Weaver granule neurons are rescued by calcium channel antagonists and antibodies against a neurite outgrowth domain of the B2 chain of laminin.

The weaver mutation impairs migration of the cerebellar granular neurons and induces neuronal death during the first two weeks of postnatal life. To elucidate the molecular mechanisms for the impaired neuronal migration, we investigated the rescue mechanisms of the weaver (wv/wv) granule neurons in vitro. We found that Fab2 fragments of antibodies against a neurite outgrowth domain of the B2 chain of laminin enhanced neurite outgrowth and neuronal migration of the weaver granule neurons on a laminin substratum and in the established cable culture system. The rescue of the weaver granule neurons by antibodies against the B2 chain of laminin may result from the neutralizing effect of these antibodies against the elevated B2 chain levels of the weaver brain. The L-type calcium channel blocker, verapamil (1-5 microM), also rescued the weaver granule neurons. High concentrations of MK-801 (10-20 microM), a glutamate receptor antagonist and voltage-gated calcium channel blocker, rescued the weaver granule neurons similar to verapamil, but low concentrations of MK-801 (1 microM) had no rescue effect. Simultaneous patch-clamp studies indicated that the weaver granule neurons did not express functional N-methyl-D-aspartate receptors further indicating that the rescue of the weaver granule neurons by MK-801 resulted from its known inhibition of voltage-gated calcium channels. The present results indicate that antibodies against the B2 chain of laminin, verapamil, and high concentrations of MK-801 protect the weaver granule neurons from the otherwise destructive action of the weaver gene. Thus, both the laminin system and calcium channel function contribute to the migration deficiency of the weaver granule neurons.

Laminin is produced by early rat astrocytes in primary culture.

The production of laminin by early rat astrocytes in primary culture was investigated by double immunofluorescence staining for laminin and the glial fibrillary acidic protein (GFAP), a defined astrocyte marker. In early cultures (3 d in vitro; 3 DIV) cytoplasmic laminin was detected in all the GFAP-positive cells which formed the major population (80%) of the nonneuronal cells present in cultures from 20-21-d embryonic, newborn, or 5-d-old rat brains. Monensin treatment (10 microM, 4 h) resulted in accumulation of laminin in the Golgi region, located using labeled wheat germ agglutinin. Laminin started gradually to disappear from the cells with the time in culture, was absent in star-shaped, apparently mature astrocytes, but remained as pericellular matrix deposits. The disappearance of cellular laminin was dependent on the age of the animal and the time in culture so that it started earlier in cultures from 5-d-old rat brains (5 DIV) and approximately following the in vivo age difference in cultures from newborn (12 DIV) and embryonic (14 DIV) rat brains. Our results indicate that laminin is a protein of early astrocytes and also deposited by them in primary culture, thus suggesting a role for this glycoprotein in the development of the central nervous system.

Identification of a neurite outgrowth-promoting domain of laminin using synthetic peptides.

We have identified a synthetic peptide derived from the B2-chain of mouse laminin, Arg-Asn-Ile-Ala-Glu-Ile-Ile-Lys-Asp-Ile (p20), which stimulates the neurite outgrowth-promoting activity of the native molecule. In organotypic cultures, neurons from newborn mouse brain or embryonic peripheral nervous system responded by extensive neurite outgrowth for native laminin or the peptide p20 in the culture medium. If rat cerebellar neurons were grown on laminin, 1-5 microM (1-5 micrograms/ml) of peptide p20 in the culture medium competed with laminin and inhibited neuronal attachment and neurite outgrowth, whereas higher concentrations (greater than 50 microM; greater than 50 micrograms/ml) had a specific neurotoxic effect. When peptide p20 was used as the culture substratum, neurite outgrowth in cerebellar cultures was up to 60% of that seen on native laminin. Our results indicate that a neurite outgrowth-promoting domain of laminin is located in the alpha-helical region of the B2-chain, and is active for both central and peripheral neurons.

Laminin in cultured mouse C1300 neuroblastoma cells: immunocytochemical localization by pre- and postembedding electron microscope procedures.

Laminin was localized in cultured mouse C1300 neuroblastoma cells by applying the peroxidase-antiperoxidase technique in preembedding electron microscopy. The results were compared to those obtained by indirect immunofluorescence and by the colloidal gold second antibody method on Epon-embedded ultrathin sections. Laminin was found in the cell membranes and within the rough endoplasmic reticulum as well as in intracytoplasmic vacuoles. Plasma membranes of the neuroblastoma cells showed a patchy localization of laminin that was apparently involved in cell-to-substrate attachment and in gap junction-like intercellular connections. Under normal conditions, the Golgi cisternae contained no laminin. Pretreatment of cells with micromolar concentrations of monensin, however, lead to an accumulation of laminin within the Golgi cisternae. These results support a role for laminin as an adhesion protein in cultured neuroblastoma cells and indicate that laminin is transported through the Golgi complex.

