Soluble KDI domain of gamma1 laminin protects adult hippocampus from excitotoxicity of kainic acid

Recent data indicate that the soluble KDI domain of gamma1 laminin promotes survival and neurite outgrowth of human central neurons in vitro (Liebkind et al.[2003] J Neurosci Res 73:637-643), and seems to neutralize both glia- and myelin-derived signals that hamper regeneration in the central nervous system (CNS) of adult mammals. We show that damage of adult rat neocortical and hippocampal areas by a stereotaxic injection of kainic acid (KA) is prevented by a preceding injection of the soluble KDI domain. In the presence of the KDI domain, both neocortical and hippocampal areas show extensive gliosis but have viable neurons and glial cells, which are absent and the areas fully destroyed after injection of KA alone. This result indicates that the KDI domain of the gamma1 laminin protects the CNS against excitotoxic insults and promotes survival of both neurons and glial cells. The KDI domain may thus be a potential drug to prevent CNS damage induced by neurodegenerative disorders, mechanical injury, or ischemia.     

Regeneration of adult rat spinal cord is promoted by the soluble KDI domain of gamma1 laminin

Regeneration in the central nervous system (CNS) of adult mammals is hampered by formation of a glial scar and by proteins released from the myelin sheaths of injured neuronal pathways. Our recent data indicate that the KDI (Lys-Asp-Ile) domain of gamma1 laminin neutralizes both glial- and myelin-derived inhibitory signals and promotes survival and neurite outgrowth of cultured human spinal cord neurons. We show that after complete transection of the adult rat spinal cord, animals receiving onsite infusion of the KDI domain via osmotic mini-pumps recover and are able to sustain their body weights and walk with their hindlimbs. Animals treated with placebo suffer from irreversible hindlimb paralysis. Microscopic and molecular analyses of the spinal cords indicate that the KDI domain reduces tissue damage at the lesion site and enables neurite outgrowth through the injured area to effect functional recovery of the initially paralyzed animals. That the KDI domain enhances regeneration of acute spinal cord injuries in the adult rat suggests that it may be used to promote regeneration of spinal cord injuries in humans.     

Interaction between serine phosphorylated IRS-1 and beta1-integrin affects the stability of neuronal processes

Tumor necrosis factor-alpha (TNFalpha) released in the brain by HIV-activated macrophages/microglia is suspected to compromise neuronal survival. Previously, we have demonstrated that activated receptor for insulin-like growth factor I (IGF-IR) protects neurons from TNFalpha-induced neuronal damage (Wang et al. [ 2006] J. Neurosci. Res. 83:7-18). Because TNFalpha triggers phosphorylation of insulin receptor substrate 1 (IRS-1) on serine residues (pS-IRS-1; Rui et al. [ 2001] J. Clin. Invest. 107:181-189), and pS-IRS-1 binds integrins (Reiss et al. [ 2001] Oncogene 20:490-500), we asked how these events affect neuronal processes. We show that beta1-integrin and pS-IRS-1 colocalize in PC12 cells and in primary cortical neurons. TNFalpha treatment elevated membrane-associated pS-IRS-1, enhanced pS-IRS-1 interaction with beta1-integrin, and attenuated cell attachment to collagen IV. In contrast, IGF-I inhibited pS-IRS-1-beta1-integrin complexes and improved cell attachment. The domain of IRS-1 involved in beta1-integrin binding mapped between amino acids 426 and 740, and the expression of 426-740/IRS-1 mutant attenuated neuronal outgrowth. Our results indicate that TNFalpha facilitates the interaction of pS-IRS-1 and beta1-integrin and destabilizes neuronal processes. IGF-I counteracts TNFalpha-mediated accumulation of pS-IRS-1-beta1-integrin complexes supporting the stability of neuronal processes.     

Estrogen and cognition: Applying preclinical findings to clinical perspectives

Regeneration of adult mammalian CNS is poor as a result of environmental factors that prevent axon growth. The major factors hampering regeneration of central axons include proteins released from the damaged myelin sheets of the injured neuronal pathways and formation of the glial scar. By using an experimental model of human CNS injury, we show that survival and neurite outgrowth of human central neurons are significantly enhanced by the soluble KDI domain of gamma1 laminin. Our results indicate that the KDI domain appears to neutralize both glia-derived inhibitory signals and inhibitory molecules released from the myelin of the adult human spinal cord. We propose that the KDI domain may enhance regeneration of injuries in the adult mammalian CNS.     

