Alcohol Vol.9, pp. 299-304, 1992

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The Effect of Long-Term Alcohol Intake on Brain NGF-Target Cells of Aged Rats LUIGI ALOE l AND PAOLA TIRASSA

Instituto di Neurobiologia, Consiglio Nazionale delle Ricerche, Viale Marx 15, 00156 Rome, Italy Received 28 O c t o b e r 1991; Accepted 26 J a n u a r y 1992 ALOE, L. AND P. TIRASSA. The effect of long-term alcohol intake on brain NGF-target cells of aged rats. ALCOHOL 9(4) 299-304, 1992.-It was reported that chronic exposure to ethanol causes a loss of hippocampal pyramidal cells and of brain cholinergic neurons in both laboratory animals and humans. In the present study, it was hypothesized that nerve growth factor (NGF), atrophic agent for the survival and maintenance of basal forebrain cholinergic neurons (FCN), might be affected by the neurodegenerative events which occur during ethanol consumption. To test this hypothesis, we used aged rats (14 months) exposed for 16 weeks to 40 g/kg per day of undiluted wine. Our experiments showed that chronic alcohol consumption causes a reduction of NGF in the hippocampus (HI) and of choline acetyltransferase (CHAT) activity in both the septum and the H1 and a reduction in the distribution of NGF-receptors (NGF-R) in the septum and nucleus of Meynert. Intracerebral injection of NGF in alcohol-exposed rats results in a return to normal levels of ChAT enzymatic activity and NGF-R expression. These experiments indicate that the damaging effect of alcohol on the FCN is also associated with impairment of central NGF-target structures. Nerve growth factor

Alcohol

Choline acetyltransferase

NUMEROUS studies have shown that ethanol consumption, both in animal models and in humans, causes neurochemical, behavioral, and neuropathological effects (3,6,8,12,20,43,47) in the central nervous system (CNS). For example, chronic ethanol intake causes a loss of neuronal cell bodies in the hippocampus, in the basal forebrain neurons (3,6,21,39) and in the cerebellum of young and adult rodents (36,37). Although the mechanism(s) underlying these deficits is poorly understood, in vitro studies have shown that ethanol potentiates GABA receptors (46), inhibits N-methyl-D-aspartate receptors (30) and reinforces the action of 5-HT serotonin receptors (18). It has also been reported that alcohol intoxication might activate the hypothalamic-pituitary adrenal axis causing hypersecretion of circulating corticosteroids, thus resulting in a wide range of behavioral and neuropathological alterations (12,20,26). Our interest in this line of research was attracted by the results of a recent study showing that neurites, which degenerate as a result of ethanol exposure, return to a normal appearance when the ethanol is withdrawn (21,26). These results, although providing evidence that nerve ceils of the CNS have the ability to overcome the neuroanatomical and neurochemical damage caused by alcohol ingestion, also raised the question of whether ethanol might affect the synthesis of neurotropic factors present in the CNS. It is known, for example, that nerve growth factor (NGF) is expressed in the hippocampus (HI) and cerebral cortex (27,28,45,48) area, re-

Aged rats

Cholinergic neurons

ceiving major cholinergic projections (5). It has also been shown that this polypeptide triggers signals which regulate the synthesis of choline acetyltransferase (CHAT) and enhance survival of lesioned forebrain cholinergic neurons (FCN) (13,19,48). In addition, prolonged intracerebral NGF treatment can improve retention of a spatial memory task in a behaviorally impaired aged rat, supporting the hypothesis that NGF may play a role in brain cholinergic pathology (19,22, 45). The present study was, therefore, aimed at exploring the effect of alcohol consumption both on NGF and on NGFtarget cells in aged rats. Recent studies, showing that ethanol causes a dose-dependent inhibition of neurite-induced outgrowth (10) and the NGF is involved in brain cholinergic deficit occurring in aged rats (13,19,22,24), encourage the pursuance of these experiments. Moreover, although the effects of ethanol on developing and adult FCN have been reported, analogous information on aged rats is not available. Therefore, our studies also aimed to assess whether aging and alcohol consumption would exacerbate the pathological signs in cholinergic brain structures. In the present study, full-strength wine was used, instead of diluted pre-prepared ethanol. It is hoped that, by comparing the results of the present studies and those reported by other investigators on alcohol-related FCN deficits in animal models and in humans (8,9,11,15), more information can be obtained on the pathophysiology of experimentally induced alcoholism.

