Retinal ganglion cells of high cytochrome oxidase activity in the rat

Retinal ganglion cells in the rat were studied using the heavy metal intensified cytochrome oxidase and horseradish peroxidase histochemical methods.The results show that a population of large retinal ganglion cells was consistently observed with the cytochrome oxidase staining method in retinas of normal rats or rats which received unilateral thalamotomy at birth.These cytochrome oxidase rich ganglion cells appeared to have large somata,3-6 primary dendrites and extensive dendritic arbors,and are comparable to ganglion cells labeled by the wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP).However,the morphological details of some of the cells revealed by the cytochrome oxidase staining method are frequently better than those shown by the HRP histochemical method.These results suggest that the mitochondrial enzyme cytochrome oxidase can be used as a simple but reliable marker for identifying and studying a population of retinal genglion cells with high metabolic rate in the rat.     

Insulin cells of pancreas extend neurites but do not arise from the neuroectoderm.

It is generally believed that during mammalian embryogenesis neurons arise only from the ectodermal germ layer, while the other two germ layers, mesoderm and endoderm, give rise to connective tissue and gut, respectively. Pancreatic islet cells, however, may be an exception to this classical cell lineage derivation. These cells, of endodermal origin, can express several neuronal antigens in addition to the peptide hormones which regulate carbohydrate metabolism. This study sought to determine whether islet cells of adult mice, in addition to displaying biochemical homology to neurons, are also capable of extending neurites, the cytoplasmic elongations that are recognized as a hallmark of the neuronal phenotype. It was found that dissociated pancreatic islet cells can extend neurite-like processes when maintained in vitro and that these processes contain neurofilament, the intermediate filament protein specific to neurons. Islet cells maintained in vitro as explants, however, did not form neurites thereby indicating that normal histotypical contacts inhibit process formation. This observation may account for the absence of process elaboration by intact islets in vivo. These results demonstrate that cells derived from the endoderm share the ability to display a characteristic neuronal phenotype with neuroectodermal cells and, furthermore, that the expression of these traits is regulated by epigenetic cues.     

LIMK1 acts downstream of BMP signaling in developing retinal ganglion cell axons but not dendrites

The actin cytoskeleton inside extending axonal and dendritic processes must undergo continuous assembly and disassembly. Some extrinsic factors modulate actin turnover through controlling the activity of LIM kinase 1 (LIMK1), which phosphorylates and inactivates the actin depolymerizing factor cofilin. Here, we for the first time examine the function and regulation of LIMK1 in vivo in the vertebrate nervous system. Upon expression of wildtype or kinase-dead forms of the protein, dendrite growth by Xenopus retinal ganglion cells (RGCs) was unchanged. In contrast, maintaining a low, but significant level, of LIMK1 function in the RGC axon is critical for proper extension. Interestingly, bone morphogenetic protein receptor II (BMPRII) is a major regulator of LIMK1 in extending RGC axons, as expression of a BMPRII lacking the LIMK1 binding region caused a dramatic shortening of the axons. Previously, we found that BMPRIIs stimulate dendrite initiation in vivo. Thus, the fact that manipulation of LIMK1 activity failed to alter dendrite growth suggests that BMPs may activate distinct signalling pathways in axons and dendrites.     

Rudimentary TCR signaling triggers default IL-10 secretion by human Th1 cells

Understanding the process of inducing T cell activation has been hampered by the complex interactions between APC and inflammatory Th1 cells. To dissociate Ag-specific signaling through the TCR from costimulatory signaling, rTCR ligands (RTL) containing the alpha1 and beta1 domains of HLA-DR2b (DRA*0101:DRB1*1501) covalently linked with either the myelin basic protein peptide 85-99 (RTL303) or CABL-b3a2 (RTL311) peptides were constructed to provide a minimal ligand for peptide-specific TCRs. When incubated with peptide-specific Th1 cell clones in the absence of APC or costimulatory molecules, only the cognate RTL induced partial activation through the TCR. This partial activation included rapid TCR zeta-chain phosphorylation, calcium mobilization, and reduced extracellular signal-related kinase activity, as well as IL-10 production, but not proliferation or other obvious phenotypic changes. On restimulation with APC/peptide, the RTL-pretreated Th1 clones had reduced proliferation and secreted less IFN-gamma; IL-10 production persisted. These findings reveal for the first time the rudimentary signaling pattern delivered by initial engagement of the external TCR interface, which is further supplemented by coactivation molecules. Activation with RTLs provides a novel strategy for generating autoantigen-specific bystander suppression useful for treatment of complex autoimmune diseases.     