Laminin-like proteins are differentially regulated during cerebellar development and stimulate granule cell neurite outgrowth in vitro

The basement membrane glycoprotein laminin-1 is a potent stimulator of neurite outgrowth. Although a variety of laminin isoforms have been described in recent years, the role of alternative laminin isoforms in neural development remains largely uncharacterized. We found that a polyclonal antibody raised against the α1, β1, and γ1 chains of laminin-1 and a monoclonal antibody raised against the α2 chain of laminin-2 detect immunoreactive material in neuronal cell bodies in the developing mouse cerebellum. In addition, laminin-1-like immunoreactivity was found in cell types throughout the cerebellum, but laminin-α2-like immunoreactivity was restricted to the Purkinje cells. Purified laminin-1 and laminin-2 stimulated neurite outgrowth in primary cultures of mouse cerebellar granule neurons to a similar extent, whereas the synthetic peptides tested appeared to be active only for cell adhesion and not for stimulation of neurite outgrowth. The E8 proteolytic fragment of laminin-1 contained full neurite outgrowth activity. The identity of laminins expressed in granule neurons was also examined by Western blotting; laminin-like complexes were associated with the cell and appeared to have novel compositions. These results suggest that laminin-like complexes play important roles in cerebellar development. J. Neurosci. Res. 54:233–247, 1998. © 1998 Wiley-Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.     

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. J. Neurosci. Res. 48:439–448, 1997. © 1997 Wiley-Liss, Inc.     

GABAergic currents in RT and VB thalamic nuclei follow kinetic pattern of alpha3- and alpha1-subunit-containing GABAA receptors

Inhibitory postsynaptic currents (IPSCs) of the thalamic reticular (RT) nucleus are dramatically slower than in the neighboring ventrobasal (VB) neurons. It has been suggested that alpha3-subunit-containing receptors underlie slow IPSCs in RT neurons, while rapid synaptic currents in the VB nucleus are due to gamma-aminobutyric acid A receptors (GABAARs), including the alpha1-subunit. In our recent study [Barberis et al. (2007) Eur. J. Neurosci., 25, 2726-2740] we have found that profound differences in kinetics of currents mediated by alpha3beta2gamma2 and alpha1beta2gamma3 receptors resulted from distinct binding and desensitization properties. However, a direct comparison between kinetics of neuronal GABAARs from RT and VB neurons and alpha3- and alpha1-subunit-containing receptors has not been made. For this purpose, current responses to ultrafast GABA applications were recorded from patches excised from neurons in VB and RT areas. Deactivation kinetics determined for RT and VB neurons closely resembled that in currents mediated by alpha3beta2gamma2 and alpha1beta2gamma2 receptors. In RT neurons, currents elicited by non-saturating [GABA] had a remarkably slow onset, a hallmark of alpha3-subunit-containing receptors. In VB and RT neurons, single-channel currents elicited by brief GABA pulses had similar characteristics to those of alpha1beta2gamma2 and alpha3beta2gamma2 receptors. However, in stationary conditions, similarity between single-channel currents in neurons and respective recombinant receptors was less apparent. We propose that the non-stationary kinetics of GABAergic currents in VB and RT nuclei mimic that of currents mediated by alpha1- and alpha3-subunit-containing receptors. The dissimilarity between stationary kinetics of neuronal and recombinant receptors probably reflects differences between GABAARs mediating phasic and tonic currents in these neurons.     

