Alcohol exposure during the brain growth spurt promotes hippocampal seizures, rapid kindling, and spreading depression.

BACKGROUND: Epilepsy is a prominent sign of brain dysfunction and a cause of substantial disability in some children with fetal alcohol syndrome. The hippocampal formation is vulnerable to alcohol-induced pathologic changes and is the source of seizure activity in a variety of epileptic conditions. This study tests the hypothesis that developmental alcohol exposure facilitates epileptic activity and promotes kindling within hippocampal circuitry. METHODS: Rat pups received either a moderate dose (2.0 g/kg) or a high dose (3.75 g/kg) of alcohol via intragastric intubation over postnatal days 4 to 10. Intubated control and suckle control groups were also included. Upon reaching adulthood (postnatal days 85-100), the rats underwent electrophysiologic testing. A double-barrel potassium-sensitive microelectrode was placed into the right dentate gyrus stratum granulosum for the recording of extracellular field potential and extracellular potassium concentration. Stimuli were delivered via an electrode positioned in the CA3 subregion of the left hippocampus. To assess whether alcohol promotes hippocampal seizures and rapid kindling, the parameters of maximal dentate activation (MDA) were measured before, during, and after a series of stimulation-induced seizures. RESULTS: Developmental exposure to the high dose of alcohol permanently altered several parameters of MDA. Time to onset of MDA and stimulus threshold for afterdischarge production were both decreased, whereas the duration of the afterdischarge was increased. Although the moderate alcohol dose reduced time to onset of MDA, it did not affect any other MDA parameters. Over the course of the repeated induced seizures, spreading depression occurred more often and with fewer stimuli in the high-dose alcohol group than in any other group. The series of repeated electrographic seizures induced rapid kindling in all of the treatment groups. However, the kindling effect was enhanced in a dose-response manner by the previous alcohol exposures. CONCLUSIONS: These findings demonstrate that exposure to alcohol during brain development can permanently alter the physiology of the hippocampal formation, thus promoting epileptic activity, enhancing kindling, and facilitating spreading depression. The relative roles of alcohol intoxication and withdrawal in these abnormal physiologic responses remain unknown.

Transfection of C6 glioma cells with glia maturation factor upregulates brain-derived neurotrophic factor and nerve growth factor: trophic effects and protection against ethanol toxicity in cerebellar granule cells.

Glial cells play active roles in neuronal survival, as well as neuroprotection against toxic insult. Recent studies suggest that the brain protein glia maturation factor (GMF) is involved in intracellular signaling in glia. This study investigated whether or not GMF plays a role in the survival-promoting and neuroprotective functions of glia. C6 glioma cells were transfected in vitro with GMF utilizing an adenovirus vector. The transfected cells overexpressed GMF intracellularly, but did not secrete the protein. The conditioned medium (CM) was obtained from the GMF-transfected cells (CM-GMF) and tested on primary neuronal cultures, consisting of cerebellar granule cells (CGC). The CGC cultures were utilized because these cultures have a background level of cell death, and the survival-promoting, i.e. neurotrophic effect, of the CM could be tested. In addition, since CGC cultures are ethanol-sensitive (ethanol enhances neuronal death), the neuroprotective effect of the CM against ethanol-induced cell death was tested also. We demonstrated that the CM-GMF had an enhanced neurotrophic effect as well as an increased neuroprotective effect against ethanol-induced cell death compared to control CM obtained from untransfected C6 cells (CM-Mock) or CM obtained from cells transfected with an unrelated gene (CM-LacZ). Because neurotrophins have trophic and protective effects, we investigated whether GMF-transfection upregulated the expression of neurotrophins in C6 cells. RT-PCR verified that GMF-transfected C6 cells had increased mRNA levels for BDNF and NGF. Immunoblotting corroborated the RT-PCR results and indicated that CM-GMF contained greater concentrations of BDNF and NGF protein compared to CM-Mock and CM-LacZ. A soluble TrkB-IgG fusion protein, which selectively binds BDNF and prevents its binding to the neuronal TrkB receptor, eliminated the neurotrophic effect of CM-GMF; whereas anti-NGF antibody was ineffective in preventing this effect, suggesting that the neurotrophic effect was due to BDNF. On the other hand, both the TrkB-IgG fusion protein and anti-NGF reduced neuroprotection, suggesting that BDNF and NGF both contribute to the neuroprotective effect of CM-GMF. In conclusion, GMF upregulates the expression of BDNF and NGF in C6 cells, and these factors exert neurotrophic and neuroprotective functions on primary neurons.