Specific detection of neuronal cell bodies: in situ hybridization with a biotin-labeled neurofilament cDNA probe.

We have used a biotinylated, 300-nucleotide cDNA probe which encodes the 68,000 MW neurofilament protein to detect neurofilament-specific mRNA in situ. The neurofilament message specifically demonstrates the neuronal cell bodies, in contrast to the usual antibody staining which detects their neurites. The hybridization is detected only in neuronal structures. Consequently, detection of the biotinylated neurofilament DNA probe by silver-intensified streptavidin-gold can be specifically used to identify neuronal cell bodies.

Involvement of non-NMDA receptors in the rescue of weaver cerebellar granule neurons and sensitivity to ethanol of cerebellar AMPA receptors in oocytes.

The cellular mechanism responsible for the death of cerebellar granule neurons in the weaver mutant mouse is still being intensely investigated. To determine if alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors are involved in producing the weaver phenotype or are altered by the weaver gene, we used (1) reverse transcription and polymerase chain reaction (RT-PCR) to detect transcripts of glutamate receptors (GluR1-4) from wild-type and mutant cerebella; (2) immunocytochemistry to establish the types of glutamate receptors present in granule neurons cultured from normal and homozygous weaver postnatal day 5-6 (P5-6) cerebella; (3) 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a blocker of glutamate (AMPA/Kainate/NMDA) receptors, and 6,7-dinitroquinoxaline-2,3-dione (NBQX), a blocker of AMPA and kainate receptors, to assess the number of neurons and the number of neurons with long neurites in cultures of homozygous weaver granule neurons; (4) two-electrode voltage clamp recordings to study AMPA glutamate receptor expression in Xenopus oocytes after injection of mRNA isolated from cerebella of normal and weaver P5-6, postnatal day 10 (P10) and postnatal day 23 (P23) mice; and (5) ethanol, which at low 1-10 mM concentrations had been shown previously to rescue homozygous weaver granule neurons in culture [Liesi et al., J. Neurosci. Res. 48 (1997) 571-579], to examine its effect on modulation of AMPA receptors expressed from mRNA. By RT-PCR, the mRNA coding for AMPA receptor subunits GluR1-4 were detected from +/+ and wv/wv cerebella, and by immunocytochemistry, GluR1, GluR2/3 and GluR4 were observed to be expressed in cultured +/+ and wv/wv granule cells. CNQX at 10 microM or NBQX at 10 microM significantly increased the number of surviving neurons and the number with long neurites as compared to wv/wv controls. In addition, CNQX was significantly more effective than NBQX. In oocytes injected with mRNA from P10 normal or weaver cerebella, the amplitudes of the responses to kainate were about equal. In contrast, the amplitudes of the kainate-activated currents in oocytes injected with weaver P23 mRNA were about twice as large as the currents observed in oocytes injected with mRNA from normal P23 cerebella, and both were larger than kainate-activated currents observed after injection of P10 normal and weaver mRNA. Kainate-activated AMPA receptor currents in oocytes injected with mRNA from P10 and P23 normal and homozygous weaver cerebella were inhibited by ethanol. There were no significant differences in the inhibition produced by ethanol on currents from P10 or P23 normal and wv/wv mRNA. Thus, P23 weaver cerebellar mRNA expressed more kainate-activated current in oocytes than P23 normal cerebellar mRNA; both normal and weaver cerebellar granule neurons express mRNA coding for functional AMPA receptors that are susceptible to ethanol inhibition.

Use of paper for treatment of a peripheral nerve trauma in the rat.

Reinnervation of the muscles and skin in the rat hindpaw was studied after transection and attempted repair of the sciatic nerve. Reconnecting the transected nerve with lens cleaning paper was at least as effective in rejoining the transected nerves as traditional microsurgical neurorraphy. Paper induced a slightly bigger fibrous scar around the site of transection than neurorraphy, but this scar did not cause impairment of functional recovery or excessive signs of neuropathic pain. We conclude that a paper graft can be used in restorative surgery of severed peripheral nerves.

Chemically modifying glass surfaces to study substratum-guided neurite outgrowth in culture.

