Localization of carbonic anhydrase in identified glial cells of the leech central nervous system: application of histochemical and immunocytochemical methods

We localized the enzyme carbonic anhydrase (CA) in frozen sections of the leech (Hirudo medicinalis) central nervous system by two histochemical techniques and the indirect immunofluorescence technique. Hansson's cobalt precipitation method and the use of 1-dimethylamino-naphthalene-5-sulfonamide (DNSA) to build a fluorescent enzyme-substrate complex showed that glial cells are the sites of CA activity in the leech. Neuropil and connective glial cells surrounding the axons had strong CA activity, whereas packet glial cells, which surround neuron cell bodies, and neurons themselves remained unstained. Glial cells reacted markedly with FITC-coupled antibodies against CA isoenzyme II, but experiments with antibodies against CA isoenzyme I showed no reaction.     

Identification of a 70 kD protein with sequence homology to squid neurofilament protein in glial cells of the leech CNS

A monoclonal antibody G39, generated against a protein extract of leech central nervous system, labels specific cell types in adult, embryonic, and regenerating preparations. The antibody stained glial cells, microglial cells, and connective tissue cells, but nor neurons or muscle on cryosections. The staining pattern resembled that of an intracellular network. Affinity purification of the antigen revealed a 70 kD protein. Peptide sequencing showed significant homology of a stretch of 15 amino acids to squid neural filament protein. The same mAb G39 delineated glial cells as they formed during development of the CNS and showed that the giant neuropil glial cells appear before those in the packets. The antigen recognized by mAb G39 represents a nonneuronal intermediate filament of the leech Hirudo medicinalis found in various cell-types such as glia, microglia, and some cells of the connective tissue. 1994 John Wiley & Sons, Inc.     

Carbohydrate-Binding Proteins in the Leech: II. Lactose-Binding Protein LL35 Is Located to Neuronal and Muscle Subsets and All Epithelial Cells

Abstract: Leech lectin 35 (LL35) is a calcium-independent galactoside-binding protein with a molecular mass of 35 kDa and binding properties similar to those of calcium-independent, galactose-specific lectins found in vertebrates, sponges, and nematodes. LL35 was initially isolated from membranes of the leech CNS; however, large amounts of this lectin were also extracted from the rest of the leech. Using affinity-purified antibodies to LL35, we report the immunocytochemical localization of LL35 in adult and embryonic leech. LL35 is developmentally regulated in epithelial, neuronal, and muscle tissue but is absent from glia. During embryogenesis, LL35 is highly expressed by a subset of sensory neurons, weakly expressed in epithelial cells, and absent from muscle. In the adult, LL35 is still present on the same sensory neurons but has become more abundant in epithelial cells lining the CNS and peripheral organs. LL35 also appeared on a muscle cell specifically located in the CNS but remained absent from peripheral muscle. The developmentally regulated distribution of LL35 in epithelial cells, neurons, and CNS muscles suggests a multifunctional role for this lectin with respect to these different cell types.     

Control of neuronal morphology and connectivity: Emerging developmental roles for gap junctional proteins

Recent evidence indicates that gap junction (GJ) proteins can play a critical role in controlling neuronal connectivity as well as cell morphology in the developing nervous system. GJ proteins may function analogously to cell adhesion molecules, mediating cellular recognition and selective neurite adhesion. Moreover, during synaptogenesis electrical synapses often herald the later establishment of chemical synapses, and thus may help facilitate activity-dependent sculpting of synaptic terminals. Recent findings suggest that the morphology and connectivity of embryonic leech neurons are fundamentally organized by the type and perhaps location of the GJ proteins they express. For example, ectopic expression in embryonic leech neurons of certain innexins that define small GJ-linked networks of cells leads to the novel coupling of the expressing cell into that network. Moreover, gap junctions appear to mediate interactions among homologous neurons that modulate process outgrowth and stability. We propose that the selective formation of GJs between developing neurons and perhaps glial cells in the CNS helps orchestrate not only cellular synaptic connectivity but also can have a pronounced effect on the arborization and morphology of those cells involved.     