Gamma 1 laminin and its biologically active KDI-domain may guide axons in the floor plate of human embryonic spinal cord

Immunocytochemistry, in situ hybridization and Matrigel-embedded cultures were used to investigate the distribution of laminins during development of the human embryonic spinal cord (7-11 weeks). Our results indicate that alpha 1, beta 1, beta 3 and gamma 1 laminins localize as punctate deposits in the floor plate region in association with commissural fibers crossing the ventral midline. In addition, the neurite outgrowth domain of gamma 1 laminin accumulates heavily in the floor plate region, in the notochord and in GFAP-immunoreactive glial fibers of the embryonic spinal cord. In culture experiments, the biologically active KDI-domain of gamma 1 laminin selectively attracted directional outgrowth of neurites from explants of the dorsal spinal cord. The spatial and temporal colocalization of punctate deposits of laminins with nerve fibers crossing the ventral midline, and the guidance of neurites by the KDI-peptide domain, indicate that laminins, specifically the gamma 1 laminin, may be involved in guidance of axons during embryonic development of the human spinal cord.     

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 μm 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. Published by Elsevier Science Ltd.     

Selective overexpression of gamma1 laminin in astrocytes in amyotrophic lateral sclerosis indicates an involvement in ALS pathology

Our earlier studies indicate that the KDI tripeptide of gamma1 laminin reverts paralysis and protects adult rat CNS from excitotoxicity of glutamate and from oxidative stress. Here we show that gamma1 laminin is selectively overexpressed in reactive astrocytes of the amyotrophic lateral sclerosis (ALS) spinal cord, with both gray and white matter astrocytes overexpressing gamma1 laminin. Intensely gamma1 laminin-positive, aggressive-looking reactive astrocytes of the lateral columns of both cervical and thoracic spinal cord surround the lateral ventral horns and roots and extend into the area of the lateral corticospinal tract. In the cervical ALS spinal cord, large numbers of strongly gamma1 laminin-immunoreactive astrocytes are also present in the dorsal columns of the ascending sensory pathways. No other laminin or any other ALS-associated protein localizes in this manner. This unique distribution of gamma1 laminin-immunoreactive astrocytes in the ALS white matter together with our recent results on the efficacy of the KDI domain as a neuronal protector strongly suggest that gamma1 laminin may be expressed by astrocytes of the ALS spinal cord as a protective measure intended to aid neuronal survival. Further comparative studies on ALS spinal cord tissues and those of the animal models of ALS are needed to clarify the specific role of gamma1 laminin and its KDI domain in ALS and its putative interactions with the additional ALS-associated factors, such as excitotoxicity, oxidative stress, and neurofilament accumulation. Most importantly, further studies are urgently needed to test the potential of the KDI tripeptide as a therapeutic treatment for ALS.     

VASE-encoded peptide modifies NCAM- and L1-mediated neurite outgrowth

Interactions between the neural cell adhesion molecule (NCAM) with NCAM-expressing neurons (trans-interaction) stimulate outgrowth of neurites. The extent of NCAM-triggered neurite outgrowth depends on the presence of 10 amino acids derived from the variable alternatively spliced exon (VASE or π-exon) in the fourth immunoglobulin-like domain of NCAM (Ig4): NCAM with VASE reduces and without VASE enhances neurite outgrowth in cis- or trans-interaction. We have investigated the role of VASE in neurite outgrowth by characterizing the receptors at the cell surface of cultured cerebellar neurons. Results from experiments with L1 and NCAM antibodies and with cerebellar neurons derived from wild-type or NCAM-deficient mice show that substrate-coated Ig4 with VASE (Ig4+) or without VASE (Ig4−) stimulates neurite outgrowth by a trans-interaction with L1 and that Ig4− promotes neurite outgrowth more strongly than Ig4+ by a transinteraction with NCAM. J. Neurosci. Res. 50:62–68, 1997. © 1997 Wiley-Liss, Inc.     

Neuronal migration in cerebellar microcultures is inhibited by antibodies against a neurite outgrowth domain of laminin.

The functional role of laminin in neuronal migration was investigated by using polyclonal antibodies or their divalent (Fab')2 fragments to a neurite outgrowth promoting domain of the B2 chain of laminin in a cerebellar microculture system widely recognized as a model for neuronal migration. We show here that these antibodies or their (Fab')2 fragments totally inhibit migration of the mouse cerebellar granule cells along the glial and other neuronal cell processes. Antibodies to native laminin or other control antibodies have no inhibitory effect. Immunocytochemical analysis of the cerebellar microcultures indicates that the functional role of these antibodies may relate to the fact that the punctate deposits of laminin and its neurite outgrowth promoting domain accumulate in between the migrating neurons and the glial cells. These data provide the first direct evidence for the functional role of laminin and its neurite outgrowth domain in neuronal migration in the mammals. They further suggest that a neuronal cell surface contact with the extracellular deposits of a neurite outgrowth domain of the B2 chain of laminin may mediate neuronal-glial interactions.     