We describe here a modification procedure for chemically fabricating neuron adhesive substrates to study the substratum-guided neurite outgrowth in culture. These substrates were fabricated by chemically attaching a synthetic peptide derived from a neurite-out-growth-promoting domain of the B2 chain of laminin. The attachment was carried out by coupling the peptide to an amine-derived glass surface using a heterobifunctional crosslinker. Hippocampal neurons were dissociated from embryonic rats and placed on the substrate at low-density in a chemically defined medium to examine the direct effect of the modified surface on their outgrowth. We observed that the neurons developed a morphology typical to that of hippocampal neurons having multiple short and single long processes within 24 h in culture. The chemical modification procedure was then combined with a UV-photo-masking technique to fabricate patterns of peptide surface on glass substrates. By culturing the hippocampal neurons on substates having alternate stripes of peptide surface and non-adhesive surface, we demonstrated substratum-controlled changes in the neuronal morphology. The modification procedure presented here can be easily achieved in the standard culture facility and should be useful in fabricating an in vitro tool for studying substratum-guided neurite outgrowth.

Serotonin-like immunoreactivity in rat carotid body.

Serotonin-like immunofluorescence was demonstrated in the glomus cells of the rat carotid body. Similar immunoreactivity was noted in mast cells in the organ, while no immunoreactive nerve fibers were seen. It is suggested that glomus cell serotonin could participate in the modulation of chemoreceptor activity.

Neurofilament proteins are constitutively expressed in F9 teratocarcinoma cells.

We examined neuronal differentiation of F9 teratocarcinoma cells using retinoic acid (RA) and cyclic AMP (cAMP) as inducing agents. Neuronal differentiation was monitored using (1) cDNA probes for the rat 68-kDa neurofilament gene, (2) RT-PCR for neurofilament genes and (3) antibodies against several neuronal differentiation markers. We found by Northern blotting that the uninduced F9 cells, grown in 10% serum, expressed mRNA for the 68-kDa neurofilament protein whereas the control cells, grown in 3% serum, failed to express detectable levels of the 68-kDa neurofilament transcripts. However, RT-PCR allowed detection of both the 68- and 200-kDa neurofilament gene transcripts in F9 cells with or without the inducing agents. Under serum deprivation, a prolonged (> 10-15 days) cultivation of the F9 cells in the presence of RA and cAMP was required for the expression of detectable levels of the 68-kDa neurofilament transcripts and immunocytochemically detectable neurofilament proteins. Treatment of the F9 cells with RA and cAMP was also required for induction of their neuronal phenotype. Immunocytochemically, the uninduced F9 cells expressed several neuronal antigens including the 68-kDa neurofilament protein, the 200-kDa neurofilament protein, neural cell adhesion molecule (N-CAM) and a neuronal specific tubulin isoform (TUJI). The control cells expressed N-CAM and TUJI, but failed to express the neurofilament proteins. A subclone, D9L2, derived from a single F9 parent cell, expressed both TUJI and neurofilament proteins, but no N-CAM molecule. The present results indicate that both the 68- and the 200-kDa neurofilament genes are constitutively active in uninduced F9 teratocarcinoma cells. Under serum deprivation both RA and cAMP are required for expression of detectable levels of neurofilament mRNA and protein. Thus, serum deprivation of the F9 cells either down-regulates the NF gene expression, stability of mRNA or degradation of the NF-proteins. Importantly, expression of a neuronal phenotype by a subpopulation of F9 cells appears to require administration of RA and cAMP, although expression of neuronal marker proteins is not dependent on these agents. Lastly, we demonstrate cloning of a novel cell line (D9L2), derived from a single F9 parent cells, capable of extending neurites and expressing several neuronal antigens under serum deprivation without the requirement of RA and cAMP. We propose that the D9L2 cell line may offer a simplified F9 cell model system to investigate the mechanisms of neuronal differentiation.

Directional neurite outgrowth and axonal differentiation of embryonic hippocampal neurons are promoted by a neurite outgrowth domain of the B2-chain of laminin.

Molecular cues involved in directional neurite outgrowth and axonal differentiation of embryonic hippocampal neurons were studied on substrates coated in a striped 5 microns pattern with synthetic peptides from a neurite outgrowth (RDIAEIIKDI, P1543) and cell attachment (CDPGYIGSR, P364) domain of the B2- and B1-chains of laminin, respectively. Both peptides supported neuronal attachment, but only the B2-chain-derived P1543 promoted expression of a mature neuronal phenotype. Directional neurite outgrowth and axonal differentiation of embryonic hippocampal neurons were selectively induced by striped substrates of the B2-chain-derived P1543. Axonal differentiation was determined by expression of a phosphorylated epitope of the 200 kDa neurofilament protein in the longer "axonal" neurite of the bipolar embryonic hippocampal neurons. Ethanol (100 mM), a neuroactive compound known to delay neuronal development, impaired both directional neurite outgrowth and expression of a phosphorylated epitope of the 200 kDa neurofilament protein on a patterned P1543 substratum. The present results provide direct evidence that a 10 amino acid peptide (P1543), derived from a neurite outgrowth domain of the B2-chain of laminin, may be an axonal guidance and differentiation factor for embryonic hippocampal neurons in vitro.

