projections from facial and hypoglossal motoneurons project into the peripheral region of their target muscles from the day of birth, and (3) facial motoneurons migrate to their destination in the brainstem thereafter, in close association with radial glial fibers.

Transient, high levels of SNAP-25 expression in cholinergic amacrine cells during postnatal development of the mammalian retina.

In the present study, we have examined the development of cholinergic amacrine cells in the retina of the Brazilian opossum, Monodelphis domestica. An antibody directed against choline acetyltransferase (ChAT) revealed that ChAT-like immunoreactivity (ChAT-IR) was first observed at 15 days postnatal (15PN). By 25PN, ChAT-IR identified two matching populations of amacrine cells in the inner nuclear and ganglion cell layer. Bromodeoxyuridine birth-dating analysis coupled with immunolabeling with the anti-ChAT antibody revealed that the cholinergic amacrine cells are born postnatally, between 2PN and 15PN. In addition, we have examined the differentiation of the cholinergic amacrine cells by using an antibody directed against a presynaptic terminal-associated protein, synaptosomal-associated protein of 25 kDa (SNAP-25). Double-labeling analysis revealed that relatively high levels of SNAP-25-IR were selectively present in cholinergic amacrine cells prior to eye opening. However, in the mature retina, high levels of SNAP-25-IR were no longer observed in the ChAT-IR amacrine cells. These results reveal a distinct period in development, prior to eye opening, when high levels of SNAP-25-IR are selectively expressed in cholinergic amacrine cells. The specificity and time course of the high levels of SNAP-25 in cholinergic amacrine cells may be critical in mediating the transient properties of these cells during visual system development.

Characterization of focal adhesion assembly in XR1 glial cells.

In the present communication, we have characterized focal adhesions in cultured glial cells derived from the Xenopus retina. Using antibodies directed against focal adhesion proteins we found that beta1 integrin immunoreactivity colocalized with talin, vinculin, and phosphotyrosine immunoreactivities in glial cells from primary cultures of Xenopus retina, as well as in the XR1 glial cell line, an immortal cell line derived from Xenopus retinal neuroepithelium. beta1 integrin immunoreactivity also colocalized with the termini of rhodamine phalloidin-labeled filamentous-actin at focal adhesions. The regulation of focal adhesion assembly was examined in XR1 glial cells using inhibitors against actin polymerization (cytochalasins) or tyrosine kinase activity (genistein). Compared to control cultures, those treated with the inhibitors exhibited a dose-dependent decrease in the proportion of cells displaying focal adhesions. Treatment with cytochalasin B also resulted in a dose-dependent decrease in cell area. Mature focal adhesions in XR1 cells with a flattened, spread morphology also were disrupted by the presence of these inhibitors. These results provide strong evidence that an intact actin cytoskeleton and tyrosine kinase activity regulate focal adhesion assembly and also play important roles in the maintenance of the integrity of focal adhesions in glial cells.

Basic fibroblast growth factor (FGF-2) induced transdifferentiation of retinal pigment epithelium: generation of retinal neurons and glia.

In the present study we report that basic fibroblast growth factor (bFGF, FGF-2) promotes the transdifferentiation of Xenopus laevis larval retinal pigment epithelium (RPE) into neural retina. Using specific antibodies we have examined the cellular composition of the regenerated retinal tissue. Our results show that, in addition to retinal neurons and photoreceptors, glial cells were also regenerated from the transdifferentiated RPE. These results were specific to FGF-2, since other factors that were tested, including acidic FGF (aFGF, FGF-1), epidermal growth factor (EGF), laminin, ECL, and Matrigel, exhibited no activity in inducing retinal regeneration. These results are the first in amphibians demonstrating the functional role of FGF-2 in inducing RPE transdifferentiation. Transplantation studies were carried out to investigate retinal regeneration from the RPE in an in vivo environment. Sheets of RPE implanted into the lens-less eyes of larval hosts transformed into neurons and glial cells only when under the influence of host retinal factors. In contrast, no retinal transdifferentiation occurred if the RPE was implanted into the enucleated orbit. Taken together, these results show that the amphibian RPE is capable of transdifferentiation into neuronal and glial cell-phenotypes and implicate FGF-2 as an important factor in inducing retinal regeneration in vitro.

Beta 1 integrins regulate axon outgrowth and glial cell spreading on a glial-derived extracellular matrix during development and regeneration.