Requests for reprints should be addressed to Luigi Aloe, Instituto di Neurobiologia, Consiglio Nazionale delle Ricerche, Viale Marx 15, 00156 Rome, Italy. 299

300

ALOE AND TIRASSA METHOD

Fourteen-month-old male Sprague-Dawley rats (mean lifespan for this strain is 20-22 months) were used for these studies. Animals were kept on a standard rat food diet and on a 12 L: 12 D h cycle until the time of sacrifice. They were housed 2 animals per cage and given free access to food and water. NGF was prepared by the method of Bocchini and Angeletti (7). Anti-NGF antibodies (anti-NGF) were prepared in rabbits and purified by "affinity chromatography as described in a previous study (42). Monoclonai antibodies against choline acetyltransferase (anti-ChAT) were purchased from Boehringer (Mannheim, Germany). Monoclonal antibodies against NGF receptors, anti-192 IgG (NGF-R), were kindly provided by Dr. E. J. Johnson (Department of Pharmacology, Washington University, St. Louis, MO). High-quality, undiluted 2-year-old commercial white wine (containing no additives), 10070 alcohol volume, was used in these studies. Rats were not deprived of food or water before treatment. Alcohol Administration

Under mild ether anesthesia, 24 aged rats (alcoholic rats), received a single intragastric wine infusion of 40 g/kg per day for 16 consecutive weeks, providing about 2007o of their total calorie intake. Also, after mild ether anesthesia and via gastric catheter, an equal number of aged rats (n = 24) received an isocaloric equivalent of sucrose (control rats). After 16 weeks, this treatment was stopped and half the rats of each group were injected into the right lateral cerebral ventricle (icv) with a single dose of 30 ag of N G F dissolved in 3 ~tl of phosphatebuffered saline (PBS). The animals were deeply anesthetized with Nembutal and the heads were mounted in a stereotaxic instrument (coord: related to bregma: - 0 . 8 0 , L: - 3 , V: 5). The remaining rats were injected with 30/~g of cytochrome-c, a molecule with physiochemical properties similar to NGF (19,45). One week later, the rats were sacrificed after injection of an overdose of Nembutal (7 mg/kg of b.wt.) and brains dissected out and used for biological, neurochemieal, immunohistochemical, and histological studies. Eight untreated rats 0 8 months of age) were also used for NGF and ChAT determination. Blood samples of 600-700/~l were collected from the orbital plexus 1 h after the last dose of wine intake, and were analyzed for alcohol content using the ethanol assay 322UV (Sigma Chemical Co., St. Louis, MO). N G F Bioassay

Fourteen aged rats which received wine (n = 7) or sucrose (n = 7) for 16 weeks as described in the previous section, were used for measuring N G F level in the brain. The entire cortex and HI were homogenated in 2: l water volume, centrifuged at 20,000 rpm for 10 rain, and the supernatant was immediately used for biological assays. Neurons dissociated from the rat superior cervical ganglion (SCG) were used, because more than 90070 are receptive to N G F (2,27,29) whereas other growth factors present in the HI, unlike NGF, have no effect on sympathetic neurons (1). Briefly, nerve cells from SCG of 17- to 18-day-old rat fetuses were mechanically dissociated and cultured in collagenated Falcon tissue culture dishes containing Dulbecco's basal medium only, or the medium with the addition of increasing amounts of NGF. In the absence of NGF, only a small number of neurite-bearing cells appeared in the culture, whereas the addition of as little as 0.02 ng of NGF yielded a detectable increase in the number of neurites. By adding increasing amounts of NGF, the number of responding cells also increased, resulting in a dose-response

curve which was used as a standard curve for evaluating the amount of NGF present in the tissue samples to be tested (2,41). The absolute concentration of NGF (nanograms per gram) was determined by extrapolation from the standard curve, generated by using known amounts of NGF. To assess whether the neurite-promoting activity was caused by NGF or by other factors known to be present in the brain (l), extracts were preincubated with 10/zg of purified NGF antibodies. All samples were tested in triplicate and presented +_ SEM. lmmunohistochemistry

Rats treated with wine (n = 5) and sucrose (n = 5) were perfused under Nembutal anesthesia via the ascending aorta with 200 ml of 0.1 M PBS, pH 7.4, containing 0.5070 sodium nitrite, followed by 500 ml of 4% paraformaldehyde in phosphate buffer (2). The brains were then removed, immersed in the same fixative for 6 more h, and then in 20070 sucrose in PBS for 24 h. Each brain was then mounted on the stage of a freezing microtome and 60 consecutive frontal sections of 40 /zm thickness were cut, spanning from the striatum (plate 14) to the caudal hypothalamus (plate 34), as listed in Paximos and Watson (34). This region includes most of the NGF-target neurons within the brain. AFter collection, sections were coded and stained in alternation: sections 1,4,7, etc., stained for CHAT; sections 2,5,8, etc., stained for NGF-R; and sections 3,6,9, etc., stained with toluidine blue for routine histological examination. For immunostalning, free-floating sections were left overnight in the first antibodies containing 0.2% Triton X-100 at 4°C. After a brief rinse, sections were

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FIG. 1. Histogram showing the NGF content extracted from the cortex and hippocampus of aged rats cxpomd to sucrose or chronic wine intragastric adiministration. Levels of NGF were determined with bioassays using rat superior cervical ganglion neurons as described in the METHODS section. Valu~ are expressed as + SEM (n = 7). *p < 0.001: Significantly different from sucrose-treated rats.