Ethanol induces cell death and cell cycle delay in cultures of pheochromocytoma PC12 cells.

Animal models have clearly established that ethanol exposure can deplete neurons in the developing nervous system. However, the mechanism by which ethanol reduces cell number is unclear. In our study, cultures of pheochromocytoma cells, a neuronal-like cell line, were maintained in media, which supported cell proliferation. Although cell numbers continued to increase in the presence of ethanol, this increase was partially inhibited by ethanol exposure. This inhibitory effect was concentration and duration dependent. Cell proliferation was still partially inhibited after removal of ethanol, but this inhibition was temporary and disappeared after a 24-hr recovery period in ethanol-free conditions. Further study indicated that ethanol partially inhibited the increase in cell numbers by two mechanisms: (1) studies with vital stains indicated that ethanol induced cell death; (2) experiments using synchronized pheochromocytoma cell cultures showed that ethanol can induce cell cycle delay, thereby lengthening the doubling time of the cells. Analysis by flow cytometry indicated that with ethanol exposure, the cells accumulated in the G1 phase of the cell cycle. Our results suggest that in the developing nervous system, ethanol may limit the numbers of proliferating, neuronal precursor cells by two simultaneous mechanisms, cell death and cell cycle delay.

The nitric oxide-cyclic GMP pathway plays an essential role in both promoting cell survival of cerebellar granule cells in culture and protecting the cells against ethanol neurotoxicity.

NMDA has two beneficial effects on primary neuronal cultures of cerebellar granule cells (CGCs) established from 10-day-old rat pups. First, NMDA is neurotrophic and will enhance survival of CGCs in culture in the absence of ethanol. Second, ethanol exposure will induce cell death in CGC cultures, and NMDA can lessen this ethanol-induced cell loss, i.e., NMDA is neuroprotective. Because NMDA can stimulate production of nitric oxide (NO), which can in turn enhance synthesis of cyclic GMP, this study tested the hypothesis that the NO-cyclic GMP pathway is essential for NMDA-mediated neurotrophism and neuroprotection. Inhibiting the synthesis of NO with N(G)-nitro-L-arginine methyl ester eliminated both the NMDA-mediated neurotrophic and neuroprotective effects. Similarly, inhibiting production of cyclic GMP with the agent LY83583 also abolished these effects. The NO generator 2,2'-(hydroxynitrosohydrazono) bisethanamine produced neurotrophic and neuroprotective effects that were similar to those induced by NMDA. Also, 8-bromo-cyclic GMP produced neurotrophic and neuroprotective effects that were quite similar to the effects produced by NMDA. In conclusion, NMDA enhances survival of cerebellar granule cells and protects the cells against ethanol-induced cell death by a mechanism(s) that involves the NO-cyclic GMP pathway.

Nerve growth factor and basic fibroblast growth factor protect rat cerebellar granule cells in culture against ethanol-induced cell death.

