Douglas J. Swanson

Modulation of ethanol neurotoxicity by nerve growth factor.

Dorsal root ganglion (DRG) neurons were cultured with varying concentrations of ethanol and NGF. At low concentrations of NGF (0.1 ng/ml) moderate initial ethanol levels (250 mg/dl) significantly suppressed neurite outgrowth. Higher NGF concentrations (5 ng/ml) protected against this neurotoxicity. At this higher NGF concentration, neuronal survival was not significantly affected by exposure to 0.25-4 g/dl ethanol, although survival was significantly diminished at 5 and 6 g/dl. Neurite outgrowth was a more sensitive indicator of ethanol neurotoxicity in this population, with significant decreases in process extension seen with 1 g/dl ethanol. When cultures were supplemented with 10 ng/ml NGF, however, process elaboration was significantly greater at 1 g/dl ethanol than that measured with 5 ng/ml NGF, and in fact did not differ from NGF controls. These studies indicate that NGF can provide neuroprotective effects against ethanol toxicity under these conditions. The results are discussed in relation to other recent reports of trophic factor neuroprotection.

Ethanol exposure affects trophic factor activity and responsiveness in chick embryo

Chick embryos were chronically exposed to either ethanol (approximately 30 mg/d) or saline, from E4-E13. Homogenate extract was prepared from forebrain tissue from E16 experimental and control embryos and was applied to cultured dorsal root ganglia (DRG). Neurotrophic activity in the forebrain extract (FBX) was significantly reduced in the ethanol-treated embryos compared to saline controls, both in terms of influences on neuronal survival and process elaboration. In addition, E8-9 DRGs from embryos exposed to ethanol from E4 were less viable in the presence of NGF than were those from controls. DRG survival in the presence of E16 FBX (from untreated embryos) was not different following ethanol treatment, but neurite production was significantly reduced. These results suggest that neurotrophic factor content and responsiveness may be appreciably altered following chronic prenatal ethanol exposure. Such alteration could underlie certain CNS anomalies seen in the fetal alcohol syndrome.

Alterations in responsiveness to ethanol and neurotrophic substances in fetal septohippocampal neurons following chronic prenatal ethanol exposure

Pregnant Long-Evans rats were maintained on three diets: a liquid diet in which ethanol accounted for 35-39% of the total calories, a similar diet with the isocaloric substitution of sucrose for ethanol, and a lab chow control diet. At gestation day 18, the fetuses were taken and cultures of septal and hippocampal neurons prepared. Neuronal survival and neurite outgrowth were compared in cultures from the three diet groups, using the following media supplements: ethanol (1.2, 1.8 or 2.4 g/dl), neurotrophic factors (nerve growth factor [NGF] with the septal cultures, basic fibroblast growth factor [bFGF] with the hippocampal cultures), or ethanol plus neurotrophic factors. Both the septal and hippocampal neurons responded to ethanol in a dose-dependent manner. The neurons from both populations from fetuses which had been exposed prenatally to ethanol, however, tolerated considerably higher ethanol concentrations before decreases in survival or outgrowth were seen. These ethanol-exposed neuronal populations were also less responsive to neurotrophic factors: in hippocampal cultures, process outgrowth was significantly enhanced by bFGF in control but not ethanol-derived cultures, and in septal and hippocampal cultures, the neurotrophic factors significantly ameliorated ethanol neurotoxicity in control cultures, but not in those from the ethanol-exposed fetuses. The possible relevance of these observations to the fetal alcohol syndrome is discussed.

Influence of chronic prenatal ethanol on cholinergic neurons of the septohippocampal system.

This study characterized the influence of full‐term gestational ethanol exposure on choline acetyltransferase (ChAT)‐immunoreactive neurons that project to the hippocampus, within the medial septal (MS) nucleus and the vertical limb of the diagonal band of Broca (DBv). On gestation days 1–22, pregnant dams were fed either a vitamin fortified ethanol‐containing liquid diet, pair fed a calorically equivalent sucrose‐containing diet, or given rat chow ad libitum. In a previous study, we found that chronic prenatal exposure to ethanol, in this manner, resulted in a significant decline in the ontogenetic upregulation of ChAT activity in the septal area during the second postnatal week, but was followed by recovery to control levels by adulthood.

Prenatal ethanol exposure alters neurotrophic activity in the developing rat hippocampus.

