Journal of Neuroscience Research 30:484-492 (1991)
Correlation Between Morphological and Biochemical Effects of Ethanol on Neuroblast-Enriched Cultures Derived From Three-Day-Old Chick Embryos S. Kentroti and A. Vernadakis Departments of Pharmacology and Psychiatry, University of Colorado School of Medicine. Denver We have shown that ethanol exposure during embryogenesis affects a variety of parameters of neuronal growth. In this study we examined the direct effects of ethanol exposure on developing neuroblasts in culture. Neuroblast-enriched cultures derived from 3-day-old whole chick embryos were grown in the presence of ethanol at doses ranging from 12.5 to 50 mM from culture day 3-14. Cholinergic and GABAergic phenotypic expression were both significantly reduced following ethanol exposure as assessed by the activities of choline acetyltransferase and glutamate decarboxylase, respectively. Morphometric analysis of the growth patterns showed significant differences between control and ethanol-treated cultures. Control cultures exhibited the characteristic pattern of growth consisting of neuronal aggregation with neuritic arborization, i.e., neuritic bundles and fasciculation. Cultures grown in ethanol from culture day 3 consisted of aggregates that measured significantly greater in size than those observed in control cultures. In addition, in ethanol-treated cultures, the primary pattern of neuritic bundles was replaced by a complex network of individual neurites radiating from the central aggregate, forming a defined “neuritic field.” Morphometric analysis revealed that both neurite number and neurite length were significantly reduced in ethanol-treated cultures. The biochemical data confirm earlier reports from this laboratory suggesting that ethanol exposure during early embryogenesis alters the normal neuronal pattern of phenotypic expression. In addition, we have presented evidence in this study that ethanol alters the morphological growth patterns of developing neurons. Although ethanol does not alter the ability of these cells to aggregate, there is a significant alteration in neuritic outgrowth. We conclude that the alterations in the growth patterns induced by ethanol hinder full neurotransmitter phenotypic expression. 0 1991 Wiley-Liss, Inc.
Key words: ethanol, neurogenesis, cholinergic expression, GABAergic expression, morphometry INTRODUCTION We have reported that ethanol markedly decreases cholinergic neuronal expression and enhances catecholaminergic neuronal expression as assessed by choline acetyltransferase (ChAT) and tyrosine hydrox ylase (TH) activities, respectively, when administered to chick embryos in ovo at early embryogenesis (Brodie and Vernadakis, 1990; Kentroti and Vernadakis, 1990). In order to delineate the effects of ethanol at a more direct cellular level, we have initiated studies using neural culture, a valuable experimental tool for studying growth phenomena, regulatory factors for growth and pharmacological manipulations (Vernadakis and Culver, 1980; Jaeger et al., 1988; Kapoor et al., 1988). Neuronal growth, locomotion, and differentiation during the development of the embryo are under the influence and guidance of cellsecreted factors and cell-cell contacts. In this study, we used neuron-enriched cultures derived from 3-day-old whole chick embryo (E3WE). The neuronal composition of these cultures has been characterized biochemically and immunocytochemically . Furthermore, this system has been shown to be composed primarily of proliferating neuroblasts (Bennett and DiLullo, 1985; Vernadakis et al., 1986), an early developmental stage in which neurons are plastic and susceptible to manipulations of their local environment (i.e., media composition, culture substrata, nonneuronal cell constituent) (Mangoura et al., 1988b; Mangoura and Vernadakis, 1988). In this study neuron-enriched cultures were exposed to a range of ethanol concentrations (12.5-50 Received January 2, 1991; revised and accepted February 5 , 1991 Address reprint requests to Dr. Susan Kentroti, Dept. of Pharmacology, University of Colorado, School of Medicine, 4200 East Ninth Ave., Denver, CO 80262.
Effects of Ethanol on Chick Embryo Cultures mM) from culture day 3 and harvested at various intervals up to day 13. Cultures were examined biochemically for cholinergic and GABAergic neuronal expression using choline acetyltransferase (ChAT) and glutamic acid decarboxylase (GAD) as respective markers. They were further evaluated morphometrically using videometric analysis to compare growth patterns that may be affected by ethanol, and thus explain the biochemical changes. Ethanol exposure of early differentiating neuroblasts markedly retarded both cholinergic and GABAergic neuronal phenotypes. Moreover, the differences between growth patterns of control and ethanol-treated cultures were striking. We have tentatively concluded that ethanol, possibly through its well-established effects on membranes (Seeman, 1972; Lyon et al., 1981) interferes with neuritic elongation and growth cone motility and, ultimately, neuron-neuron intercommunications important for their phenotypic expression. We propose, therefore, that normal neuronal phenotypic expression is dependent to a certain degree on neuronal organization and adequate cell-cell contacts.
METHODS Animals All experiments were performed on white Leghorn chick embryos (SPAFAS, Norwich, CT). Embryos were grown in an automatic forced air incubator (Robbins), which automatically turned the eggs. The chamber was maintained at 37-38.5"C and 80-90% relative humidity.
Cell Cultures Neuron-enriched cultures derived from 3-day-old whole chick embryos (E3WE) were prepared as previously described (Vernadakis et al., 1986; Mangoura et al., 1988a). Briefly, 3-day-old whole chick embryos were freed of any attached membranous elements (allantois, yolk) and mechanically dissociated by sieving through a 48-pm nylon mesh in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). Approximately lo6 cells were plated per 35 mm polystyrene Petri dish (Lux) precoated with polyL-lysine (MW 30,000-70,000). All cultures were incubated at 37°C in an atmosphere of 7.5% CO, and 92.5% air, saturated with water. The neuronal composition and normal growth patterns of E3WE grown in DMEM + 10% FBS have been described in detail in studies from this laboratory (Vernadakis et al., 1986; Mangoura et al., 1988a,b). Ethanol Treatment Cultures were grown for 3 days in DMEM + 10% FBS in the absence of ethanol. Prior to ethanol administration, on day 3, five cultures were harvested and
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frozen for assay of ChAT, GAD, and protein to serve as a common control at time zero. The media was aspirated from the remaining cultures and replaced with DMEM + 10% FBS with or without ethyl alcohol (USP) in doses ranging from 12.5-50 mM. Ethanol-treated cultures were placed in the incubator above a pan containing 100 mM ethanol in water and sealed in a plastic bag. The media was exchanged every 3 days and samples were taken periodically to determine the media concentration of ethanol. The assay of ethanol concentration in the media was performed enzymatically by Dr. Rodney Baker (Alcohol Research Center, University of Colorado School of Medicine) and was shown to be maintained at the appropriate concentrations throughout the culture period. Five cultures per group were mechanically harvested on culture days 6, 10, and 13 using a teflon cell scraper. Cells were frozen at -20°C for assay of the activity of ChAT and GAD. In addition, cultures from each group were fixed at culture days 6 and 10, stained histochemically for acetylcholinesterase (Karnovsky and Roots, 1964) and analyzed morphometrically .