Neuronal cell loss is one of the most debilitating effects of fetal ethanol exposure. Cultures of cerebellar granule cells are a useful model to investigate ethanol neurotoxicity, because ethanol depletes cell numbers in these cultures, which also occurs in vivo. The primary goal of the present study was to identify and characterize agents that can ameliorate the ethanol-induced cell death that occurs in this culture system. Growth factors, such as nerve growth factor (NGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and insulin-like growth factor-I (IGF-I) can prevent neuronal degeneration after toxic insult in various experimental paradigms. These growth factors were investigated in the current study to determine whether or not they can mitigate ethanol-induced death of cerebellar granule cells in culture. Results indicate that NGF and bFGF significantly reduced the ethanol-induced cell loss. Both the NGF- and bFGF-mediated neuroprotection required protein and RNA synthesis, because actinomycin D (RNA synthesis inhibitor) and cycloheximide (protein synthesis inhibitor) blocked their neuroprotective effects. In addition to its neuroprotective effect, bFGF also had a neurotrophic effect and could enhance cell survival in the absence of ethanol exposure. NGF did not have a neurotrophic effect. Neither EGF nor IGF-I was neuroprotective, although the latter did have a substantial neurotrophic effect. In conclusion, bFGF and NGF have long been recognized for their role in enhancing neuronal cell survival and differentiation. This study suggests that these growth factors can also provide neuroprotection against ethanol-induced cell death.

Ethanol reduces expression of the nerve growth factor receptor, but not nerve growth factor protein levels in the neonatal rat cerebellum.

The cerebellum is especially vulnerable to ethanol's neurotoxic effects during development, and ethanol exposure during the brain growth spurt will deplete cerebellar neurons. The mechanisms undertying this neuronal cell loss remain elusive. Nerve growth factor (NGF) is a neurotrophin that promotes cell survival in various brain areas, and there is evidence that NGF may play a role in the developing cerebellum. This study examined whether ethanol exposure of the neonatal rat cerebellum altered the levels of either NGF or the expression of p75 and trkA, which are two components of the NGF receptor. Ethanol exposure had no effect on NGF levels in the neonatal cerebellum, as determined by an NGF-specific ELISA. Immunohistochemical labeling techniques indicated that both the p75 and trkA NGF receptors were expressed on Purkinje cell dendrites in the developing cerebellum, with posterior lobules expressing higher levels of p75 and trkA NGF receptor, compared with anterior lobules. Ethanol exposure of neonatal rats reduced the expression of both p75 and trkA NGF receptors on the Purkinje cell dendrites. These results suggest that ethanol could interfere with neurotrophic support of Purkinje cells by reducing the levels of available NGF receptor.

Ethanol exposure reduces the density of the low-affinity nerve growth factor receptor (p75) on pheochromocytoma (PC12) cells.

Although ethanol is detrimental to the developing nervous system, the mechanism(s) by which ethanol produces neuronal damage is (are) not clear. One potential mechanism is ethanol-induced inhibition of neurotrophic support. This study utilized an in vitro model, pheochromocytoma PC12 cells, to examine the effect of ethanol on the nerve growth factor (NGF) receptor. NGF binding studies indicated that ethanol exposure (400 mg/dl for 4 days) reduced the density of the low-affinity (p75) NGF receptor on PC12 cells, but had no effect on the density of the high-affinity NGF receptor. The equilibrium dissociation constants (Kd) for both the low-affinity and high-affinity NGF receptors were unaffected by ethanol. Low-affinity NGF binding is mediated by the p75 component of the NGF receptor. Quantification of p75 by immunoprecipitation revealed that ethanol reduced the level of p75 in PC12 cells. However, Northern analysis indicated that the p75 mRNA was not reduced by ethanol exposure, raising the possibilities that ethanol inhibited translation of p75 or incorporation of the p75 protein into the plasma membrane. This work is consistent with the hypothesis that ethanol's detrimental effects may be produced in part by inhibition of neurotrophic support at the receptor level.

Nerve growth factor synthesis by intestinal epithelial cells.