Differential distribution of laminins in Alzheimer disease and normal human brain tissue

Immunocytochemistry, Western blotting, and RT-PCR were used to identify the isoforms of laminin expressed in the Alzheimer disease, but not in normal human brain tissue. We found that alpha 1 laminin was heavily over-expressed in Alzheimer disease frontal cortex, and localized in reactive astrocytes of the grey and white matter, and as punctate deposits in the senile placques of the Alzheimer brain tissue. Antibodies against the C-terminal neurite outgrowth domain of the gamma 1 laminin demonstrated expression of the gamma 1 laminin in GFAP-immunoreactive reactive astrocytes of the Alzheimer disease frontal cortex. The gamma 1 laminin was also heavily over-expressed in reactive astrocytes of both grey and white matter. Although antibodies against the C-terminal neurite outgrowth domain failed to localize gamma 1 laminin in senile plaques, antibodies against the N-terminal domains of the gamma 1 laminin demonstrated gamma 1 laminin as punctate deposits in the senile plaques. The present results indicate that enhanced and specialized expression patterns of alpha 1 and gamma 1 laminins distinctly associate these two laminins with the Alzheimer disease. The fact that domain specific antibodies localize both alpha1 and gamma 1 laminins in the senile plaques as punctate deposits and in astrocytes of both the gray and white matter indicate that these laminins and their specific domains may have distinct functions in the pathophysiology of the Alzheimer disease.     

Synthetic NGF peptide derivatives prevent neuronal death via a p75 receptor-dependent mechanism

Cyclized peptides corresponding to β-loop regions of NGF were purified by HPLC and assayed for neurotrophic activity using DRG neurons. Peptides with the highest activity corresponded to loop region 29–35, a domain likely to interact with the p75 receptor. Unexpectedly, activity was confined to late-eluting HPLC fractions containing peptide multimers and primarily promoted neuronal survival without neurite outgrowth. Directed synthesis of dimer and monomer cyclized peptides demonstrated that dimers acted as partial NGF agonists in that they had both survival-promoting and NGF-inhibiting activity while monomer and linear peptides were inactive. Dimer activity was not affected by the Trk inhibitor K252a but was blocked by p75 receptor antibody and absent using p75 null mutant neurons. These studies suggest that region 29–35 peptide derivatives inhibit neuronal death via a structure- and p75-dependent mechanism. J. Neurosci. Res. 48:1–17, 1997. © 1997 Wiley-Liss, Inc.     

Cooperation between nerve growth factor and laminin or fibronectin in promoting sensory neuron survival and neurite outgrowth

Chick embryo dorsal root ganglion (DRG) neurons were purified by differential adhesion to plastic. The purified neurons were used to study the cooperation between nerve growth factor (NGF) and laminin or fibronectin in promoting neuron survival and neurite outgrowth. NGF alone supported the survival of only 20% embryonic day 10 (E10) cells, of which only 40-50% had neurites. Treatment of the substrate with fibronectin or laminin increased survival in the presence of NGF up to 80% of the seeded neurons, all of which showed extensive neurite outgrowth. Survival and neurite outgrowth were also enhanced by the combined effects of elevated potassium and laminin. In contrast to E8-10 cells, 85% of E16 neurons survived in the basal culture conditions, i.e. without additional NGF, fibronectin or laminin, although neurite outgrowth was enhanced by all 3 proteins. Antisera to NGF, laminin and fibronectin, each independently decreased survival and neurite outgrowth of DRG neurons, totally with E9 and partially with E16 cells. The results suggest that the cooperative actions of extracellular matrix proteins and NGF are essential for survival and neurite outgrowth of embryonic DRG neurons and that these neuronal requirements change during development.     