Immunohistochemical demonstration of nociceptors in the ligamentous structures of the lumbar spine.

Substance P, a physiologically potent neuropeptide is known to participate in the sensory, and especially nociceptive, transmission of neural impulses. On histologic grounds, the nerve terminals of the sinuvertebral nerve formerly have been suggested to be sensory in character and to mediate the low-back pain syndrome. Samples of paramedullary ligamentous structures were collected on disc operations. A positive immunoreaction as an indicator of substance P was confirmed in some nerve terminals of the posterior longitudinal ligament. Neither the yellow ligament nor the intervertebral disc showed such nociceptive-type nerves.

The resistance of glyoxylic acid induced catecholamine fluorescence to sodium borohydride reduction.

The borohydride reduction of glyoxylic acid induced fluorescence in noradrenergic and DOPA-minergic nervous structures and in amines in model experiments was studied. Both DOPAmine and noradrenaline fluorescences were resistant to borohydride reduction differing thus from the formaldehyde-induced fluorescence. Thus when the specificity of glyoxylic acid induced fluorescence is in doubt, other tests than borohydride reduction of the fluorescence must be employed.

Neuronal migration on laminin involves neuronal contact formation followed by nuclear movement inside a preformed process.

Neuronal migration was investigated in rodent cerebellum in vitro and in vivo. Time-lapse video recording showed that cultured neurons migrated on laminin by first extending neurites that formed contacts with other neurons. This was followed by movement of the cell nucleus inside the preformed process. No guidance cues other than laminin were required. When the rodent premigratory (E18-P0) cerebellum was examined by immunocytochemistry, the radial glial cells were found to have extracellular punctate deposits of laminin along their fibers. Such punctate deposits of laminin were more numerous in the premigratory cerebellum than during the peak of neuronal migration (e.g., at 7-10 days postnatally). At the same time (E18-P0) L1 antigen- and neurofilament-positive, presumably granule cell processes extend radially from the external granule cell layer (EGL). These results imply that neuronal migration on laminin in vitro involves neuronal contact formation followed by nuclear movement inside a preformed process. That this mode of neuronal migration may occur in vivo is indicated by the fact that L1 antigen- and neurofilament-positive "pioneer neurites" colocalize with the punctate deposits of laminin deposited along the radial glial processes in the premigratory EGL. Taken together these results imply that the established glial dependency of the granule cell migration may in fact be dependency of the granule cells and their pioneer neurites on the punctate deposits of laminin produced and laid down by the glial cells.

Ethanol-exposed central neurons fail to migrate and undergo apoptosis.

Prenatal exposure of human brain to ethanol impairs neuronal migration and differentiation and causes mental retardation. The present results indicate that the adverse effects of ethanol on brain development may be partly due to the ethanol-induced disturbance of neuronal interaction with laminin, a protein involved in neuronal migration and axon guidance. This report shows that physiological concentrations (IC50 = 28 mM) of ethanol inhibit neurite outgrowth and neuronal migration of the rat cerebellar granule neurons on a laminin substratum. The ethanol-treated granule neurons undergo apoptosis, degrade their laminin substratum, and appear to release and bind increased amounts of the B2-chain-derived peptides along their surfaces. A protease inhibitor aprotinin, and the NMDA receptor channel, and voltage-gated calcium channel antagonist MK801 partially protect cerebellar granule neurons from ethanol-induced neurotoxicity. These results imply that ethanol-treated granule neurons resemble the granule neurons of the homozygous weaver mouse cerebellum with respect to their apoptosis, laminin expression, and partial rescue by approtinin and MK-801. Thus, ethanol may influence neuronal survival and neurite outgrowth via molecular pathways similar to those involved in neuronal death in other neurodegenerative processes of the central nervous system.

Do neurons in the vertebrate CNS migrate on laminin?