Localization of the myomodulin-like immunoreactivity in the leech CNS

The distribution of myomodulinlike immunoreactivity in the leech CNS was determined using an antiserum raised against Aplysia myomodulin. Segmental ganglia contained approximately 60 immunoreactive neurons. In addition, numerous fibers containing immunoreactive varicosities were found throughout the neuropil. Using a combination of Lucifer Yellow injections and immunocytochemistry, we identified neurons including the anterior Pagodas (AP), annulus erector (AE), motor neurons, Leydig, longitudinal muscle motoneurons (L), S cells, and coupling interneurons, all of which are active during the touch-elicited shortening reflex. FMRF-amide-like immunoreactivity in three of these cells (L, AP, and AE) was previously demonstrated. Specific staining for myomodulin was abolished by preadsorption of the antiserum with synthetic myomodulin, but not with FMRF-amide. These results suggest a potential role for myomodulin in both intrinsic and extrinsic modulation of the leech touch-elicited shortening reflex. Further, it is possible that several neurons mediating this reflex contain multiple neuromodulatory peptides. © 1996 John Wiley & Sons, Inc.     

Heterotypic binding between neuronal membrane vesicles and glial cells is mediated by a specific cell adhesion molecule

By means of a multistage quantitative assay, we have identified a new kind of cell adhesion molecule (CAM) on neuronal cells of the chick embryo that is involved in their adhesion to glial cells. The assay used to identify the binding component (which we name neuron-glia CAM or Ng-CAM) was designed to distinguish between homotypic binding (e.g., neuron to neuron) and heterotypic binding (e.g., neuron to glia). This distinction was essential because a single neuron might simultaneously carry different CAMs separately mediating each of these interactions. The adhesion of neuronal cells to glial cells in vitro was previously found to be inhibited by Fab' fragments prepared from antisera against neuronal membranes but not by Fab' fragments against N-CAM, the neural cell adhesion molecule. This suggested that neuron-glia adhesion is mediated by specific cell surface molecules different from previously isolated CAMs . To verify that this was the case, neuronal membrane vesicles were labeled internally with 6-carboxyfluorescein and externally with 125I-labeled antibodies to N-CAM to block their homotypic binding. Labeled vesicles bound to glial cells but not to fibroblasts during a 30-min incubation period. The specific binding of the neuronal vesicles to glial cells was measured by fluorescence microscopy and gamma spectroscopy of the 125I label. Binding increased with increasing concentrations of both glial cells and neuronal vesicles. Fab' fragments prepared from anti-neuronal membrane sera that inhibited binding between neurons and glial cells were also found to inhibit neuronal vesicle binding to glial cells. The inhibitory activity of the Fab' fragments was depleted by preincubation with neuronal cells but not with glial cells. Trypsin treatment of neuronal membrane vesicles released material that neutralized Fab' fragment inhibition; after chromatography, neutralizing activity was enriched 50- fold. This fraction was injected into mice to produce monoclonal antibodies; an antibody was obtained that interacted with neurons, inhibited binding of neuronal membrane vesicles to glial cells, and recognized an Mr = 135,000 band in immunoblots of embryonic chick brain membranes. These results suggest that this molecule is present on the surfaces of neurons and that it directly or indirectly mediates adhesion between neurons and glial cells. Because the monoclonal antibody as well as the original polyspecific antibodies that were active in the assay did not bind to glial cells, we infer that neuron- glial interaction is heterophilic, i.e., it occurs between Ng-CAM on neurons and an as yet unidentified CAM present on glial cells.     

Effects of Increased Extracellular K on the Elemental Composition and Water Content of Neuron and Glial Cells in Leech CNS

Elemental (Na, Cl, K) and water contents of leech (Macrobdella decora) neurons and glial cells were determined under steady-state exposure to 4, 10, and 20 mM KCl concentrations (bathing media) using x-ray microanalysis for quantitative digital imaging of frozen hydrated and dried cryosections. Effects of furosemide, 5-hydroxytryptamine (5-HT), and ouabain on elemental distribution changes, induced by exposure to 20 mM K, were also determined. Results demonstrated that packet glial cells and neurons accumulated substantial amounts of K that appeared evenly distributed throughout the cytoplasm. Cell water content also increased as a function of increased cytoplasmic K so that the net effect was an unchanged wet-weight K concentration (expressed as millimoles per kilogram wet weight). Dry-weight Na and Cl concentration (expressed as millimoles per kilogram dry weight) increased slightly in glial cells; however, because cell water increased, both Na and Cl (wet-weight) concentrations decreased. Neurons, in contrast, had no significant change in either Na or K on a wet-weight basis, so a relatively constant Na/K ratio was maintained despite a small, but significant, increase in K (dry weight) and cell water. These increases, like those in packet glia, were a function of exposure to different concentrations of extracellular space K. These changes were completely abolished by 10(-4) M ouabain. Neither furosemide nor 5-HT appeared to affect neuronal or glial K wet-weight concentrations. These data show that both glial cells and neurons can act as substantial reservoirs for K while maintaining stable K concentrations (by altering cell water content and elemental composition). This process appears to depend on a functioning Na+, K+-ATPase system.     