Domain-specific antibodies against the B2 chain of laminin inhibit neuronal migration in the neonatal rat cerebellum

Although the spatial and temporal patterns of neuronal migration have been analyzed in great detail, little direct evidence is available as to what extracellular matrix molecules are involved. Because there is indirect evidence implicating the extracellular matrix protein laminin in neuronal migration, we investigated the effects of antibodies against a synthetic peptide derived from a neurite outgrowth domain of the B2 chain of laminin on neuronal migration in living cerebellar slices. We show by using infrared video microscopy that divalent Fab₂ fragments of these antibodies inhibit granule neuronal movement in living slices of (P8) rat cerebellum. This inhibition of neuronal movement manifests itself by cessation of both radial and horizontal translocations of nuclei inside the granule neuronal processes. Fab₂ fragments of antibodies against the intact (native) laminin molecule or Fab₂ fragments from the preimmune serum do not affect nuclear translocation. Immunocytochemistry shows binding of the divalent Fab₂ fragments of the B2 chain-specific antibodies to the Purkinje and Bergmann glial cell areas, and as punctate deposits in between the cells of the external granule cell layer. Native laminin antibodies bind to the basement membranes, and binding of the Fab₂ fragments from the preimmune sera cannot be demonstrated. These results indicate that neuronal migration in the postnatal rat cerebellum in vivo involves nuclear translocation that can be inhibited by antibodies against a neurite outgrowth domain of the B2 chain of laminin. Thus, migration of cerebellar granule neurons may depend on the interaction between a neurite outgrowth domain of the B2 chain of laminin and neuronal cytoskeleton involved in nuclear movement. © 1995 Wiley-Liss, Inc.     

Endomorphin-2-immunoreactive fibers selectively appose serotonergic neuronal somata in the rostral ventral medial medulla

The rostral portion of the ventral medial medulla (RVM) is a crucial site for the supraspinal antinociceptive actions of opioids. Previous studies have reported that serotonergic antagonists block the analgesia induced by microinjection of morphine into the RVM (Hammond and Yaksh [1984] Brain Res 298:329-337) and that spinally projecting serotonergic RVM neurons express mu-opioid receptors (MOR) (Kalyuzhny et al. [1996] J Neurosci 16:6490-6503; Wang and Wessendorf [1999] J Comp Neurol 404:183-196). In addition, axons immunoreactive for the endogenous MOR ligand endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) (EM-2) have been reported to be in the RVM (Martin-Schild et al. [1999] J Comp Neurol 405:450-471; Pierce and Wessendorf [2000] J Chem Neuroanat 18:181-207). In the present study we examined the relationship of EM-2-immunoreactive (EM-2-ir) axons to serotonergic and nonserotonergic RVM neurons in rats, including neurons projecting to the dorsal spinal cord. We also examined the origins of EM-2-ir in the RVM. Using unbiased methods we estimated the total number of cells in the RVM to be 1.50 x 10(4) and of these up to 70% were retrogradely labeled from the dorsal spinal cord. EM-2-ir fibers apposed both serotonergic and nonserotonergic RVM neuronal profiles. However, serotonergic profiles were significantly more likely to be apposed than nonserotonergic profiles. Thus, although serotonergic neurons comprise a minority of RVM neurons (23% of the total RVM neurons), they appear to be selectively apposed by EM-2-ir fibers. We also found that hypothalamic EM-2-ir neurons, but not EM-2-ir neurons, in the nucleus of the solitary tract projected their axons to the RVM.     

Stage-specific and opposing roles of BDNF, NT-3 and bFGF in differentiation of purified callosal projection neurons toward cellular repair of complex circuitry

Cellular repair of neuronal circuitry affected by neurodegenerative disease or injury may be approached in the adult neocortex via transplantation of neural precursors ("neural stem cells") or via molecular manipulation and recruitment of new neurons from endogenous precursors in situ. A major challenge for potential future approaches to neuronal replacement will be to specifically direct and control progressive differentiation, axonal projection and connectivity of neural precursors along a specific neuronal lineage. This goal will require a progressively more detailed understanding of the molecular controls over morphologic differentiation of specific neuronal lineages, including neurite outgrowth and elongation, in order to accurately permit and direct proper neuronal integration and connectivity. Here, we investigate controls over the morphologic differentiation of a specific prototypical lineage of cortical neurons: callosal projection neurons (CPN). We highly enriched CPN to an essentially pure population, and cultured them at three distinct stages of development from embryonic and postnatal mouse cortex by retrograde fluorescence labelling, followed by fluorescence-activated cell sorting. We find that specific peptide growth factors exert direct stage-specific positive and negative effects over the morphologic differentiation and process outgrowth of CPN. These effects are distinct from the effects of these growth factors on CPN survival [Catapano et al. (2001)J. Neurosci., 21, 8863-8872]. These data may be critical for the future goal of directing lineage-specific neuronal differentiation of transplanted or endogenous precursors/"stem cells" toward cellular repair of complex cortical circuitry.     