Morphometry Morphological parameters of growth in E3WE neuron-enriched cultures were quantitated using a videometric system. Images obtained through the microscope are captured on computer by a Targa M8 board. The images are then relayed to a video monitor from which measurements are made and analyzed by Image-Pro version 2.0 software. Morphological parameters examined for morphometric measurements include: (1) area of neuronal aggregate, (2) area of neuritic outgrowth field, (3) length of neuritic bundles, and (4) number of neuritic bundles. Growth patterns in neuronal cultures may differ among aggregates within the same culture or between cultures from different treatment groups. The following criteria are used in quantitatively analyzing growth patterns in these aggregates. The culture is first assessed for total number of aggregates per 35 mm culture dish. Cultures with numbers in excess of 50 aggregates are counted by selecting 15 random fields for analysis. In cultures with 50 or less aggregates, the entire culture dish is systematically analyzed. Area is expressed as units that are equivalent to pixels. Only aggregates with areas of 1,000 units or greater are included in measurements. In measuring area, only those cells that form a tightly packed aggregate are included in the trace; in other words, cells that may occur as an outgrowth of loosely dispersed cells in the periphery are not considered to compose the main aggregate. Neuronal aggregates differ widely with respect to outgrowth of neurites. Figures 3C and 4B illustrate two different patterns of neuritic outgrowth in E3WE cultures. In Figure 3C, the pattern consists of a relatively
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few (< 15) neuritic bundles emanating from the central aggregate. These aggregates are analyzed for number of neuritic bundles as well as total length of neuritic processes. Only neuritic processes of 25 units or greater are measured. Figure 4B illustrates an aggregate with a complex network of radiating neurites that form a neuritic field. In this example, it is the area of the neuritic field that is measured. In instances of neuronal aggregates with a combination of the two patterns of outgrowth, both the area of the neuritic field and the length and number of single neuritic bundles that occur outside of the neuritic field are computed. The data obtained for aggregate area, neurite number, and neurite length are expressed in histogram form with the x-axis signifying the frequency of occurrance and the y-axis representing specific ranges of: (1) aggregate area, (2) neurite number, or ( 3 ) neurite length. In contrast, neuritic field area is expressed as a mean ? s.e.m. for each treatment group.
Choline Acetyltransferase Activity Choline acetyltransferase (ChAT) activity was measured by the radiometric method of Fonnum (1975) as modified in our laboratory for cell culture (Sakellaridis et al., 1986; Kentroti and Vernadakis, 1990). This method employs a reaction catalyzed by tissue ChAT in which [3H]acetyl-CoA and unlabelled choline react to yield [3H]acetylcholine.The labelled acetylcholine is extracted by sodium tetraphenylboron into the toluene of the scintillation cocktail, whereas the substrate acetylCoA remains undetectable in the aqueous phase. Aliquots of supernatant were incubated for 30 min at 37°C in buffer substrate consisting of 8 mM choline-I, 19.6 pM [3H]acetyl-CoA, 100 pM eserine sulfate, 50 mM PO,, 20 mM EDTA, and 300 mM NaCl. The reaction was terminated by the addition of 10 mM PO, buffer, pH 7.4. Radiolabelled acetylcholine was extracted into the organic phase with Na-tetraphenylboron solution in 3heptanone (5g/L). ChAT activity is expressed as nmoles [3H]acetylcholine formed/h/mg protein.
2-mercaptoethanol and 50 mM KH,PO, buffer, pH 7.0, containing 1 mM EDTA and 0.5% Triton X-100, 1.25 mM P5P, 5 mM glutamate, and 0.25 pCi L-[l-',C]glutamic acid in 2% ethanol. Tubes were incubated for 20 min at 37°C and the liberated CO, collected on a piece of Whatman #1 paper presaturated with 0.2 ml hyamine hydroxide. The reaction was terminated with 10% TCA and the tubes incubated for an additional 90 min. Activity is expressed as nmoles I4CO2formed/20 min/mg protein.
Histochemistry Prior to videometric analysis, cultures from each treatment group were fixed for 5 min at -20°C in absolute methanol and stained for the activity of acetylcholinesterase using the method of Karnovsky and Roots (1964). This technique was used as morphological marker to identify both cholinergic and cholinoceptive neuronal populations. Further, this method increases cellular resolution for the purposes of morphometric analysis. Protein Assay Protein content in the supernatant was as determined according to the method of Lowry et al. (1951) using standards prepared with bovine serum albumin. Statistics All data collected in quantitative analyses were statistically described using the student's t tests for comparison of means.
RESULTS Biochemical Analysis of the Effects of Ethanol Choline acetyltransferase activity. The developmental profiles of ChAT in both control and ethanoltreated cultures are illustrated in Figure 1 and 2A. As previously reported (Vernadakis et al., 1986), in control cultures, ChAT activity rises slowly up to day 6 (Fig. 1) and remains at this level up to day 13, the end of the Glutamic Acid Decarboxylase Activity experimental period. Thus cholinergic neurons remain Glutamate decarboxylase (GAD), the enzyme that relatively active in culture for up to 2 weeks. In contrast, catalyzes the formation of GABA from L-glutamic acid, cultures treated with ethanol 12.5 to 50 mM (Fig. 1) was assayed using the method of Quinn and Cagan exhibited a marked dose-dependent decline in ChAT ac(1980), as modified in our laboratory for cell culture tivity beginning with day 6 (Figs. 1, 2A). (Mangoura and Vernadakis, 1988). The method is based Glutamic acid decarboxylase activity. As also on the conversion of L-l-'4C-glutamic acid to GABA reported previously (Mangoura and Vernadakis, 1988), and I4CO2, which is trapped and measured. Cell pellets the developmental profile of GAD activity in control were homogenized in 50 mM KH,PO, buffer, pH 7.0, cultures exhibits a rapid rise up to day 10 followed by a containing 1 mM aminoethylisothiouronium hydrobro- marked decline by day 12 (Fig. 2B). Thus it appears that mide (AET), 1 mM EDTA, and 0.2 mM pyridoxal phos- under these experimental conditions, GABAergic neuphate (P5P). GAD activity in the supernatant was deter- rons do not survive for extensive periods. Exposure to mined by adding reaction mixture consisting of 2.5 mM ethanol further exacerbated this neuronal decline: GAD
Effects of Ethanol on Chick Embryo Cultures
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Fig. 1. Activity of choline acetyltransferase (ChAT) in neuron-enriched cultures derived from 3-day-old whole chick embryos and grown in control medium (DMEM + lO%FBS) or medium supplemented with ethanol (12.5-50 mM). Ethanol (EtOH) was added to medium on culture day 3 and cells were harvested on culture days 6 and 10. ChAT activity is expressed as nmoles acetylcholine (Ach) formed/h/mg protein. Points with bracketed lines represent means SEM of 4-6 samples.