Proteome modifications of the medicinal leech nervous system under bacterial challenge

Once considered as lacking intrinsic immune mechanisms, the CNS of vertebrates is now known to be capable of mounting its own innate immune response. Interestingly, while invertebrates have been very useful in the interpretation of general vertebrate innate immunity mechanisms, only scarce data are available on the immune response of nervous tissue within this group. This study provides new data on the innate immune response of medicinal leech Hirudo medicinalis CNS. We identified several spots in 2-D gels of leech CNS proteins that showed specific changes following bacterial challenge, thus demonstrating the ability of the leech nervous system to mount a response to an immune stress. Protein identifications were based on comparison of sequence data with publicly available databases and a recently established leech ESTs database. The broad nature of the identified proteins suggests a clear involvement of cytoskeletal rearrangements, endoplasmic reticulum stress, modulation of synaptic activity and calcium mobilization, all during the first 24 hours of this response. Moreover, several of these proteins are specifically expressed in glial cells, suggesting an important role for glial cells in the immune response of the leech nervous system, similar to what has been observed in vertebrates.     

Synemin Isoforms in Astroglial and Neuronal Cells from Human Central Nervous System

The intermediate filament (IF) synemin gene encodes three IF proteins (H 180, M 150, L 41 kDa) with overlapping distributions. Synemin M was present early with vimentin and nestin. Synemin H was found later in the nervous system and mesodermic derivatives concomitantly with angiogenesis and the migration of neural crest cells. Synemin L appeared later in neurons. A series of in vitro cell cultures were done to identify the linkage between synemin isoforms and specific cell types of the central nervous system (CNS). The neurons and glia from the brains of humans and rats were cultured and double immunostaining done with antibodies against the H/M or L synemin isoforms and neural cell types (βIII-tubulin or NeuN) or astrocyte intermediate filaments (GFAP or vimentin). In neurons of the CNS, synemin H/M were co-expressed with GFAP, vimentin or nestin in glial cells, whereas synemin L was found in neurons.     

Antigenic and functional characterization of a rat central nervous system-derived cell line immortalized by a retroviral vector

We have immortalized rat central nervous system (CNS) cells of primary cultures of rat optic nerve with murine leukemia virus psi-2,SV-40-6, which is defective in assembly and contains the SV-40 large T antigen and neomycin resistance genes, to produce a cell line that we named A7. After drug selection, greater than 90% of the growing cells expressed nuclear SV-40 large T cells and a fraction of these contained the astrocyte-specific marker, glial fibrillary acidic protein. The majority of these cells also expressed surface marker A4 (specific for neural tube derivatives), Ran 2, p185 (the 185-kD phosphoprotein product of the neu oncogene), and fibronectin, but did not express the astrocyte enzymes glutamine synthetase and monoamine oxidase B. Surface markers characteristic of glial progenitors (A2B5) and oligodendrocytes (galactocerebroside) were not detected. After two rounds of cell cloning, subclone A7.6-3 expressed Ran 2, fibronectin, and the neural cell adhesion molecule (N-CAM) but not glial fibrillary acidic protein and A4. The A7 cell line and subclones also displayed certain functions of type 1 astrocytes: the conditioned medium of these cells had a potent mitogenic activity for glial progenitor cells which could be neutralized by anti-platelet-derived growth factor antibodies and monolayers of these cells supported the growth of embryonic hypothalamic neurons. We conclude that a retrovirus containing SV-40 large T antigen can immortalize rat CNS cells and that such immortalized glial cells retain at least two important functions of type 1 astrocytes: the ability to secrete platelet-derived growth factor and to support the growth of embryonic CNS neurons. Moreover, such stable immortalized clonal cell lines can be used to study gene regulation in glial cells.     