In adult rat brain the extracellular matrix glycoprotein, laminin, is found only in basement membranes, but is transiently expressed by astrocytes after brain injury. Here, I show that laminin also appears in immature brain cells during CNS development, and that its presence coincides with phases of neuronal migration. In early embryos, laminin is seen throughout the whole thickness of the forming brain, and is apparently synthesized by the cells, as judged by its intracytoplasmic localization. As development proceeds, intracellular laminin becomes restricted to the periventricular regions while punctate deposits of laminin follow the course of vimentin-positive radial glial fibers. In most brain regions, the adult pattern of laminin expression is achieved by birth. In the post-natal rat cerebellum, however, laminin is detected in external granule cells, in Purkinje cells, and in punctate deposits along the radial Bergmann glial fibers. By day 24 after birth, when the migration of external granule cells is complete, all laminin immunoreactivity disappears from these structures. The transient expression of laminin in regions where neurons are migrating raises the possibility that laminin plays a role in neuronal migration during CNS development.

Laminin-immunoreactive glia distinguish regenerative adult CNS systems from non-regenerative ones.

Most regions of the adult mammalian central nervous system (CNS) do not support axonal growth and regeneration. Laminin, expressed by cultured astrocytes and known to promote neurite outgrowth of cultured neurons, is normally present in brain basement membranes, and only transiently induced in adult brain astrocytes by injury. Here I provide three lines of evidence which suggest that the continued expression of laminin by astrocytes may be a prerequisite for axonal growth and regeneration in adult CNS. Firstly, laminin is continuously present in astrocytes of adult rat olfactory bulb apparently in close association with the olfactory nerve axons. Secondly, laminin is continuously expressed by astrocytes in adult frog brain, and sectioning of the optic tract further increases laminin immunoreactivity in astrocytes of the optic tectum during the period of axonal regeneration. Lastly, laminin appears normally in astrocytes of the frog and goldfish optic nerves which regenerate, but not in astrocytes of the rat or chick optic nerves which do not regenerate. The selective association of laminin with axons that undergo growth and regeneration in vivo is consistent with the possibility that astrocytic laminin provides these central nervous systems with their regenerative potential.

Extracellular matrix and neuronal movement.

During brain development, both neuronal migration and axon guidance are influenced by extracellular matrix molecules present in the environment of the migrating neuronal cell bodies and nerve fibers. Glial laminin is an extracellular matrix protein which these early brain cells preferentially attach to. Extracellular glycosaminoglycans are suggested to function in restricting neuronal cell bodies and axons from certain brain areas. Since laminin is deposited along the radial glial fibers and along the developing nerve pathways in punctate form, the punctate assemblies may be one of the key factors in routing the developing neurons in vivo. This review discusses the role of laminin in neuronal movement given the present concept of the extracellular matrix molecules and their proposed interactions.

Laminin and fibronectin in normal and malignant neuroectodermal cells.

Laminin and fibronectin, the major noncollagenous matrix glycoproteins, were studied in connection with normal brain cells and neuroectodermal cell lines. Laminin, a Mr 900,000 dalton matrix glycoprotein and an essential component of basement membranes, was found to be produced by cultured cells of several malignant cell lines of neuroectodermal origin. In cultured mouse C1300 neuroblastoma line cells laminin was localized, by immunoelectron microscopy, to the rough endoplasmic reticulum and, to sites of cell-to-cell and cell-to-substratum adhesion. Further experiments on the intracellular transport of this glycoprotein in C1300 cells confirmed that laminin is, at least partially, transported through the Golgi pathway. These results favor a role for laminin in attachment and cellular interactions of malignant neuronal cells. Laminin was also found in connection with neurons and glial cells from mammalian brain. In primary cultures from developing rat brain the vast majority of non-neuronal cells (80%) expressed immunoreactivity for the glial fibrillary acidic protein, a cytoskeletal protein specific for astrocytes. During the first week in culture all the glial fibrillary acidic protein-positive cells, with the exception of mature-looking star-shaped astrocytes, exhibited immunoreactivity for laminin. The intracellular laminin disappeared gradually after a few weeks in culture, but an extensive laminin matrix persisted and seemed to be localized on the upper surface of the non-neuronal cells. The neurofilament-positive neurons were negative for laminin. Pretreatment of the cultures with the ionophore monensin, caused accumulation of laminin-immunoreactivity within the Golgi region, which confirmed that laminin is, indeed, produced by cultured astrocytes and secreted through the Golgi complex. No fibronectin immunoreactivity was found in the majority of glial cells. However, under culture conditions where fibronectin was omitted from the culture medium there was, in the primary cultures, a minor population of glial fibrillary acidic protein-positive flat glial cells that exhibited intracytoplasmic immunofluorescence for fibronectin. In the presence of fibronectin in culture medium no fibronectin-positive glial cells could be detected. It thus appears that laminin, and to a minor extent fibronectin, are proteins that normal glial cells are capable of producing under specific conditions. Laminin and fibronectin were localized in adult rat brain in capillary and meningeal structures.(ABSTRACT TRUNCATED AT 400 WORDS)