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Morphometric Analysis of the Effects of Ethanol General morphology. Photomicrographs in Figures 3 and 4 illustrate the neuronal growth patterns that were evaluated using videometric analysis. Figure 3A,C show two typical neuronal aggregates that possess thick neuritic bundles. Figures 3B and 4B illustrate two typical neuronal aggregates exhibiting large neuritic fields lacking the typical neuritic bundles. The most striking neuritic growth is illustrated in Figure 4B, an ethanol-treated culture with a well-developed neuritic field pattern of growth. Neuronai aggregate area. As described previously, the characteristic growth pattern of neurons in E3WE cultures is one of neuronal aggregation and neuritic arborization (Vernadakis et al., 1986). Figure 3A,C illustrate a typical neuronal aggregate from E3WE at 10 days in culture, with long neuritic bundles radiating from the central cell mass. In some cases, aggregates are interconnected by means of these large neuritic bundles. The primary growth pattern in ethanol-supplemented medium (12.5-50 mM) continues to be one of neuronal aggregation. The most significant difference occurs in comparing aggregate size (area) between treatment groups and controls (Fig. 5A); 55% of aggregates in control cultures measured between 2,500 and 5,000
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Fig. 2. Activities of choline acetyltransferase (ChAT) (A) or glutamate decarboxylase (GAD) (B) in neuron-enriched cultures derived from 3-day-old whole chick embryos and grown in control medium (DMEM + 10% FBS) or medium supplemented with ethanol (50 mM). Ethanol was added to the medium on culture day 3 and cells harvested on culture days 6, 10 and 13. ChAT activity is expressed as nmoles acetylcholine (Ach) formed/h/mg protein, and GAD activity is expressed as nmoles CO, formed/h/mg protein. Points with bracketed lines represent means ? SEM of 4-6 samples. units2 with 25% measuring > 5000 units2. In cultures treated with 12.5 mM ethanol, only 31% of aggregates exhibited these dimensions with 56% measuring greater than 5,000 units2. Measurements from cultures treated with 50 mM ethanol more closely approximated those obtained in controls (Fig. 5A) in that 57% of aggregates fell in the 2,500-5,000 unit2 range with only 14% measuring greater than 5,000 units2. Of interest is the observation that no significant difference in the total number of aggregateskulture dish was observed between control and 12.5 mM ethanol (11 and 16, respectively). However, cultures treated with 50 mM ethanol averaged 33% fewer aggregateskulture dish as compared control cultures (7 and l l , respectively). Neuritic number. The typical neuritic growth pattern observed in control cultures consisted of numerous neurites organized into bundles of varying thickness, em-
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Fig. 3 . Photomicrographs of neuronal aggregates from neuron-enriched cultures derived from 3-day-old whole chick embryos: grown in control medium and fixed at culture day 6 (A); grown in medium supplemented with ethanol (12.5 mM) and fixed at culture day 6 (B); grown in control medium and fixed at culture day 10 (C);and stained histochemically for acetyl-
cholinesterase. Control aggregates exhibit characteristic outgrowth of neurites in bundles of varying thickness radiating from the central aggregate. Neuritic outgrowth in the ethanoltreated culture at 6 days consisted predominantly of individual neurites which formed a mesh. Magnification, x 806.
anating from the central neuronal aggregate. As discussed in Methods, only neurites measuring > 25 units were measured. Upon quantification of these neuritic bundles, it was observed that their number was significantly greater in control than in ethanol-treated cultures (7.1 t 1.3 vs. 3.0 ? 0.6, respectively). Figure 5B illustrates the distribution of neurite number per aggregate with respect to treatment in culture. The occurrance of aggregates with > 4 neuritic bundledaggregate is significantly higher in controls versus ethanol-treated cultures. Neuritic length. Calculation of total neuritic length revealed that control cultures exhibited a bellshape distribution with 86% of neurites measuring between 20 and 250 units. Ethanol-treated cultures exhibited a very different distribution with respect to neurite length (Fig. 6). In cultures treated with 12.5 mM ethanol, neurite length was evenly distributed between 20 and > 400 units with a significant (74%) decrease in the
total number of neurites as compared with controls. Growth of cultures in 50 mM ethanol reduced the total number of neurites by 89% with the length distribution being similar to that observed in controls (78% of neurites measured between 20 and 200 units in length). Pattern of neuritic outgrowth. Growth of E3WE cultures in 12.5 mM ethanol resulted in a unique pattern of outgrowth, distinct from that of the neuritic bundles observed in controls. Whereas the occurrence of bundles of neuritic processes was decreased in ethanol-treated cultures, the occurrence of large fields of individual neurites was greatly increased, giving the appearance of a neuritic arbor. Figure 4B illustrates this distinctive growth pattern, which consists of an intricate network of individual neuritic processes radiating in a weblike pattern from the central aggregate. Because of the complexity of this growth pattern, it has been referred to as a neuritic field and can be expressed as a neuritic area
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Fig. 4. Photomicrographs of a neuronal aggregate from neuron-enriched culture derived from 3-day-old whole chick embryos and grown in medium supplemented with 12.5 mM ethanol. Culture was fixed at day 10, stained immunohistochemically for acetylcholinesterase, and photographed using (A) low power ( X 161), dark-field micrography (to include the entire aggregate); or (B) high power ( X 806), bright-field micrography in order to show the intricate outgrowth pattern of neurites. In contrast to control cultures (Fig. 3A,C), ethanoltreated cultures exhibit outgrowth consisting of individual neurites enamating from the central aggregate in a mesh-like pattern.
rather than length. Figure 7 compares the total area of neuritic fields in ethanol-treated versus control cultures. Both 12.5 and 50 mM ethanol increased the area of the neuritic outgrowth field by 225% and 210%, respectively. Typically, in ethanol-treated cultures, the neuritic outgrowth field emanates directly from the central aggregate with little or no organization of neuritic processes into bundles. In contrast, in control cultures neuritic fields often arise as offshoots secondary to neuritic bundles (Fig. 3A). These results, then, show a quantifiable difference in the growth patterns of neuron-enriched cultures derived from E3WE in response to supplementation of the growth medium with ethanol. Thus at low doses, ethanol increases aggregate area and shifts the neuritic outgrowth pattern from that of organized neuritic bundles to that of a weblike neuritic field.
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Fig. 5. Histogram showing the distribution of neuronal aggregates with respect to: (A) aggregate size (area), bars represent the total number of aggregates within each size range; and (B) the number of neuritedaggregate. Cultures were derived from E3WE, grown in the absence (control) or presence of ethanol (12.5 or 50 mM) and measured at day 10. All aggregates growing on the culture dishes were counted: control cultures contained 11-12 aggregateddish, cultures treated with 12.5 mM ethanol contained 15-16 aggregateddish and cultures treated with 50 mM ethanol contained 7-8 aggregatesldish. Units for measuring aggregate area in this experiment are defined as pixels2 (2500 pixels2 = 100 prn’).
DISCUSSION The results of the experiments reported in this study indicate that ethanol exerts (1) severe neuronotoxic effects as expressed by a decrease in two neuronal phenotypes, cholinergic and GABAergic, and (2) dramatically alters the neuronal growth pattern characteristic of the E3WE cultures. The aim of this discussion is to attempt to correlate the changes in neurotransmitter neuronal phenotypes with the morphometric changes produced by ethanol. The decrease in cholinergic and GABAergic neuronal phenotypic expression produced by ethanol (Figs. 1,2) supports our previous studies in ovo, where we found that ethanol administered at embryonic days 1-3,
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Fig. 6. Histogram showing the number of neurites (frequency) exhibiting the various length measurements. Neuritic bundles emanating from the central aggregate were examined in 10day-old cultures derived from E3WE, grown in the absence (control) or presence of ethanol (12.5 or 50 mM). Neurites from each aggregate on the culture dish were measured. Length units in this study are defined as pixels (50 pixels = 10 Km).