MAP4 counteracts microtubule catastrophe promotion but not tubulin-sequestering activity in intact cells

Microtubules are polar polymers that continually switch between phases of elongation and shortening, a property referred to as dynamic instability. The ubiquitous microtubule associated protein 4 (MAP4) shows rescue-promoting activity during in vitro assembly of microtubules (i.e., promotes transitions from shortening to elongation), but its regulatory role in intact cells is poorly defined. Here, we demonstrate that ectopic MAP4 promotes outgrowth of extended MTs during beta1-integrin-induced cell spreading. An inducible cotransfection protocol was employed to further analyze the regulatory role of MAP4 in human leukemia cells with microtubules partially destabilized by either ectopic tubulin-sequestering proteins or proteins that promote catastrophes (i.e., transitions from elongation to shortening). Coexpression of proteins that sequester free tubulin heterodimers with different efficiencies was found to abolish microtubule stabilization by MAP4. In contrast, however, the microtubule-stabilizing activity of MAP4 was found to suppress the activities of two distinct and specific catastrophe promoters, namely, XKCM1 and a nonsequestering truncation derivative of Op18/stathmin. These observations reveal specificity in the microtubule-stabilizing activity of MAP4 that differentiates between two mechanistically distinct types of MT destabilization.     

Sensory transduction and electrical signaling in guard cells

Guard cells are a valuable model system for the study of photoreception, ion transport, and osmoregulation in plant cells. Changes in stomatal apertures occur when sensing mechanisms within the guard cells transduce environmental stimull into the ion fluxes and biosynthesis of organic solutes that regulate turgor. The electrical events mediating sensory transduction in guard cells can be characterized with a variety of electrophysiological recording techniques. Recent experiments applying the patch clamp method to guard cell protoplasts have demonstrated activation of electrogenic pumps by blue and red light as well as the presence of potassium channels in guard cell plasmalemma. Light activation of electrogenic proton pumping and the ensuing gating of voltage-dependent ion channels appear to be components of sensory transduction of the stomatal response to light. Mechanisms underlying stomatal control by environmental signals can be understood by studying electrical events associated with ion transport.     

Retinal axons inXenopus laevis recognise differences between tectal and diencephalic glial cells in vitro

Explants of retina from Xenopus laevis were cultured on monolayers of tectal and diencephalic glial cells in order to determine whether the glia, normally encountered by optic nerve fibres as they grow to the optic tectum, can influence the growth of these neurons in any way. Explants of nasal retina produced prolific radial outgrowth patterns on both tectal and diencephalic monolayers. Explants of temporal retina produced similar outgrowth patterns on diencephalic glia, but on tectal glia the outgrowth was restricted and fibres were fasciculated in short, fat bundles.     

Retinal ganglion cell-derived sonic hedgehog signaling is required for optic disc and stalk neuroepithelial cell development

The development of optic stalk neuroepithelial cells depends on Hedgehog (Hh) signaling, yet the source(s) of Hh protein in the optic stalk is unknown. We provide genetic evidence that sonic hedgehog (Shh) from retinal ganglion cells (RGCs) promotes the development of optic disc and stalk neuroepithelial cells. We demonstrate that RGCs express Shh soon after differentiation, and cells at the optic disc in close proximity to the Shh-expressing RGCs upregulate Hh target genes, which suggests they are responding to RGC-derived Shh signaling. Conditional ablation of Shh in RGCs caused a complete loss of optic disc astrocyte precursor cells, resulting in defective axon guidance in the retina, as well as conversion of the neuroepithelial cells in the optic stalk to pigmented cells. We further show that Shh signaling modulates the size of the Pax2(+) astrocyte precursor cell population at the optic disc in vitro. Together, these data provide a novel insight into the source of Hh that promotes neuroepithelial cell development in the mammalian optic disc and stalk.     

Pituitary cell type-specific electrical activity, calcium signaling and secretion

All secretory anterior pituitary cells exhibit spontaneous and extracellular calcium-dependent electrical activity, but differ with respect to the patterns of firing and associated calcium signaling and hormone secretion. Thus, somatotrophs and lactotrophs fire plateau-bursting action potentials spontaneously and without coupling to calcium release from intracellular stores, which generate calcium signals of sufficient amplitude to keep steady hormone release. In these cells, both spontaneous electrical activity and basal hormone secretion can be further amplified by activation of Gq/11 and Gs-coupled receptors and inhibited by Gi/o/z-coupled receptors. In contrast, gonadotrophs fire single, high-amplitude spikes with limited ability to promote calcium influx and exocytosis, whereas activated Gq/11-coupled receptors in these cells transform single-action potential spiking into the plateau-bursting type of electrical activity and trigger periodic high-amplitude calcium signals and exocytosis of prestored secretory vesicles. Here, we review biochemical and biophysical aspects of spontaneous and receptor-controlled electrical activity, calcium signaling, and hormone secretion in pituitary cells.     

