Päivi Liesi

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.

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.

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.

Molecular gradient along the axon pathway is not required for directional axon growth.

We show that axon guidance of embryonic hippocampal neurons is promoted by pathways of a decapeptide (RDIAEIIKDI) derived from a neurite outgrowth domain of the γ1 chain of laminin‐1. This guidance is directly dependent on: (1) a concentration difference of the decapeptide between the peptide pathway and its surrounding areas, and (2) the optimal surface geometry of the decapeptide pathway. These results indicate that axon guidance of central neurons may proceed along a preferred substratum pathway without a concentration gradient of the guiding molecule along this pathway, or without a repulsive molecule next to the axon pathway. J. Neurosci. Res. 53:114–124, 1998. Published 1998 Wiley‐Liss, Inc. This article was prepared by a group consisting of both United States government employees and non‐United States government employees, and as such is subject to 17 U.S.C. Sec.105.

BAPTA-AM and ethanol protect cerebellar granule neurons from the destructive effect of the weaver gene.

The mechanisms by which the weaver gene (Reeves et al., 1989; Patil et al., 1995) inhibits neurite extension and/or induces death of the granule neurons in homozygous weaver mouse cerebellum are not presently understood. Here we show that BAPTA‐AM and ethanol, which either reduce cytosolic levels of free calcium or prevent calcium entry, promote neurite outgrowth of the weaver neurons similar to the L‐type calcium channel blocker verapamil (Liesi and Wright, 1996). Importantly, BAPTA‐AM, ethanol, and verapamil not only restore neurite outgrowth of the weaver neurons but adjust their depolarized resting membrane potentials to the levels of normal neurons. These results indicate that calcium‐dependent mechanisms mediate the action of the weaver gene and that the weaver neurons can be normalized by blocking this calcium effect. We further report that BAPTA‐AM and verapamil also have a neuroprotective effect on normal neurons exposed to high concentrations of ethanol. We suggest that verapamil should be evaluated as a drug for treatment of alcohol‐induced brain damage and neurodegenerative disorders. J. Neurosci. Res. 48:571–579, 1997.

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.

Weaver cerebellar granule neurons show altered expression of NMDA receptor subunits both in vivo and in vitro

Biochemical, immunocytochemical, and molecular biological techniques were used to investigate the expression of N-methyl-D-aspartate (NMDA) receptor subunits in migration-deficient weaver mouse cerebellum in vivo and in primary cultures of the vermal weaver granule neurons with or without a rescue by verapamil. We found that both NMDAR1(zeta1) message and protein were expressed by the weaver granule neurons in situ. Immunocytochemical and biochemical analyses indicated that granule neurons of the weaver cerebellum expressed R1(zeta1) and R2A(epsilon1) subunits but showed little expression of the R2B(epsilon2) subunit. In weaver cerebellum, the R2B(epsilon2) subunit was primarily expressed in nerve fibers of the internal granule cell layer and white matter. Reverse-transcriptase-polymerase chain reaction followed by sequence analysis of the R1(zeta1) subunit indicated that the zeta1 subunit amplicons of both normal and weaver cerebella were identical, and that splice variants with exon 22 (1-2) and with or without exon 5 (a/b) or exon 21 (1-4) were detectable. The R2A(epsilon1), and R2B(epsilon2) subunits of the normal and weaver mouse cerebellum revealed no primary structural differences between the normal and weaver NMDA receptor subunits or the cloned mouse NMDA receptor subunits. In vermal cultures, normal granule neurons expressed all three NMDA receptor subunits (zeta1, epsilon1, and epsilon2), whereas the weaver neurons failed to express the epsilon2 subunit. Rescue of the weaver neurons by verapamil induced expression of the epsilon2 protein along the granule neuronal surfaces. The present results suggest that lack of the epsilon2 subunit in the weaver cerebellum may relate to the lack of functional NMDA receptors and/or to the migratory failure of the weaver granule neurons. Our data further suggest that NMDA receptor-mediated neurotoxicity is an unlikely mediator of neuronal death of the weaver granule neurons. In fact, down-regulation of the NMDA receptor expression and function may be a protective measure of the weaver granule neurons to reduce calcium entry via these receptors.

Neurite outgrowth guidance in vitro

Patterned substrates bound with a synthetic peptide were used to guide the outgrowth of hippocampal and cerebellar neurons in culture. Patterns of parallel lines were formed on glass substrates by coating the surfaces with an aminosilane chemical and irradiating them with deep UV light through a photomask. The patterns were then used to differentially attach a synthetic peptide derived from a neurite-outgrowth domain of laminin onto the surfaces. Hippocampal and cerebellar neurons were dissected from embryonic rats and grown on the substrates in chemically defined media. Both hippocampal and cerebellar neurons initially developed morphologies by reflecting the surface patterns in culture. With continued culture periods, the hippocampal neurons extended their longer neuritic processes mainly along the patterned lines, while their shorter processes showed random outgrowth. Our in vitro demonstration clearly shows an ability of growing neurites, putatively axons, for detecting a step gradient formed of substrate-bound laminin peptide. Substratum-guided axonal growth will be essential for the formation of well-organized small neuronal networks in culture.