die and Vernadakis 1990; Brodie et al., 1990). Thus proliferating neuroblasts, whether in vivo or in vitro, appear to be vulnerable to ethanol insult. The morphological changes in growth patterns of the ethanol-treated cultures suggest that ethanol may retard normal patterns of neuronal growth, thus reducing phenotypic expression. As previously described in detail (Vernadakis et al., 1986; Mangoura et al., 1988a,b), the prevailing pattern of growth in E3WE cultures plated on poly-L-lysine is one of neuronal aggregation with emanating neuritic bundles of varying length and thickness which frequently interconnect aggregates or make contacts with flat cells. In the present study using videometric analysis, we measured the aggregate size (Fig. 5A) neurite number (Fig. 5B),neurite length (Fig. 6), and area of the neuritic field (Fig. 7) and compared these growth parameters between control and ethanol-treated cultures. In ethanol-treated cultures, the neuritic field with a neuritic arbor was the most consistant growth pattern, whereas in controls the characteristic pattern of medium-size aggregates with long neuritic bundles prevailed. Based on these morphometric observations, we speculate that ethanol interferes with neuritic growth, sprouting, and migration. An abundance of literature explores the numerous factors that may be involved in neuritic sprouting, including extracellular matrix factors (Akers et al., 1981; Krystosek and Seeds, 1981, 1984; Edelman, 1983; Monard et al., 1983), factors (including neurotransmitters) secreted by neurons themselves (see reviews, Varon et al., 1979; Varon and Adler, 1981; Berg, 1984), factors secreted by glial cells (see for review, Vernadakis, 1988). In our case several of these possibilities may partially explain the effects of ethanol: (1) lack of glial cells accompanying ethanol-treatment, (2) that ethanol inhibited production of substances involved in neuritic growth, and (3) changes in Ca+ flux produced by ethanol. We and others have shown that ethanol retards glial proliferation and differentiation (Davies and Vernadakis, 1984, 1986; Kennedy and Muerji, 1986a,b); consequently, these E3WE cultures grown on poly-L-lysine do not show an abundance of glial cells. However, there may be enough glial cells in the control cultures to account for neurite elongation and migration. Studies by Mattson and Kater (1987) and Mattson et a1 (1988) have shown that intracellular calcium can regulate the pattern of neuritic growth in a variety of different, identified neurons depending upon changes in calcium influx. Furthermore, ethanol, in a variety of experimental paradigms, inhibits calcium influx. Ethano1 inhibits voltage-dependent 45Ca+ influx . into PC12 cells (Messing et al., 1986), synaptosomes (Harris and Hood, 1980; Stokes and Harris, 1982), and also in PC12, ethanol inhibits muscarine-stimulated elevation of intracellular free Ca+ , which corresponded with the inhi+
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Fig. 7. Barogram showing the area of the neuritic field outgrowth in 10-day-old cultures derived from ESWE and grown in the absence (control) or presence of ethanol (EtOH; 12.5 or 50 mM).Every aggregate growing on the culture dish surface was assessed for neuritic field area. Units represent pixels' and 2500 pixels2 = 100 pm'. Bars represent the mean +- SEM of 5-9 fields/group. "P < 0.001 versus control.
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a period of active neuronal proliferation (Bennett and DiLullo, 1985), markedly retards neuronal phenotypic expression (Kentroti and Vernadakis, 1990, 1991; Bro-
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Effects of Ethanol on Chick Embryo Cultures
bition of 3H-norepinephrine release (Rabe and Weight, 1988). Based on these reports, we propose that the decrease in neurite elongation in ethanol-treated cultures may be caused by (1) an inhibition of Ca+ influx, and/or (2) a decrease of possible neurite elongation-promoting molecules. The phenomenon of fasciculation during neurogenesis as well as the topographic organization of growing axons (Straznicky et al., 1979; Holt and Harris, 1983) suggest that axon-axon interactions play a major role in pathway guidance and mapping in the developing nervous system. Several studies have reported that ethanol interferes with production of neurotrophic factors that may be essential to this process. For example, Riopelle and Cameron (1984) reported that developing neurons can display an autocrine phenotype, e.g., production of a substrate-attachedfactor, which enhances neuronal survival and neurite extension. In another study, Dow and Riopelle (1985) reported that ethanol inhibited process formation in cultures derived from chick embryo sensory and spinal cord grown on several biological substrates. Alternatively, ethanol may interfere with the uptake or binding of substances from the nutrient medium and serum that are important for neurite growth, elongation, and migration. This theory is supported by studies from our laboratory in which cotreatment of embryos with NGF, EGF, or the neurotrophic peptides, GHRH or SRIF, attenuated the cholinotoxic effects of ethanol (Brodie et al., 1990; Kentroti and Vernadakis, 1990). Low concentrations of glutamate have also been demonstrated to stimulate neurite outgrowth and elongation (Pearce et al., 1987; Bulloch, 1989). Our findings that ethanol retards GABAergic neuronal expression and decreases activity of glutamine synthetase (Davies and Vernadakis, 1984) supports the notion that ethanol interferes with glutamic acid-glutamine-GABAcompartmentation, which, as proposed by Hertz (1979), plays an important role in aminergic neuronal homeostasis. Finally, the effect of ethanol on maturing GABAergic neurons may reflect the general inhibitory effects of ethanol on GABAergic function (Ticku, 1980; Stokes and Harris, 1982; Hams and Sinclair, 1984a,b). Based on our morphological, morphometric, and biochemical findings, we propose that normal neuronal phenotypic expression is dependent to a certain degree on neuronal organization and adequate cell-cell contacts. The long neurites observed in the control cultures may be the cellular messenger for tropic factors and ionic signals. This growth pattern being less defined in the ethanol-treated cultures hinders full neuronal expression and differentiation. It is not surprising, then, that addition of factors to the chick embryo environment or in culture attenuates these neuronotoxic effects of ethanol (Brodie et al., 1990; Kentroti and Vernadakis, 1990). +
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Krystosek A, Seeds NW (1984): Peripheral neurons and Schwann cells secrete plasminogen activator. J Cell Biol 98:773-776. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951): Protein determination with the Folin phenol reagent. J Biol Chem 193: 265 -275. Lyon RC, McComb JA, Schreurs J , Goldstein DB (1981): A relationship between alcohol intoxication and the disordering of brain membranes by a series of short-chain alcohols. J Pharmac Exp Ther 218:669-675. Mangoura D, Sakellaridis N, Vernadakis A (1990): Evidence for plasticity in neurotransmitter expression in neuronal cultures derived from 3-day-old chick embryo. Dev Brain Res 51 :93-101. Mangoura D, Vernadakis A (1988): GABAergic neurons in cultures derived from three-, six-, or eight-day-old chick embryo: A biochemical and immunocytochemical study. Dev Brain Res 40:25-35. Mangoura D, Sakellaridis N, Vernadakis A (1988a): Cholinergic neurons in cultures derived from three-, six-, or eight-day-old chick embryo: A biochemical and immunocytochemical study. Dev Brain Res 40:37-46. Mangoura D, Sakellaridis N, Vernadakis A (1988b): Factors influencing neuronal growth in primary cultures derived from 3day-old chick embryos. Int J Develop Neurosci 6:89-102. Mattson MP, Kater SB (1987): Calcium regulation of neurite elongation and growth cone motility. J Neurosci 7:4034-4043. Mattson MP, Taylor-Hunter A, Kater SB (1988): Neurite outgrowth in individual neurons of a neuronal population is differentially regulated by calcium and cyclic AMP. J Neurosci 8:17041711. Messing RO, Carpenter CL, Diamond I, Greenberg DA (1986): Ethanol regulates calcium channels in clonal neural cells. Proc Natl Acad Sci USA 83:6213-6215. Monard D, Niday E, Limat A, Solomon F (1983): Inhibition of protease activity can lead to neurite extension in neuroblastoma cells. Prog Brain Res 58:359-364. Pearce IA, Cambray-Deakin MA, Burgoyne RD (1987): Glutamate acting on NMDA receptors stimulates neurite outgrowth from cerebellar granule cells. FEBS Lett 223: 143-147.