Extract made from hippocampus of rat pups exposed prenatally to an ethanol-supplemented diet was found to contain more neurotrophic activity at postnatal day 21 than that from animals exposed to control diets, when quantified in a dorsal root ganglion bioassay. This apparent upregulation was specific to hippocampal extract (cerebellar and forebrain/midbrain extracts were also assessed), and to this age (P1, P7, P14 and P60 extracts were also tested). It was suggested that this upregulation may be indicative of, or secondary to, trauma resulting from fetal ethanol exposure. It is speculated that such departures from the normal developmental timetable could contribute to anomalies seen in the fetal alcohol syndrome.

Ethanol neurotoxicity in vitro: effects of GM1 ganglioside and protein synthesis inhibition

Cultures of septal and hippocampal neurons from fetal rat and dorsal root ganglion neurons from embryonic chick were pretreated with GM1 ganglioside or cycloheximide and then supplemented with toxic concentrations of ethanol. GM1 provided significant protection against ethanol neurotoxicity in each population. The inhibition of protein synthesis by cycloheximide, however, which protects against cell death resulting from withdrawal of neurotrophic factor support, did not ameliorate ethanol-induced neuronal loss.

Chronic ethanol alters CNS cholinergic and cerebellar development in chick embryos

Chick embryos were given daily injections of ethanol (approximately 30 mg/day) either chronically from embryonic days 4 to 15 (E4-E15) or E18, or acutely from E15 to E18. Untreated and saline-injected embryos served as controls. Although morphological indicators of developmental age were not different among groups, chronic ethanol reduced weights of several brain regions. Similar to rodent models of prenatal ethanol exposure, chronic ethanol treatment reduced cerebellar Purkinje cell numbers compared to controls. Chronic but not acute ethanol exposure resulted in a significant reduction in choline acetyltransferase activity in the optic tectum (OT) and forebrain (FB) compared with controls. This study demonstrates that the chick embryo is a viable model to investigate the effects of ethanol exposure on CNS development. Unlike the mammalian fetus, the avian embryo is isolated from maternal interactions and may prove more useful in investigating the mechanisms by which ethanol directly influences brain development.

Neurotrophic Activity in Embryonic Chick Brain: Early Appearance and Differential Regional Distribution

Homogenate extracts were prepared from various regions of the embryonic chick brain from E6 to E19. The extracts were applied to cultured dorsal root ganglia (DRGs), sympathetic ganglion cells, PC12 cells and E5 spinal cord neurons. Potent neurotrophic activity with respect to DRGs was found in an extract from the forebrain (FBX) from the earliest stage assayed (E6). This activity was comparable to that of nerve growth factor (NGF), and it was not appreciably altered during later stages. By E8, extracts prepared from the optic lobe also induced responsiveness, although to a lesser extent than the FBX and NGF. At E12-19, extracts from the hippocampus (HCX) and cerebellum (CBX) were also tested. Activity found in the HCX resembled that in the FBX, but responsiveness to the CBX was similar to that in control (unsupplemented) cultures. The responsiveness of sympathetic neurons in the FBX was comparable to that with NGF. The FBX induced only moderate differentiation of PC12 cells, and spinal cord neurons were not responsive. Antibodies to NGF and basic fibroblast growth factor did not diminish the influence of the FBX, and the FBX and NGF when combined had synergistic effects. The results demonstrate potent neurotrophin-like activity in the embryonic brain at much earlier stages than had previously been seen, with differential regional and temporal distributions.

Comparative responsiveness of early chick neural tube neurons to muscle-conditioned medium, laminin, NGF and fibronectin

Dissociates from the metencephalic basal plate of early chick embryo neural tubes containing the trigeminal (V) motor nucleus were cultured on substrates conditioned with appropriate target-derived muscle conditioned medium (MCM), laminin (LAM), MCM with nerve growth factor (NGF) in the medium, and fibronectin (FN). Comparisons were made of neuronal survival, the number of neurons with processes, and the length of processes elaborated. It was found that both MCM and LAM significantly enhanced survival and neuritic production from this population when compared to controls grown on a collagen-polyornithine substrate, but MCM surpassed LAM in these measures. When the neurons were grown on an MCM-conditioned substrate with an NGF-supplemented medium, no improvements were produced over the MCM or the NGF conditions alone. Therefore, the two do not appear to act in synergy, as NGF and LAM have been shown to do. FN produced no enhancement of any of the measures taken from this population. An ELISA analysis revealed no detectable LAM in the early target MCM. These results indicate that the specific responsiveness of this early neural tube population to its target MCM is not mediated by LAM, but the growth-enhancing component acts in a similar manner, although its influence is more potent.