In the present study we have investigated functional roles for beta 1 integrin receptors in regulating axon outgrowth, and glial cell adhesion and spreading in the Xenopus retina. The XR1 glial cell line, isolated from Xenopus retinal neuroepithelium, deposits a proteinaceous extracellular matrix (ECM) with potent neurite outgrowth promoting activity. To investigate a potential role of the integrins as cellular receptors for these glial cell-derived ECM components, embryonic and regenerating retinal explants were cultured in the presence of polyclonal antibodies directed against the beta 1 integrin receptor complex. The IgGs and Fabs of the anti-beta 1 integrin antibody strongly inhibited ganglion cell axon outgrowth on the glial cell-derived ECM, although axons grew freely across the surfaces of glial cells surrounding the explants. The antibodies also inhibited outgrowth on purified laminin containing substrates in a dose-dependent fashion. In addition, the anti-beta 1 antibodies were effective at inhibiting the spreading of glial cells that migrated out from the embryonic explants, and also inhibited attachment and spreading of Xenopus XR1 glial cells on ECM substrates. These results show that the beta 1 integrins play important functional roles in axon outgrowth during development and regeneration, and also serve in regulating retinal glial cell attachment and spreading in vitro, and thus are likely to play similar roles in vivo.

Perturbation of the developing Xenopus retinotectal projection following injections of antibodies against beta1 integrin receptors and N-cadherin.

We have examined the function of beta1 integrin receptors and N-cadherin in the development of the Xenopus retinotectal projection. In vivo perturbation experiments were performed by injecting antibodies directed against beta1 integrin receptors and N-cadherin into the embryonic optic pathway. The antibodies were present during the initial development of the retinal projection, when the axons of the ganglion cells are migrating through the optic tract and terminating within the optic tectum. When injected individually, the antibodies were insufficient to cause obvious pathfinding errors. However, when injected together, the antibodies caused specific abnormalities in the development of the retinotectal projection. Pathfinding errors most commonly observed included ectopically projecting axons within the optic tract region, meandering and splaying of axons in the optic tectum, and the induction of prominent ipsilateral projections. IgGs and Fab' fragments of the antibodies produced pathfinding errors; these defects were not observed in animals injected with control antibodies. These in vivo results show that beta1 integrin receptors and N-cadherin have important roles during the development of the visual projection and provide evidence that a balance between cell-cell and cell-matrix adhesion may be critical for the normal development of the vertebrate visual system.

Postnatal development and the differential expression of presynaptic terminal-associated proteins in the developing retina of the Brazilian opossum, Monodelphis domestica.

In the present study we have characterized the postnatal (PN) development of the retina in the Brazilian opossum, Monodelphis domestica. Monodelphis, a small, pouchless marsupial, undergoes a protracted period of postnatal development. Using bromodeoxyuridine immunohistochemistry, we have investigated postnatal neurogenesis of the retina. In addition, we have examined the differentiation of the retina by using antibodies directed against the presynaptic terminal-associated proteins synaptotagmin, Rab3A, synaptophysin and synaptosomal-associated protein-25 (SNAP-25), and have characterized their spatial and temporal distribution during postnatal development. This study is the first systematic comparison of the developmental expression of multiple presynaptic terminal-associated proteins in relation to retinal neurogenesis and differentiation. At birth (1PN), the Monodelphis retina was relatively undifferentiated morphologically and birthdating analysis revealed mitotically active cells throughout the retina. The 8PN retina was organized into two cellular layers: an outer region of mitotically active neuroepithelial cells and an inner region of postmitotic cells. The inner plexiform layer formed between 5PN and 10PN, and exhibited unique patterns of immunoreactivity with the antibodies used in this analysis. By 25PN the retina was well laminated, and synaptotagmin-, Rab3A-, synaptophysin- and SNAP-25-like immunoreactivities exhibited distinct and specific patterns within the plexiform layers, although they had not yet achieved their mature, adult patterns. These results indicate that each of these proteins exhibits developmentally regulated changes in its cellular localization, and therefore may play important roles during morphogenesis and synaptogenesis of the vertebrate retina.

Developmental distribution of GFAP and vimentin in the Brazilian opossum brain.