Quinn MR, Cagan RH (1980): Subcellular distribution of glutamate decarboxylase in rat olfactory bulb: High content in dendrodendritic synaptosomes. J Neurochem 35583-590. Rabe CS, Weight FF (1988): Effects of ethanol on neurotransmitter release and intracellular free calcium in PC12 cells. J Pharmacol Exp Ther 244:417-422. Riopelle RJ, Cameron DA (1984): Neurite-promoting factors from embryonic neurons. Dev Brain Res 15:265-274. Sakellaridis N, Mangoura D, Vernadakis A (1986): Effects of opiates on the growth of neuron-enriched cultures from chick embryonic brain. Int J Develop Neurosci 4:293-302. Seeman P (1972): The membrane actions of anesthetics and tranquilizers. Pharmac Rev 24:583-655. Stokes JA, Harris RA (1982): Alcohols and synaptosomal calcium transport. Molecular Pharmacol 22:99-104. Straznicky C, Gaze RM, Horden TJ (1979): Selection of appropriate medial branch of the optic tract by fibres of ventral retinal origin during development and in regeneration: An autoradiographic Xenopus. J Embryo1 Exp Morphol 50:253-267. Ticku MK (1980): The effects of acute and chronic ethanol administration and its withdrawal on GABA receptor binding in rat brain. Br J Pharmacol 70:403-410. Varon S, Adler R (1981): Trophic and specifying factors directed to neuronal cells. Adv Cell Neurobiol 2: 115-163. Varon S , Manthorpe M, Adler R (1979): Cholinergic neuronotrophic factors: I. Survival, neurite outgrowth and choline acetyltransferase activity in monolayer cultures from chick embryo ciliary ganglia. Brain Res 173:29-45. Vernadakis A (1988): Neuron-Glia Interrelations. Int Rev Neurobiol 30:149-224. Vernadakis A, Culver B (1980): Neural tissue culture: A biochemical tool. In Kumar S (ed): “Biochemistry of the Brain.” New York: Pergamon Press, pp 407-452. Vernadakis A, Sakellaridis N, Mangoura D (1986): Growth patterns of primary cultures dissociated from 3-day-old chick embryos: Morphological and biochemical comparisons. J Neurosci Res 16:397-407.
Correlation Between Morphological and Biochemical Effects of Ethanol on Neuroblast-Enriched Cultures Derived From Three-Day-Old Chick Embryos S. Kentroti and A. Vernadakis Departments of Pharmacology and Psychiatry, University of Colorado School of Medicine. Denver We have shown that ethanol exposure during embryogenesis affects a variety of parameters of neuronal growth. In this study we examined the direct effects of ethanol exposure on developing neuroblasts in culture. Neuroblast-enriched cultures derived from 3-day-old whole chick embryos were grown in the presence of ethanol at doses ranging from 12.5 to 50 mM from culture day 3-14. Cholinergic and GABAergic phenotypic expression were both significantly reduced following ethanol exposure as assessed by the activities of choline acetyltransferase and glutamate decarboxylase, respectively. Morphometric analysis of the growth patterns showed significant differences between control and ethanol-treated cultures. Control cultures exhibited the characteristic pattern of growth consisting of neuronal aggregation with neuritic arborization, i.e., neuritic bundles and fasciculation. Cultures grown in ethanol from culture day 3 consisted of aggregates that measured significantly greater in size than those observed in control cultures. In addition, in ethanol-treated cultures, the primary pattern of neuritic bundles was replaced by a complex network of individual neurites radiating from the central aggregate, forming a defined “neuritic field.” Morphometric analysis revealed that both neurite number and neurite length were significantly reduced in ethanol-treated cultures. The biochemical data confirm earlier reports from this laboratory suggesting that ethanol exposure during early embryogenesis alters the normal neuronal pattern of phenotypic expression. In addition, we have presented evidence in this study that ethanol alters the morphological growth patterns of developing neurons. Although ethanol does not alter the ability of these cells to aggregate, there is a significant alteration in neuritic outgrowth. We conclude that the alterations in the growth patterns induced by ethanol hinder full neurotransmitter phenotypic expression. 0 1991 Wiley-Liss, Inc.
Key words: ethanol, neurogenesis, cholinergic expression, GABAergic expression, morphometry INTRODUCTION We have reported that ethanol markedly decreases cholinergic neuronal expression and enhances catecholaminergic neuronal expression as assessed by choline acetyltransferase (ChAT) and tyrosine hydrox ylase (TH) activities, respectively, when administered to chick embryos in ovo at early embryogenesis (Brodie and Vernadakis, 1990; Kentroti and Vernadakis, 1990). In order to delineate the effects of ethanol at a more direct cellular level, we have initiated studies using neural culture, a valuable experimental tool for studying growth phenomena, regulatory factors for growth and pharmacological manipulations (Vernadakis and Culver, 1980; Jaeger et al., 1988; Kapoor et al., 1988). Neuronal growth, locomotion, and differentiation during the development of the embryo are under the influence and guidance of cellsecreted factors and cell-cell contacts. In this study, we used neuron-enriched cultures derived from 3-day-old whole chick embryo (E3WE). The neuronal composition of these cultures has been characterized biochemically and immunocytochemically . Furthermore, this system has been shown to be composed primarily of proliferating neuroblasts (Bennett and DiLullo, 1985; Vernadakis et al., 1986), an early developmental stage in which neurons are plastic and susceptible to manipulations of their local environment (i.e., media composition, culture substrata, nonneuronal cell constituent) (Mangoura et al., 1988b; Mangoura and Vernadakis, 1988). In this study neuron-enriched cultures were exposed to a range of ethanol concentrations (12.5-50 Received January 2, 1991; revised and accepted February 5 , 1991 Address reprint requests to Dr. Susan Kentroti, Dept. of Pharmacology, University of Colorado, School of Medicine, 4200 East Ninth Ave., Denver, CO 80262.
Effects of Ethanol on Chick Embryo Cultures mM) from culture day 3 and harvested at various intervals up to day 13. Cultures were examined biochemically for cholinergic and GABAergic neuronal expression using choline acetyltransferase (ChAT) and glutamic acid decarboxylase (GAD) as respective markers. They were further evaluated morphometrically using videometric analysis to compare growth patterns that may be affected by ethanol, and thus explain the biochemical changes. Ethanol exposure of early differentiating neuroblasts markedly retarded both cholinergic and GABAergic neuronal phenotypes. Moreover, the differences between growth patterns of control and ethanol-treated cultures were striking. We have tentatively concluded that ethanol, possibly through its well-established effects on membranes (Seeman, 1972; Lyon et al., 1981) interferes with neuritic elongation and growth cone motility and, ultimately, neuron-neuron intercommunications important for their phenotypic expression. We propose, therefore, that normal neuronal phenotypic expression is dependent to a certain degree on neuronal organization and adequate cell-cell contacts.
METHODS Animals All experiments were performed on white Leghorn chick embryos (SPAFAS, Norwich, CT). Embryos were grown in an automatic forced air incubator (Robbins), which automatically turned the eggs. The chamber was maintained at 37-38.5"C and 80-90% relative humidity.