In vitro neurite extension by granule neurons is dependent upon astroglial-derived fibroblast growth factor

When grown in the absence of astroglial cells, purified mouse cerebellar granule neurons survive less than 36 hr and do not extend neurites. Here we report that low concentrations of basic fibroblast growth factor (bFGF, 1-25 ng/ml) maintained the viability and promoted the differentiation of purified granule neurons. The effect of bFGF on granule cell neurite outgrowth was dose dependent. Neurite outgrowth was stimulated markedly in the presence of 1-25 ng/ml bFGF, but effects were not seen below 1 ng/ml or above 50 ng/ml. When affinity-purified antibodies against bFGF (1-5 micrograms/ml) were added either to purified granule cells or to co-cultures of neurons and astroglial cells, process extension by granule neurons was severely impaired. The inhibition of neurite outgrowth in the presence of anti-bFGF antibodies was reversed by the addition of 25 ng/ml of exogenous bFGF. In addition to neuronotrophic effects, bFGF influenced the rate of growth of the astroglial cells. This result depended on whether the astroglia were grown in isolation from neurons, where low doses of bFGF (10-25 ng) stimulated glial growth, or in coculture with neurons, where much higher doses of bFGF (100-250 ng/ml) were needed for glial mitogenesis. Immunoprecipitation of lysates from 35S-labeled cerebellar astroglial cells with anti-bFGF antibodies revealed a single band after SDS-PAGE at 18,000 Da, the molecular weight of bFGF. These results indicate that glial cells synthesize bFGF and are possibly an endogenous source of bFGF in cerebellar cultures. Thus, astroglial cells synthesize soluble factors needed for neuronal differentiation.     

Immune responses to RNA-virus infections of the CNS

A successful outcome for the host of virus infection of the central nervous system (CNS) requires the elimination of the virus without damage to essential non-renewable cells, such as neurons. As a result, inflammatory responses must be tightly controlled, and many unique mechanisms seem to contribute to this control. In addition to being important causes of human disease, RNA viruses that infect the CNS provide useful models in which to study immune responses in the CNS. Recent work has shown the importance of innate immune responses in the CNS in controlling virus infection. And advances have been made in assessing the relative roles of cytotoxic T cells, antibodies and cytokines in the clearance of viruses from neurons, glial cells and meningeal cells.     

Attachment to an endogenous laminin-like protein initiates sprouting by leech neurons

Leech neurons in culture sprout rapidly when attached to extracts from connective tissue surrounding the nervous system. Laminin-like molecules that promote sprouting have now been isolated from this extracellular matrix. Two mAbs have been prepared that react on immunoblots with a approximately equal to 220- and a approximately equal to 340-kD polypeptide, respectively. These antibodies have been used to purify molecules with cross-shaped structures in the electron microscope. The molecules, of approximately equal to 10(3) kD on nonreducing SDS gels, have subunits of approximately equal to 340, 220, and 160-180 kD. Attachment to the laminin-like molecules was sufficient to initiate sprouting by single isolated leech neurons in defined medium. This demonstrates directly a function for a laminin-related invertebrate protein. The mAbs directed against the approximately equal to 220-kD chains of the laminin-like leech molecule labeled basement membrane extracellular matrix in leech ganglia and nerves. A polyclonal antiserum against the approximately equal to 220-kD polypeptide inhibited neurite outgrowth. Vertebrate laminin did not mediate the sprouting of leech neurons; similarly, the leech molecule was an inert substrate for vertebrate neurons. Although some traits of structure, function, and distribution are conserved between vertebrate laminin and the invertebrate molecule, our results suggest that the functional domains differ.     