Nerve growth factor (NGF) exists in the gut of adult rats. The cells responsible for NGF synthesis in the gut remain unknown. IEC-6 and Caco-2 cells, established cell culture models of intestinal epithelial cells, were studied to determine whether intestinal epithelial cells, were studied to determine whether they synthesize and release NGF. Conditioned media from both IEC-6 and Caco-2 cells stimulated neurite outgrowth in both rat pheochromocytoma (PC-12) cells and sensory neurons derived from embryonic chick dorsal root ganglia (DRG). The addition of anti-NGF antibody blocked neurite outgrowth in PC-12 cells and partially blocked outgrowth in DRG cells. An NGF-enzyme-linked immunosorbant assay readily detected immunoreactive NGF in conditioned media from both cell lines, whereas cellular extracts from IEC-6, Caco-2, and isolated rat intestinal epithelial cells had low levels of immunoreactivity. Caco-2 monolayers primarily secreted NGF from the basolateral compartment, and interleukin-1 enhanced its secretion. IEC-6, Caco-2, and isolated rat intestinal epithelial cells expressed NGF mRNA as determined by reverse transcription polymerase chain reaction. These observations suggest that intestinal epithelial cells are capable of NGF synthesis.

NMDA prevents alcohol-induced neuronal cell death of cerebellar granule cells in culture.

Neuronal cell loss is one of the most debilitating effects of alcohol exposure during development of the nervous system. In this study, primary cultures of neuronal cells (cerebellar granule cells) were used to examine mechanisms of alcohol-induced neuronal cell death. Previously, we established that (Pantazis et al., Alcohol Clin Exp Res 17:1014-1021, 1993): (1) alcohol exposure caused neuronal cell death in cultures of cerebellar granule cells and this cell loss was both time-dependent and dose-dependent; and (2) the vulnerability of cerebellar granule cells to alcohol-induced loss changed with the length of time the cells were in culture before initiating alcohol exposure-that is, younger cultures (1 day in vitro) were much more susceptible to alcohol-induced neuronal cell death than older cultures (4 or 7 days in vitro). The primary goal of the present study was to examine the potential role of the NMDA receptor in alcohol-induced death of cerebellar granule cells in culture. Experiments were performed to test the hypothesis that the alcohol-induced death of cerebellar granule cells can be prevented or reduced by NMDA treatment. Our results indicate that stimulation of the NMDA receptor has a neuroprotective effect and can significantly reduce the alcohol-induced neuronal cell death of newly established cerebellar granule cell cultures. This neuroprotective effect of NMDA is blocked by 2-amino-5-phosphonovalerate, a competitive inhibitor of the NMDA receptor, confirming that this neuroprotective effect is mediated via the NMDA receptor. This is the first report that alcohol's neurotoxic effect can be ameliorated by activation of the NMDA receptor.

Fetal alcohol syndrome: the vulnerability of the developing brain and possible mechanisms of damage.

Fetal alcohol exposure has multiple deleterious effects on brain development, and represents a leading known cause of mental retardation. This review of the effects of alcohol exposure on the developing brain evaluates results from human, animal and in vitro studies, but focuses on key research issues, including possible mechanisms of damage. Factors that affect the risk and severity of fetal alcohol damage also are considered.

Vulnerability of cerebellar granule cells to alcohol-induced cell death diminishes with time in culture.

This study examined the effects of alcohol exposure on the viability of cerebellar granule cells in culture. Continuous alcohol exposure, starting 1 day after the cultures were established, significantly reduced granule cell numbers, even with a single day of exposure to an alcohol concentration as low as 100 mg/dl. The depletion of cerebellar granule cells by alcohol was concentration-dependent (greater loss of cells at higher alcohol concentrations) and duration-dependent (greater loss of cells at longer exposure durations). The loss of granule cells also depended on the number of days the granule cells were in culture before alcohol exposure. Alcohol was significantly more effective in reducing the cell numbers of newly established granule cell cultures (1 day in vitro) compared with older cultures (4 or 7 days in vitro). Cell cycle analysis established that the cerebellar granule cells did not proliferate in culture, indicating that alcohol exposure did not reduce cell numbers by interfering with cell proliferation in this system. Instead, alcohol-induced killing of the granule cells was the most likely mechanism to account for the depletion of granule cells in vitro. Granule cell cultures are a useful in vitro model system to study the cellular and molecular aspects of neuronal cell depletion associated with fetal alcohol exposure. The potential role of the N-methyl-D-aspartate receptor in this alcohol-induced neuronal cell death is discussed.