Cell Cultures Neuron-enriched cultures derived from 3-day-old whole chick embryos (E3WE) were prepared as previously described (Vernadakis et al., 1986; Mangoura et al., 1988a). Briefly, 3-day-old whole chick embryos were freed of any attached membranous elements (allantois, yolk) and mechanically dissociated by sieving through a 48-pm nylon mesh in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). Approximately lo6 cells were plated per 35 mm polystyrene Petri dish (Lux) precoated with polyL-lysine (MW 30,000-70,000). All cultures were incubated at 37°C in an atmosphere of 7.5% CO, and 92.5% air, saturated with water. The neuronal composition and normal growth patterns of E3WE grown in DMEM + 10% FBS have been described in detail in studies from this laboratory (Vernadakis et al., 1986; Mangoura et al., 1988a,b). Ethanol Treatment Cultures were grown for 3 days in DMEM + 10% FBS in the absence of ethanol. Prior to ethanol administration, on day 3, five cultures were harvested and
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frozen for assay of ChAT, GAD, and protein to serve as a common control at time zero. The media was aspirated from the remaining cultures and replaced with DMEM + 10% FBS with or without ethyl alcohol (USP) in doses ranging from 12.5-50 mM. Ethanol-treated cultures were placed in the incubator above a pan containing 100 mM ethanol in water and sealed in a plastic bag. The media was exchanged every 3 days and samples were taken periodically to determine the media concentration of ethanol. The assay of ethanol concentration in the media was performed enzymatically by Dr. Rodney Baker (Alcohol Research Center, University of Colorado School of Medicine) and was shown to be maintained at the appropriate concentrations throughout the culture period. Five cultures per group were mechanically harvested on culture days 6, 10, and 13 using a teflon cell scraper. Cells were frozen at -20°C for assay of the activity of ChAT and GAD. In addition, cultures from each group were fixed at culture days 6 and 10, stained histochemically for acetylcholinesterase (Karnovsky and Roots, 1964) and analyzed morphometrically .
Morphometry Morphological parameters of growth in E3WE neuron-enriched cultures were quantitated using a videometric system. Images obtained through the microscope are captured on computer by a Targa M8 board. The images are then relayed to a video monitor from which measurements are made and analyzed by Image-Pro version 2.0 software. Morphological parameters examined for morphometric measurements include: (1) area of neuronal aggregate, (2) area of neuritic outgrowth field, (3) length of neuritic bundles, and (4) number of neuritic bundles. Growth patterns in neuronal cultures may differ among aggregates within the same culture or between cultures from different treatment groups. The following criteria are used in quantitatively analyzing growth patterns in these aggregates. The culture is first assessed for total number of aggregates per 35 mm culture dish. Cultures with numbers in excess of 50 aggregates are counted by selecting 15 random fields for analysis. In cultures with 50 or less aggregates, the entire culture dish is systematically analyzed. Area is expressed as units that are equivalent to pixels. Only aggregates with areas of 1,000 units or greater are included in measurements. In measuring area, only those cells that form a tightly packed aggregate are included in the trace; in other words, cells that may occur as an outgrowth of loosely dispersed cells in the periphery are not considered to compose the main aggregate. Neuronal aggregates differ widely with respect to outgrowth of neurites. Figures 3C and 4B illustrate two different patterns of neuritic outgrowth in E3WE cultures. In Figure 3C, the pattern consists of a relatively
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few (< 15) neuritic bundles emanating from the central aggregate. These aggregates are analyzed for number of neuritic bundles as well as total length of neuritic processes. Only neuritic processes of 25 units or greater are measured. Figure 4B illustrates an aggregate with a complex network of radiating neurites that form a neuritic field. In this example, it is the area of the neuritic field that is measured. In instances of neuronal aggregates with a combination of the two patterns of outgrowth, both the area of the neuritic field and the length and number of single neuritic bundles that occur outside of the neuritic field are computed. The data obtained for aggregate area, neurite number, and neurite length are expressed in histogram form with the x-axis signifying the frequency of occurrance and the y-axis representing specific ranges of: (1) aggregate area, (2) neurite number, or ( 3 ) neurite length. In contrast, neuritic field area is expressed as a mean ? s.e.m. for each treatment group.
Choline Acetyltransferase Activity Choline acetyltransferase (ChAT) activity was measured by the radiometric method of Fonnum (1975) as modified in our laboratory for cell culture (Sakellaridis et al., 1986; Kentroti and Vernadakis, 1990). This method employs a reaction catalyzed by tissue ChAT in which [3H]acetyl-CoA and unlabelled choline react to yield [3H]acetylcholine.The labelled acetylcholine is extracted by sodium tetraphenylboron into the toluene of the scintillation cocktail, whereas the substrate acetylCoA remains undetectable in the aqueous phase. Aliquots of supernatant were incubated for 30 min at 37°C in buffer substrate consisting of 8 mM choline-I, 19.6 pM [3H]acetyl-CoA, 100 pM eserine sulfate, 50 mM PO,, 20 mM EDTA, and 300 mM NaCl. The reaction was terminated by the addition of 10 mM PO, buffer, pH 7.4. Radiolabelled acetylcholine was extracted into the organic phase with Na-tetraphenylboron solution in 3heptanone (5g/L). ChAT activity is expressed as nmoles [3H]acetylcholine formed/h/mg protein.
2-mercaptoethanol and 50 mM KH,PO, buffer, pH 7.0, containing 1 mM EDTA and 0.5% Triton X-100, 1.25 mM P5P, 5 mM glutamate, and 0.25 pCi L-[l-',C]glutamic acid in 2% ethanol. Tubes were incubated for 20 min at 37°C and the liberated CO, collected on a piece of Whatman #1 paper presaturated with 0.2 ml hyamine hydroxide. The reaction was terminated with 10% TCA and the tubes incubated for an additional 90 min. Activity is expressed as nmoles I4CO2formed/20 min/mg protein.
Histochemistry Prior to videometric analysis, cultures from each treatment group were fixed for 5 min at -20°C in absolute methanol and stained for the activity of acetylcholinesterase using the method of Karnovsky and Roots (1964). This technique was used as morphological marker to identify both cholinergic and cholinoceptive neuronal populations. Further, this method increases cellular resolution for the purposes of morphometric analysis. Protein Assay Protein content in the supernatant was as determined according to the method of Lowry et al. (1951) using standards prepared with bovine serum albumin. Statistics All data collected in quantitative analyses were statistically described using the student's t tests for comparison of means.