Repair of the central nervous system: Lessons from lesions in leeches

In contrast to the limited repair observed in the mammalian central nervous system (CNS), injured neurons in the leech reliably regenerate synapses and restore function with remarkable accuracy at the level of individual neurons. New and recent results reveal important roles for microglial cells and extracellular matrix components, including laminin, in repair. Tissue culture experiments have permitted isolation of neurons and manipulation of their environment, providing insights into the influence of substrate, electrical activity, and other cells, including microglia, on axon growth and synapse formation. The results account for distinctive features of successful repair in the adult leech, where axonal sprouting and target selection can be influenced by unequal competition between neurons. Differences between the formation of connections during embryonic development and repair in the adult include dissimilarities in the roles of glia and microglia in adults and embryos, suggesting that axon growth during regeneration in the CNS is not simply a recapitulation of processes observed during embryonic development. It may be possible in the future to improve mammalian CNS regeneration by recruiting cells whose counterparts in the leech have been identified as instrumental in repair. © 1995 John Wiley & Sons, Inc.     

Distribution and partial characterization of CREB-like immunoreactivity in the medicinal leech Hirudo

The presence and distribution of immunoreactivity to the cyclic AMP response element binding protein (CREB) were determined in the central nervous system (CNS) and in peripheral tissues of the medicinal leech Hirudo. Western blots revealed several CREB-immunoreactive (CREB-IR) bands including one whose molecular weight (43–44 kDa) was similar to mammalian CREB. The 43–44 kDa CREB-like protein was detected in nuclear extracts of the ventral nerve cord and was not observed following preincubation of the primary antiserum with the epitope sequence. CREB-like immunoreactivity was detected in extracts from each of six regions of the leech CNS, and in extracts from leech body wall musculature, crop, intestine, jaw musculature, pharynx, and salivary tissues. Whole mounts of leech ganglia revealed specific CREB-IR in a restricted population of neurons distributed throughout the leech CNS. Apparent homologues to a pair of CREB-IR dorsolateral neurons were observed in most ganglia along the ventral nerve cord. Several CREB-IR neurons exhibited segmental specificity. A number of neurons stained with an antiserum to the cyclic AMP response element modulator (CREM). These neurons showed no overlap in location with CREB-IR neurons, and this staining was not eliminated with a preabsorption control. Possible roles for a CREB-like protein in the leech are discussed. Electronic Publication     

Selective interaction of peripheral and central nervous system cells with two distinct cell-binding domains of fibronectin

Mechanisms of cell interaction with fibronectin have been studied with proteolytic fibronectin fragments that have well-defined ligand binding properties. Results of a previous study (Rogers, S. L., J. B. McCarthy, S. L. Palm, L. T. Furcht, and P. C. Letourneau, 1985, J. Neurosci., 5:369-378) demonstrated that (a) central (CNS) and peripheral (PNS) nervous system neurons adhere to, and extend neurites on a 33-kD carboxyl terminal fibronectin fragment that also binds heparin, and (b) neurons from the PNS, but not the CNS, have stable interactions with a 75-kD cell-binding fragment and with intact fibronectin. In the present study domain-specific reagents were used in inhibition assays to further differentiate cell surface interactions with the two fibronectin domains, and to define the significance of these domains to cell interactions with the intact fibronectin molecule. These reagents are (a) a soluble synthetic tetrapeptide Arg-Gly-Asp-Ser (RGDS; Pierschbacher, M. D., and E. Ruoslahti, 1984, Nature (Lond.), 309:30-33) representing a cell-binding determinant in the 75-kD fragment, and (b) an antibody raised against the 33-kD fragment that binds specifically to that fragment. Initial cell attachment to, and neurite extension upon, fibronectin and the two different fragments was evaluated in the presence and absence of the two reagents. Attachment of both PNS and CNS cells to intact fibronectin was reduced in the presence of RGDS, the former more so than the latter. In contrast, the antibody to the 33-kD fragment did not affect attachment of PNS cells to fibronectin, but significantly decreased attachment of CNS cells to the molecule. RGDS inhibited attachment of CNS cells to the molecule. RGDS inhibited attachment of both cell types to the 75-kD fragment to a greater degree than it did attachment to the intact molecule. Cell interaction with the 33-kD fragment was not affected by RGDS. Reduction of neurite lengths (determined after 24 h of culture) by the domain-specific reagents paralleled the reduction in initial adhesion to each substratum. Therefore, it appears that (a) both PNS and CNS cells have receptors for each cell-binding domain of fibronectin, (b) the receptor(s) for the two domains are distinct, with attachment to the 33-kD fragment being independent of RGDS, and (c) the relative importance of each domain to cell interaction with intact fibronectin is different for CNS and PNS cells.     