Alcohol reduces the number of pheochromocytoma (PC12) cells in culture.

Pheochromocytoma (PC12) cells were used as an in vitro neuronal cell model to examine detrimental effects of alcohol on cell numbers. Alcohol exposure (100, 200, 400, and 800 mg/dl) reduced PC12 cell numbers in a dose-dependent manner. Cells that were treated with nerve growth factor (NGF) incurred less severe reductions in numbers compared with cells that were never treated with NGF. Because NGF stops proliferation of many of the PC12 cells and differentiates them into neuronal-like cells, these data suggest that differentiated, nonproliferating cells are less vulnerable to alcohol-induced reductions in cell numbers. In a subsequent experiment using only undifferentiated PC12 cells, alcohol reduced cell number of both proliferating and nonproliferating cultures; however, the reductions in proliferating cultures were more severe than in nonproliferating cultures. Two mechanisms may account for alcohol-induced reductions of PC12 cell numbers--inhibition of proliferation and killing of cells. PC12 cell cultures are a useful model system to examine mechanism(s) underlying alcohol's depletion of neuronal-like cells.

Effectiveness of teaching radiologic image interpretation in gross anatomy. A long-term follow-up.

This prospective study was designed to gauge the effectiveness of teaching radiologic interpretation during a gross anatomy course for first-year medical students by measuring short- and long-term ability to identify normal anatomic structures on radiologic diagnostic images. The evaluation required students to identify normal anatomic structures on radiographs, computed tomographs, ultrasonograms, and magnetic resonance images (MRIs). The assessments were made before (pre-test) and during (post-test) the experimental radiology portion of the gross anatomy course. The students were then retested 14 to 17 months later (long term). The pre-test correct response rate of 17% improved to 88% on the post-tests. After 14 to 17 months, the students had a 74% correct response rate on the same images and anatomic structures. This high level of long-term retention documents the effectiveness of integrating diagnostic radiologic imaging into normal gross anatomy instruction.

Isolated hepatic granulomas from mice infected with Schistosoma mansoni contain nerve growth factor.

Nerve growth factor (NGF) is a neurotrophic factor essential for the development and maintenance of specific neuronal cell populations. In addition, NGF has biological effects on inflammatory cells. The aim of this study was to determine whether NGF is present in chronic inflammation, using isolated hepatic granulomas from mice infected with Schistosoma mansoni as the model. The schistosome granuloma is a complex T-cell-mediated immune response to the egg. Intact granulomas were isolated from the livers of infected mice and examined for the presence of NGF. In homogenized granuloma samples, radioimmunoassay and immunoblotting analyses detected an immunoreactive NGF that had the same molecular mass as that of purified murine NGF (13 kDa). Isolated granulomas cultured in vitro released soluble factor(s) with NGF-like neurite-promoting activity in a rat pheochromocytoma (PC12) bioassay. This activity was partially inhibited by a blocking anti-NGF antibody. There were two potential sources of this NGF-like neurite-promoting activity, either the schistosome egg or the host inflammatory response. Since neither isolated eggs nor soluble egg antigen had neurite-promoting activity, the inflammation was the source of this activity. The inability of the anti-NGF antibody to inhibit completely the granuloma-induced neurite outgrowth in the bioassay signifies that NGF is not the only neurotrophic factor present in these granulomas. The presence of NGF within the granulomas may indicate that NGF has a role in the granulomatous response.

Gross anatomy instruction with diagnostic images.

A new module of instruction for diagnostic imaging with emphasis on cross-sectional anatomy was developed within the existing course of gross anatomy for freshmen medical students. Two lectures introduced radiation protection, radiology history, and the basic principles of computed tomography, magnetic resonance imaging, ultrasound, and radiograph image production. Six radiographic anatomy correlation sessions allowed student-teacher interaction while studying viewbox images in a "hands on" fashion. Relevant exhibits complemented cadaver dissection. Testing established that significant improvement occurred in the students' ability to identify anatomic structures on diagnostic images.