RESULTS Biochemical Analysis of the Effects of Ethanol Choline acetyltransferase activity. The developmental profiles of ChAT in both control and ethanoltreated cultures are illustrated in Figure 1 and 2A. As previously reported (Vernadakis et al., 1986), in control cultures, ChAT activity rises slowly up to day 6 (Fig. 1) and remains at this level up to day 13, the end of the Glutamic Acid Decarboxylase Activity experimental period. Thus cholinergic neurons remain Glutamate decarboxylase (GAD), the enzyme that relatively active in culture for up to 2 weeks. In contrast, catalyzes the formation of GABA from L-glutamic acid, cultures treated with ethanol 12.5 to 50 mM (Fig. 1) was assayed using the method of Quinn and Cagan exhibited a marked dose-dependent decline in ChAT ac(1980), as modified in our laboratory for cell culture tivity beginning with day 6 (Figs. 1, 2A). (Mangoura and Vernadakis, 1988). The method is based Glutamic acid decarboxylase activity. As also on the conversion of L-l-'4C-glutamic acid to GABA reported previously (Mangoura and Vernadakis, 1988), and I4CO2, which is trapped and measured. Cell pellets the developmental profile of GAD activity in control were homogenized in 50 mM KH,PO, buffer, pH 7.0, cultures exhibits a rapid rise up to day 10 followed by a containing 1 mM aminoethylisothiouronium hydrobro- marked decline by day 12 (Fig. 2B). Thus it appears that mide (AET), 1 mM EDTA, and 0.2 mM pyridoxal phos- under these experimental conditions, GABAergic neuphate (P5P). GAD activity in the supernatant was deter- rons do not survive for extensive periods. Exposure to mined by adding reaction mixture consisting of 2.5 mM ethanol further exacerbated this neuronal decline: GAD
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Fig. 1. Activity of choline acetyltransferase (ChAT) in neuron-enriched cultures derived from 3-day-old whole chick embryos and grown in control medium (DMEM + lO%FBS) or medium supplemented with ethanol (12.5-50 mM). Ethanol (EtOH) was added to medium on culture day 3 and cells were harvested on culture days 6 and 10. ChAT activity is expressed as nmoles acetylcholine (Ach) formed/h/mg protein. Points with bracketed lines represent means SEM of 4-6 samples.
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Morphometric Analysis of the Effects of Ethanol General morphology. Photomicrographs in Figures 3 and 4 illustrate the neuronal growth patterns that were evaluated using videometric analysis. Figure 3A,C show two typical neuronal aggregates that possess thick neuritic bundles. Figures 3B and 4B illustrate two typical neuronal aggregates exhibiting large neuritic fields lacking the typical neuritic bundles. The most striking neuritic growth is illustrated in Figure 4B, an ethanol-treated culture with a well-developed neuritic field pattern of growth. Neuronai aggregate area. As described previously, the characteristic growth pattern of neurons in E3WE cultures is one of neuronal aggregation and neuritic arborization (Vernadakis et al., 1986). Figure 3A,C illustrate a typical neuronal aggregate from E3WE at 10 days in culture, with long neuritic bundles radiating from the central cell mass. In some cases, aggregates are interconnected by means of these large neuritic bundles. The primary growth pattern in ethanol-supplemented medium (12.5-50 mM) continues to be one of neuronal aggregation. The most significant difference occurs in comparing aggregate size (area) between treatment groups and controls (Fig. 5A); 55% of aggregates in control cultures measured between 2,500 and 5,000
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Fig. 2. Activities of choline acetyltransferase (ChAT) (A) or glutamate decarboxylase (GAD) (B) in neuron-enriched cultures derived from 3-day-old whole chick embryos and grown in control medium (DMEM + 10% FBS) or medium supplemented with ethanol (50 mM). Ethanol was added to the medium on culture day 3 and cells harvested on culture days 6, 10 and 13. ChAT activity is expressed as nmoles acetylcholine (Ach) formed/h/mg protein, and GAD activity is expressed as nmoles CO, formed/h/mg protein. Points with bracketed lines represent means ? SEM of 4-6 samples. units2 with 25% measuring > 5000 units2. In cultures treated with 12.5 mM ethanol, only 31% of aggregates exhibited these dimensions with 56% measuring greater than 5,000 units2. Measurements from cultures treated with 50 mM ethanol more closely approximated those obtained in controls (Fig. 5A) in that 57% of aggregates fell in the 2,500-5,000 unit2 range with only 14% measuring greater than 5,000 units2. Of interest is the observation that no significant difference in the total number of aggregateskulture dish was observed between control and 12.5 mM ethanol (11 and 16, respectively). However, cultures treated with 50 mM ethanol averaged 33% fewer aggregateskulture dish as compared control cultures (7 and l l , respectively). Neuritic number. The typical neuritic growth pattern observed in control cultures consisted of numerous neurites organized into bundles of varying thickness, em-
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Fig. 3 . Photomicrographs of neuronal aggregates from neuron-enriched cultures derived from 3-day-old whole chick embryos: grown in control medium and fixed at culture day 6 (A); grown in medium supplemented with ethanol (12.5 mM) and fixed at culture day 6 (B); grown in control medium and fixed at culture day 10 (C);and stained histochemically for acetyl-
cholinesterase. Control aggregates exhibit characteristic outgrowth of neurites in bundles of varying thickness radiating from the central aggregate. Neuritic outgrowth in the ethanoltreated culture at 6 days consisted predominantly of individual neurites which formed a mesh. Magnification, x 806.
anating from the central neuronal aggregate. As discussed in Methods, only neurites measuring > 25 units were measured. Upon quantification of these neuritic bundles, it was observed that their number was significantly greater in control than in ethanol-treated cultures (7.1 t 1.3 vs. 3.0 ? 0.6, respectively). Figure 5B illustrates the distribution of neurite number per aggregate with respect to treatment in culture. The occurrance of aggregates with > 4 neuritic bundledaggregate is significantly higher in controls versus ethanol-treated cultures. Neuritic length. Calculation of total neuritic length revealed that control cultures exhibited a bellshape distribution with 86% of neurites measuring between 20 and 250 units. Ethanol-treated cultures exhibited a very different distribution with respect to neurite length (Fig. 6). In cultures treated with 12.5 mM ethanol, neurite length was evenly distributed between 20 and > 400 units with a significant (74%) decrease in the
total number of neurites as compared with controls. Growth of cultures in 50 mM ethanol reduced the total number of neurites by 89% with the length distribution being similar to that observed in controls (78% of neurites measured between 20 and 200 units in length). Pattern of neuritic outgrowth. Growth of E3WE cultures in 12.5 mM ethanol resulted in a unique pattern of outgrowth, distinct from that of the neuritic bundles observed in controls. Whereas the occurrence of bundles of neuritic processes was decreased in ethanol-treated cultures, the occurrence of large fields of individual neurites was greatly increased, giving the appearance of a neuritic arbor. Figure 4B illustrates this distinctive growth pattern, which consists of an intricate network of individual neuritic processes radiating in a weblike pattern from the central aggregate. Because of the complexity of this growth pattern, it has been referred to as a neuritic field and can be expressed as a neuritic area
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Fig. 4. Photomicrographs of a neuronal aggregate from neuron-enriched culture derived from 3-day-old whole chick embryos and grown in medium supplemented with 12.5 mM ethanol. Culture was fixed at day 10, stained immunohistochemically for acetylcholinesterase, and photographed using (A) low power ( X 161), dark-field micrography (to include the entire aggregate); or (B) high power ( X 806), bright-field micrography in order to show the intricate outgrowth pattern of neurites. In contrast to control cultures (Fig. 3A,C), ethanoltreated cultures exhibit outgrowth consisting of individual neurites enamating from the central aggregate in a mesh-like pattern.
rather than length. Figure 7 compares the total area of neuritic fields in ethanol-treated versus control cultures. Both 12.5 and 50 mM ethanol increased the area of the neuritic outgrowth field by 225% and 210%, respectively. Typically, in ethanol-treated cultures, the neuritic outgrowth field emanates directly from the central aggregate with little or no organization of neuritic processes into bundles. In contrast, in control cultures neuritic fields often arise as offshoots secondary to neuritic bundles (Fig. 3A). These results, then, show a quantifiable difference in the growth patterns of neuron-enriched cultures derived from E3WE in response to supplementation of the growth medium with ethanol. Thus at low doses, ethanol increases aggregate area and shifts the neuritic outgrowth pattern from that of organized neuritic bundles to that of a weblike neuritic field.