Peripheral motoneuron interactions with laminin and Schwann cell-derived neurite-promoting molecules: developmental regulation of laminin receptor function

Schwann cells synthesize several neurite outgrowth-promoting molecules and localize them in either the extracellular matrix (ECM; e.g., laminin) or on the plasma membrane (e.g., L1/NgCAM and N-cadherin). Neurite outgrowth by embryonic chick ciliary ganglion (CG) neurons in response to these Schwann cell molecules largely depends on several specific neuronal cell surface receptors: integrin beta 1-class ECM receptors, L1/NgCAM, and N-cadherin (Bixby et al.: Journal of Cell Biology 107:353-361 1988). To address whether neuronal ECM receptors are regulated independently of cell surface adhesion molecules, we studied the ability of dissociated CG neurons from different developmental ages to extend neurites rapidly on 1) substrates coated with the ECM glycoprotein laminin (either from Schwann cell-conditioned medium or purified from the Engelbreth-Holm-Swarm sarcoma) or 2) the surfaces of Schwann cells or Schwannoma (RN22) cells. CG neurons gradually lost the ability between embryonic day 8 (E8) and E14 to attach to and extend neurites in an integrin-dependent fashion on purified laminin or Schwann cell-derived laminin. The inability of E14 CG neurons to respond to laminin was partially reversed after explantation for 2.5 days in vitro, which increased the percentage of responsive neurons approximately ten-fold. E14 neurons remained capable of extending neurites rapidly on the surfaces of Schwann and Schwannoma cells. Thus, the inability of E14 neurons to respond to laminin reflects a specific loss of laminin receptor function, while other receptors, most likely N-cadherin and L1/NgCAM, remain capable of promoting neurite outgrowth on Schwann cell surfaces. Since integrin beta 1-class heterodimers have been shown to function directly as receptors mediating neuronal attachment and process outgrowth on laminin, our results imply that the expression or function of laminin-binding integrin heterodimers is regulated during the development of CG neurons. The apparent loss of integrin receptor function occurs during the period when the axons of CG neurons innervate their targets. Substantial integrin receptor function is recovered when target contact is disrupted by explantation. Thus, the functions of integrin-class receptors in CG neurons may be regulated by target contact.     

Specific effects of ethanol on neurite-promoting proteoglycans of neuronal origin

Quantitative analysis of proteoglycan synthesis and release by neurons indicated that of the total incorporation of 35SO4 and [3H]glucosamine into proteoglycan, approximately 75% was chondroitin sulphate while approximately 25% was heparan sulphate. Using a biological assay it has been shown that heparan sulphate proteoglycans (HSPGs) in conditioned medium promote neurite outgrowth from sensory neurons when complexed to a laminin substrate. Culture media conditioned in the presence of ethanol did not enhance neurite formation over control levels. Co-incubation of neurons with beta-D-xyloside, an inhibitor of proteoglycan synthesis, reduced neurite outgrowth after 20 h in culture and the combination of ethanol and beta-D-xyloside produced no further inhibition than with either ethanol or beta-D-xyloside used alone. If the laminin substrate was coated with medium conditioned by neurons, the direct inhibitory effects on process formation seen when ethanol was co-incubated with neurons were no longer observed. Ethanol inhibited the incorporation of 35SO4 and [3H]glucosamine into HSPG by neurons while having little or no effect on incorporation into chondroitin sulphate. These results suggest that inhibition of neuronal synthesis of HSPGs by ethanol is responsible for the decrease in neurite promoting activity of medium conditioned in the presence of ethanol.