Localization of the nerve growth factor receptor on fetal human Schwann cells in culture.

Previous studies have established that Schwann cells (SC) in culture express an NGF receptor. In this study, cultures of fetal human SC were established from fetal nerves and various light microscopic (LM) and electron microscopic (EM) techniques were used to localize the NGF receptor on the SC. Results indicate that NGF receptor is localized to the plasma membrane of the SC. Quantitative digital analysis determined that the distal portion of the SC process had high concentrations of NGF receptor. The possible functional significance of this latter observation is discussed in terms of SC migration and ensheathment of axons.

Detection of a nerve growth factor receptor on fetal human Schwann cells in culture: absence of the receptor on fetal human astrocytes.

Cultures of human Schwann cells and astrocytes were established from fetal nerves and brains respectively. The human Schwann cells in culture expressed a nerve growth factor (NGF) receptor as determined by indirect immunofluorescence, autoradiography, and immunoprecipitation. In contrast, the human astrocytes in culture did not have an NGF receptor. Cultures of rat Schwann cells and astrocytes were also established for comparison, with similar results. The rat Schwann cells had an NGF receptor whereas the astrocytes did not. The functional significance of this NGF receptor on Schwann cells, as well as the lack of the receptor on astrocytes, is discussed.

Nerve growth factor, not laminin, is the major neurite-promoting component in medium conditioned by mouse L929 fibroblast cells.

In this study the neurite-promoting activity present in conditioned medium (LCM) from mouse fibroblast L929 cells was examined. The biological and biochemical properties of nerve growth factor (NGF) synthesized by L929 cells and released into the LCM were studied. In addition it was established that the extracellular matrix protein laminin is synthesized by L929 cells and is also present in LCM. Although NGF is a well-recognized neurite-promoting factor, recent studies have suggested that laminin is also a neurite-promoting factor. In this paper the possibility that NGF and laminin were both responsible for the neurite-promoting activity of LCM was examined. It was determined that NGF, not laminin, is the major neurite-promoting component in LCM. This was established using pheochromocytoma (PC12) cells as a biological test system and also utilizing antibodies to both NGF and laminin. The possible functional significance of NGF synthesis by fibroblast cells is discussed.

Schwann cell-conditioned medium supports neurite outgrowth and survival of spinal cord neurons in culture.

The effect of Schwann cell-conditioned medium (SCM) on the development in vitro of spinal cord neurons was studied. Spinal cord neurons from 18-day-old rat embryos were cultured in serum-free conditioned medium obtained from confluent rat Schwann cells. In cultures fed SCM, the cells developed typical neuronal morphology and were identified by indirect immunofluorescence using a monoclonal antibody to neurofilament protein. SCM stimulated neurite outgrowth and supported survival of spinal cord neurons. Preliminary characterization suggests that the neurotrophic factor in SCM appears to be a protein with a molecular weight greater than 8000 daltons.

Rat astrocytes and Schwann cells in culture synthesize nerve growth factor-like neurite-promoting factors.

Neurite-promoting activity in feeding medium conditioned by rat astrocytes and Schwann cells in culture was examined. The conditioned medium (CM) from both types of glial cultures stimulated extensive neurite outgrowth from embryonic chick dorsal root ganglia (DRG) as well as pheochromocytoma (PC12) cells. Both the DRG and PC12 cells also produce neurite outgrowth in the presence of nerve growth factor (NGF). With the DRG, the neurite growth rates observed with the glial cell CM were identical to growth rates seen with NGF. Although anti-NGF antibody did not inhibit the neurite outgrowth produced by either of the glial CM, a nerve growth factor radioreceptor assay did detect an NGF-like molecule in both CM. Since the extensive neurite outgrowth stimulated by the glial CM was not mimicked by pure laminin alone, we conclude that the glial neurite promoting factors are distinct from laminin.