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Fig. 5. Histogram showing the distribution of neuronal aggregates with respect to: (A) aggregate size (area), bars represent the total number of aggregates within each size range; and (B) the number of neuritedaggregate. Cultures were derived from E3WE, grown in the absence (control) or presence of ethanol (12.5 or 50 mM) and measured at day 10. All aggregates growing on the culture dishes were counted: control cultures contained 11-12 aggregateddish, cultures treated with 12.5 mM ethanol contained 15-16 aggregateddish and cultures treated with 50 mM ethanol contained 7-8 aggregatesldish. Units for measuring aggregate area in this experiment are defined as pixels2 (2500 pixels2 = 100 prn’).
DISCUSSION The results of the experiments reported in this study indicate that ethanol exerts (1) severe neuronotoxic effects as expressed by a decrease in two neuronal phenotypes, cholinergic and GABAergic, and (2) dramatically alters the neuronal growth pattern characteristic of the E3WE cultures. The aim of this discussion is to attempt to correlate the changes in neurotransmitter neuronal phenotypes with the morphometric changes produced by ethanol. The decrease in cholinergic and GABAergic neuronal phenotypic expression produced by ethanol (Figs. 1,2) supports our previous studies in ovo, where we found that ethanol administered at embryonic days 1-3,
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Fig. 6. Histogram showing the number of neurites (frequency) exhibiting the various length measurements. Neuritic bundles emanating from the central aggregate were examined in 10day-old cultures derived from E3WE, grown in the absence (control) or presence of ethanol (12.5 or 50 mM). Neurites from each aggregate on the culture dish were measured. Length units in this study are defined as pixels (50 pixels = 10 Km).
die and Vernadakis 1990; Brodie et al., 1990). Thus proliferating neuroblasts, whether in vivo or in vitro, appear to be vulnerable to ethanol insult. The morphological changes in growth patterns of the ethanol-treated cultures suggest that ethanol may retard normal patterns of neuronal growth, thus reducing phenotypic expression. As previously described in detail (Vernadakis et al., 1986; Mangoura et al., 1988a,b), the prevailing pattern of growth in E3WE cultures plated on poly-L-lysine is one of neuronal aggregation with emanating neuritic bundles of varying length and thickness which frequently interconnect aggregates or make contacts with flat cells. In the present study using videometric analysis, we measured the aggregate size (Fig. 5A) neurite number (Fig. 5B),neurite length (Fig. 6), and area of the neuritic field (Fig. 7) and compared these growth parameters between control and ethanol-treated cultures. In ethanol-treated cultures, the neuritic field with a neuritic arbor was the most consistant growth pattern, whereas in controls the characteristic pattern of medium-size aggregates with long neuritic bundles prevailed. Based on these morphometric observations, we speculate that ethanol interferes with neuritic growth, sprouting, and migration. An abundance of literature explores the numerous factors that may be involved in neuritic sprouting, including extracellular matrix factors (Akers et al., 1981; Krystosek and Seeds, 1981, 1984; Edelman, 1983; Monard et al., 1983), factors (including neurotransmitters) secreted by neurons themselves (see reviews, Varon et al., 1979; Varon and Adler, 1981; Berg, 1984), factors secreted by glial cells (see for review, Vernadakis, 1988). In our case several of these possibilities may partially explain the effects of ethanol: (1) lack of glial cells accompanying ethanol-treatment, (2) that ethanol inhibited production of substances involved in neuritic growth, and (3) changes in Ca+ flux produced by ethanol. We and others have shown that ethanol retards glial proliferation and differentiation (Davies and Vernadakis, 1984, 1986; Kennedy and Muerji, 1986a,b); consequently, these E3WE cultures grown on poly-L-lysine do not show an abundance of glial cells. However, there may be enough glial cells in the control cultures to account for neurite elongation and migration. Studies by Mattson and Kater (1987) and Mattson et a1 (1988) have shown that intracellular calcium can regulate the pattern of neuritic growth in a variety of different, identified neurons depending upon changes in calcium influx. Furthermore, ethanol, in a variety of experimental paradigms, inhibits calcium influx. Ethano1 inhibits voltage-dependent 45Ca+ influx . into PC12 cells (Messing et al., 1986), synaptosomes (Harris and Hood, 1980; Stokes and Harris, 1982), and also in PC12, ethanol inhibits muscarine-stimulated elevation of intracellular free Ca+ , which corresponded with the inhi+
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Fig. 7. Barogram showing the area of the neuritic field outgrowth in 10-day-old cultures derived from ESWE and grown in the absence (control) or presence of ethanol (EtOH; 12.5 or 50 mM).Every aggregate growing on the culture dish surface was assessed for neuritic field area. Units represent pixels' and 2500 pixels2 = 100 pm'. Bars represent the mean +- SEM of 5-9 fields/group. "P < 0.001 versus control.
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Effects of Ethanol on Chick Embryo Cultures
bition of 3H-norepinephrine release (Rabe and Weight, 1988). Based on these reports, we propose that the decrease in neurite elongation in ethanol-treated cultures may be caused by (1) an inhibition of Ca+ influx, and/or (2) a decrease of possible neurite elongation-promoting molecules. The phenomenon of fasciculation during neurogenesis as well as the topographic organization of growing axons (Straznicky et al., 1979; Holt and Harris, 1983) suggest that axon-axon interactions play a major role in pathway guidance and mapping in the developing nervous system. Several studies have reported that ethanol interferes with production of neurotrophic factors that may be essential to this process. For example, Riopelle and Cameron (1984) reported that developing neurons can display an autocrine phenotype, e.g., production of a substrate-attachedfactor, which enhances neuronal survival and neurite extension. In another study, Dow and Riopelle (1985) reported that ethanol inhibited process formation in cultures derived from chick embryo sensory and spinal cord grown on several biological substrates. Alternatively, ethanol may interfere with the uptake or binding of substances from the nutrient medium and serum that are important for neurite growth, elongation, and migration. This theory is supported by studies from our laboratory in which cotreatment of embryos with NGF, EGF, or the neurotrophic peptides, GHRH or SRIF, attenuated the cholinotoxic effects of ethanol (Brodie et al., 1990; Kentroti and Vernadakis, 1990). Low concentrations of glutamate have also been demonstrated to stimulate neurite outgrowth and elongation (Pearce et al., 1987; Bulloch, 1989). Our findings that ethanol retards GABAergic neuronal expression and decreases activity of glutamine synthetase (Davies and Vernadakis, 1984) supports the notion that ethanol interferes with glutamic acid-glutamine-GABAcompartmentation, which, as proposed by Hertz (1979), plays an important role in aminergic neuronal homeostasis. Finally, the effect of ethanol on maturing GABAergic neurons may reflect the general inhibitory effects of ethanol on GABAergic function (Ticku, 1980; Stokes and Harris, 1982; Hams and Sinclair, 1984a,b). Based on our morphological, morphometric, and biochemical findings, we propose that normal neuronal phenotypic expression is dependent to a certain degree on neuronal organization and adequate cell-cell contacts. The long neurites observed in the control cultures may be the cellular messenger for tropic factors and ionic signals. This growth pattern being less defined in the ethanol-treated cultures hinders full neuronal expression and differentiation. It is not surprising, then, that addition of factors to the chick embryo environment or in culture attenuates these neuronotoxic effects of ethanol (Brodie et al., 1990; Kentroti and Vernadakis, 1990). +
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