Immunohistochemical demonstration of proteinase inhibitor alpha-1-antichymotrypsin in normal human central nervous system

The presence of alpha-1-antichymotrypsin, a serine proteinase inhibitor with a high affinity for cathepsin G, is demonstrated in the normal human central nervous system (CNS) by immunohistochemical techniques. Paraffin-embedded normal human CNS tissue from five adult, two fetal, one neonatal and three newborn autopsies were stained with monospecific rabbit antibodies to human alpha-1-antichymotrypsin using biotinylated goat anti-rabbit antibodies and an avidinbiotin-peroxidase complex. Positive immunostaining was seen in neurons and glial cells in the cerebral cortex, basal ganglia, hippocampus, cerebellum, brainstem, and spinal cord of the adults. The epithelium of the adult choroid plexus had the most intense staining in apical granular organelles corresponding in position to lysosomes or secretory granules. Ependymal cells, particularly those near the choroid plexus, were immunostained. The fetal CNS had no alpha-1-antichymotrypsin staining. Limited staining of choroid plexus, ependyma, and frontal lobe was found in the newborns. Immunostaining in the neonatal temporal lobe was only found in the choroid-plexus epithelium. These observations establish a widespread distribution of this proteinase inhibitor in the normal human CNS. Developmental regulation of this inhibitor in the human CNS is also indicated.     

Involvement of low-density lipoprotein receptor-related protein and ABCG1 in stimulation of axonal extension by apoE-containing lipoproteins

Apolipoprotein E (apoE)-containing lipoproteins (LpE) are produced by glial cells in the central nervous system (CNS). When LpE are supplied to distal axons, but not cell bodies, of CNS neurons (retinal ganglion cells) the rate of axonal extension is increased. In this study we have investigated the molecular requirements underlying the stimulatory effect of LpE on axonal extension. We show that enhancement of axonal growth by LpE requires the presence of the low-density lipoprotein receptor-related protein-1 (LRP1) in neurons since RNA silencing of LRP1 in neurons, or antibodies directed against LRP, suppressed the LpE-induced axonal extension. In contrast, an alternative LRP1 ligand, α2-macroglobulin, failed to stimulate axonal extension, suggesting that LpE do not exert their growth-stimulatory effect solely by activation of a LRP1-mediated signaling pathway. In addition, although apoE3-containing LpE enhanced axonal extension, apoE4-containing LpE did not. Over-expression of ABCG1 in rat cortical glial cells resulted in production of LpE that increased the rate of axonal extension to a greater extent than did expression of an inactive, mutant form of ABGC1. Furthermore, reconstituted lipoprotein particles containing apoE3, phosphatidylcholine and sphingomyelin, but not cholesterol, stimulated axonal extension, suggesting that sphingomyelin, but not cholesterol, is involved in the stimulatory effect of LpE. These observations demonstrate that LpE and LRP1 promote axonal extension, and suggest that lipids exported to LpE by ABCG1 are important for the enhancement of axonal extension mediated by LpE.     

Immunohistochemical localization of S-100 protein subunits (alpha and beta) in dorsal root ganglia of the rat

The distribution of S-100 protein and their subunits (alpha and beta) in lumbar dorsal root ganglia of adult rat was investigated immunohistochemically using monoclonal antibodies against the S-100 protein, alpha-subunit and beta-subunit of S-100 protein. The conventional S-100 protein antibody stained both neurons (large and intermediate in size; 20.3% and 41 +/- 3.2 microns of diameter) and glial cells (satellite cells and Schwann cells). The immunoreaction for the alpha-subunit was observed in the perikarya of some large and intermediate sized neurons (17.2%, 45.6 +/- 6.1 microns of diameter), satellite cells and Schwann cells, whereas the beta-subunit immunoreactivity was found principally in glial cells, and in a scarce number of large and intermediate sized neurons (2.8%, 43.3 +/- 5 microns of diameter) Our results demonstrate that a subpopulation of large and intermediate sized neurons of lumbar DRG contain alpha- and beta-subunits of S-100 protein, being alpha-subunit predominant. Furthermore, the satellite glial and Schwann cells contain also the two subunits but mainly beta-subunit. These data confirm previous studies about the presence of S-100 protein in neurons of the central and peripheral nervous system.