Macrophages and T cells do not express Mlsa determinants

In order to test the tissue distribution of Mlsa determinants, we have generated highly purified stimulator cell populations. First, Mlsa expression in bone marrow derived macrophages (M phi) of Mlsa genotype was tested in primary MLR and on Mlsa-specific T cell hybridomas (THy). Second, a similar experimental approach was used to analyze thioglycolate, peptone or Con A elicited peritoneal M phi. In all cases, these M phi cell populations were able to generate an excellent alloresponse, whereas no functional Mlsa determinants could be detected. Third, to further investigate whether the expression of Mlsa is lymphocyte specific, but dependent on expression of class II molecules, we have transfected I-Ek alpha and beta cDNA into a panel of thymomas of Mlsa genotype. Although we achieved a high level of surface I-Ek expression in all of these T cell tumors, none of them was able to trigger the Mlsa-specific THy. These results strongly suggest that Mlsa expression is limited to B cells. It is likely that Mlsa is a tissue-specific self-peptide that associates with class II molecules.     

Detection of brain-derived neurotrophic factor-like activity in fibroblasts and Schwann cells: inhibition by antibodies to NGF

mRNA coding for brain-derived neurotrophic factor (BDNF) has been detected in cultured L929 fibroblasts, rat dermal fibroblasts, and sciatic nerve Schwann cells, as well as in rat skin. Medium conditioned by cultured fibroblasts and Schwann cells also stimulates neurite growth from retinal explants and promotes the survival in culture of BDNF-responsive sensory neurons; biological activity is abolished by antibodies raised against NGF. These results suggest that molecules with BDNF-like activity may be produced by cells in the peripheral nervous system and that the BDNF-like activity in fibroblasts and Schwann cells is derived from molecules immunologically related to NGF. In support of this concept, antibodies against NGF have been found to reduce the biological activity of recombinant BDNF in culture and to cross-react with BDNF on Western blots.     

Retinal FABP principally localizes to neurons and not to glial cells

The presence of fatty acid-binding protein (FABP) in the embryonic chick retina may be linked to the demand for polyunsaturated fatty acids in this developing neural tissue. There is a decline in the overall level of FABP as the retina matures, suggesting a role for FABP in cellular differentiation. However, this pattern is not present in the chick brain, indicating a unique function for FABP in the retina. Immunohistochemical staining of paraffin sections of chick retina from embryonic day 21 revealed immunopositive photoreceptor inner segments, outer nuclear layer, radial processes in the inner nuclear layer, a subpopulation of cells in the ganglion cell layer, and inner limiting membrane. This pattern suggested that FABP positive cells were photoreceptors, Müller (glial) cells, and possibly ganglion cells. Staining of sections for glutamine synthetase, an enzyme specific for Müller cells, was similar but not identical to the pattern observed with FABP; thus identification of these cells as FABP-positive was not conclusive. However, in retinal cells dissociated from day E14 embryos and cultured for one week, staining with FABP was more intense in the neurons than in the flat cells (presumed to be derived from the Müller cells). Retinal FABP thus appears to be localized predominantly in neurons, and may serve to sequester fatty acids in preparation for neurite outgrowth as the retinal cells differentiate.Abbreviations FABP Fatty Acid-Binding Protein - PUFA Polyunsaturated Fatty Acid     

Hamster T cells participate in MHC alloimmune reactions but do not effect virus-induced cytotoxic activity

The participation of hamster T cells in a variety of putative MHC-determined reactions was studied utilizing a well-characterized, highly selective goat anti-hamster thymocyte (GHT) serum. Hamster lymphoid cell suspensions treated with GHT lose much of their capacity to induce local graft-versushost reactions and to function as responder cells in mixed lymphocyte reactions. In contrast to the participation of hamster T cells in alloimmune reactions (MLR and GVHR), virus-induced, cytotoxic activity in hamsters undergoing acute virus infection is not T-cell-mediated. This latter finding was rather surprising in view of the major role played by cytotoxic T effector cells in comparably infected mice and rats. These results suggest that, although hamsters are able to respond to putative class II MHC disparities in allogeneic reactions, MHC-encoded molecules, presumably class I, are not utilized for induction of effective cytotoxic activity in response to acute virus infection in this species. The implications of these findings are discussed in relation to our present understanding of the hamster MHC.     

Stimulating axonal regeneration of mature retinal ganglion cells and overcoming inhibitory signaling

Like other neurons of the central nervous system (CNS), retinal ganglion cells (RGCs) are normally unable to regenerate injured axons and instead undergo apoptotic cell death. This regenerative failure leads to lifelong visual deficits after optic nerve damage and is partially attributable to factors located in the inhibitory environment of the forming glial scar and myelin as well as to an insufficient intrinsic ability for axonal regrowth. In addition to its ophthalmological relevance, the optic nerve has long been used as a favorable paradigm for studying regenerative failure in the CNS as a whole. Findings over the last 15 years have shown that, under certain circumstances, mature RGCs can be transformed into an active regenerative state enabling these neurons to survive axotomy and to regenerate axons in the optic nerve. Moreover, combinatorial treatments overcoming the inhibitory environment of the glial scar and optic nerve myelin, together with approaches activating the intrinsic growth program, can further enhance the amount of regeneration in vivo. These findings are encouraging and open the possibility that clinically meaningful regenerationmay become achievable in the future.