Cells of glial origin are involved in the morphogenesis of the mammalian central nervous system (CNS). Characterization of glial-associated proteins during neurogenesis and differentiation may aid in understanding the complexity of CNS development. We have utilized immunoblotting and immunohistochemistry to characterize the developmental profiles of glial fibrillary acidic protein (GFAP) and vimentin (VIM) in the brain of the Brazilian opossum, Monodelphis domestica. Typical of marsupials, CNS morphogenesis and neurogenesis in the opossum extend well into the postnatal period. Opossum GFAP and VIM were found as single bands at molecular weights consistent with those reported for other species, thus indicating conservation of the VIM and GFAP proteins through mammalian evolution. Differential developmental trends were observed for both proteins with relative VIM levels decreasing and GFAP levels increasing with age. Vimentin-like immunoreactivity (VIM-IR) was present at day 1 of postnatal life throughout the brain. The density of VIM-IR was maximal at 10 and 15 days postnatal (especially in radial glial elements) and decreased slightly by 25 days postnatal. In the adult brain, VIM-IR was markedly reduced compared to that of younger ages. In contrast, GFAP-like immunoreactivity (GFAP-IR) in the brain of Monodelphis increased dramatically with age. No GFAP-IR was observed in the 1 and 5 day postnatal brains. By 25 days postnatal, the pattern of GFAP-IR in the brainstem resembled that of the adult. In the forebrain, more GFAP-IR was present than at younger ages. The adult distribution of GFAP-IR was very similar to that reported for other mammalian species. These results indicate that GFAP and VIM are reciprocally related during periods of morphogenesis and differentiation of the opossum brain.

Imaging F-actin in fixed glial cells with a combined optical fluorescence/atomic force microscope.

A prototype combined optical fluorescence/atomic force microscope (OFAFM) designed for use in neurobiology and related disciplines has been constructed and used to study filamentous actin (F-actin) and other cellular structures in fixed Xenopus retinal glial cells (XR1 glial cell line). F-actin was readily observed by both fluorescence and AFM. AFM images of nuclei and other cellular structures were also obtained. The OFAFM consists of an AFM with an interferometer detection mechanism mounted on an inverted optical microscope. Integration of optical and scanned probe imaging methods provides a unique and useful approach to studying glial (and other) cell structure and function.

Actin filament dynamics in living glial cells imaged by atomic force microscopy.

Observation of filamentous actin (F-actin) in living cells is currently limited to the resolution of the light microscope. Higher resolution procedures require sample fixation and preclude dynamic studies. The atomic force microscope (AFM) can image and manipulate samples at very high, sometimes atomic resolution by scanning a fine tip over the surface of interest and detecting physical interactions between the tip and sample. This study demonstrates that F-actin can be readily resolved in living cells with the AFM and that the dynamic properties of F-actin are easily observed.

Characterization of the neurosecretory activity of hypothalamic beta-endorphin-containing neurons in primary culture.

To determine the neurosecretory activity of hypothalamic beta-endorphin (beta EP)-containing neurons, rat fetal hypothalamic cells were mechanically dispersed and maintained in primary cultures for periods up to 24 days; their electrophysiological properties and regulation by depolarization, calcium and sodium channel-active agents were studied. Under culture conditions, the majority of the cells were immunopositive to neurofilament antibody, and a significant number (7-10%) were reactive to beta EP antibody. Cultured cells were often electrically excitable and possessed voltage-activated ionic conductances. In culture, there was a progressive increase in immunoreactive beta EP (IR-beta EP) in both cells and media, reaching maximum values at 12-16 days. The majority of IR-beta EP in both cells and media corresponded to [125I]beta EP on gel chromatography and was similar to the form previously found in the hypothalamus. These findings suggest viability of the beta EP neurons and continuing synthesis of IR-beta EP during the culture period. To evaluate the influence of membrane depolarization on IR-beta EP release, the cells were challenged with 56 mM potassium. This treatment induced a significant increase in medium IR-beta EP. The depolarization-induced IR-beta EP release was dependent upon calcium, since a calcium channel blocker, verapamil (0.1 microM), prevented the release; also a calcium ionophore, A23187 (1 microM), stimulated IR-beta EP release in the cultures. Activation of the sodium channel by veratridine (100 microM) also increased the medium content of IR-beta EP, and this effect was blocked by tetrodotoxin (1 microM). These results suggest that the beta EP neurons in primary culture respond to the well defined physiological challenges and that the culture system can be used in determining the regulation of hypothalamic beta EP activity.