A L C O H O L E F F E C T ON B R A I N - N G F T A R G E T C E L L S

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treated with biotinylate antimouse IgG, washed again, and incubated with avidin-biotin-HRP complex using the Vectastain A B C Kit o f Vector Laboratory following the manufacturer's instructions. For the evaluation o f immunoreactivity, anatomically comparable sections o f the septum (ST), striatum, and nucleus basalis of Meynert (NB) o f each experimental group were selected, and immunostained neurons were counted. C h A T and NGF-labeled cell bodies were first quantified independently by two observers, one of w h o m was blind to the experimental design. The results were subsequently evaluated using an image analysis m o r p h o m e t r y program (Zeiss Vidas System, Germany). Minimal and maximal neuron areas were determined by measuring the smallest and largest C h A T or N G F - R positive neurons (ranging from 6-18/~m of diameter) present in the selected brain field.

FIG. 3. ChAT immunoreactivity in the septum of aged rats treated for 16 consecutive weeks with sucrose (A) or wine (B). Note loss of ChAT immunoreactivity and decrease in neuronal size in medial (m) and diagonal band of Broca (dB) following alcohol intake. Bar = 200 t~m.

not tabulated• Only 3 alcohol- and 2 sucrose-treated aged rats died during 16 weeks of treatment. No somatic or behavioral differences were noticed between the two groups. The average body weight was 516 g for alcoholic rats (varying from 484526 g) and 487 g for controls (varying from 467-507 g). The initial body weight was 345-368 g for both groups. Likewise, no gross pathology was observed in the brain, digestive tract (including the liver), or lymphoid tissues after wine or sucrose consumption. Sucrose treatment alone did not cause any changes in NGF or ChAT activity levels as compared to the untreated control tissues (results not shown). Biological assays showed that the level o f N G F in the HI of alcoholic rats was reduced by as much as 44% (1.5 ng in

Histology Sections were stained with toluidine blue and examined under a standard Zeiss microscope.

Biochemical Determinations C h A T enzymatic activity was determined in wine (n = 5) or sucrose-treated rats (n = 5) in the entire cortex, HI, striaturn, ST, and cerebellum. After an overdose of CO2, brain tissues were quickly dissected out and C h A T activity determined according to the method described by Fonnum (14). The values are expressed as units o f enzymatic activity per milligram o f protein. A unit of C h A T was defined as a micromole of acetylcholine formed per minute at 37°C.

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Statistical Analysis The data are presented as the mean :!: SEM. The significance of differences between groups was assessed by Student's t test or analysis o f variance.

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8 RESULTS

The blood alcohol concentration (in samples collected 1 h after the last day's alcohol consumption) varied greatly from one sample to another, ranging from 15-220 m g / d L and was

A

B

FIG. 4. ChAT immunoreactivity in two representative sections of the NB neurons of aged rats treated for 16 consecutive weeks with sucrose (A) or wine (B). Note loss of immunoreactivity of neurons in (B) (see also Table 1). Bar = 2501am.

302

ALOE AND T1RASSA

sucrose-treated versus 0.8 ng in wine-treated rats), although, in the cortex, no differences were observed (Fig. 1). The induced neurite outgrowth was inhibited by NGF antibodies, suggesting that it was caused by NGF and not by other growth factors present in the HI (1). Figure 2 summarizes ChAT enzymatic activity in forebrain cholinergic tissues of wine-treated and untreated rats. This cholinergic marker is reduced in the septum ( - 2 3 % , p < 0.01), the HI ( - 1 5 % ; p = 0.02), and the cortex ( - 9 % , p = not significant}, whereas, in the striatum, no differences were observed. Morphometric examination carried out on CHAT- and NGF-R-immunostained FCN showed that neurons and neurite profiles localized in the ST and NB of wine-exposed rats are less densely immunoreactive (Figs. 3 and 41. Following alcohol consumption, many ChAT-positive neurons in these brain regions appear vacuolated and shrunken in comparison so the FCN of sucrose-treated rats. Likewise, FCN of the medial septal area expressing N G F - R showed loss of immunoreactivity following chronic wine consumption (not shown). The number of densely stained ChAT immtmoreactive neurons in the ST is reduced by 26~/0 (382 vs. 514) and by 16% (157 vs. 186) in the NB of alcohol-exposed rats (see Table 1). No neuronal loss was observed following alcohol intake. Morphometric and microdensiometric analysis performed by means of an automatic image analyzer showed changes in size and distribution of ChAT-positive cells in the septum of wine-treated rats. As shown in Fig. 5, the number of small (20-50 t~m area) neurons of the septum of alcoholic rats increase (black bars), whereas the number of large cells (70-200 pm21 decreases. A significant but less pronounced decrease was also present in the area of the NB (data not shown1. The reduced immunoreactivity and ChAT enzymatic activity caused by wine consumption are re-established following icy administration of NGF. Furthermore, after NGF treatment, most of the neurons in the ST and NB showed tapering varicoses and darkly immunoreactive neurites, which were not present after chronic wine uptake. DISCUSSION

The main object of this investigation was to study the role of chronic wine intake on NGF and NGF-target cells in the CNS of aged rats. Studies published in recent years have shown that chronic alcohol intake in both young and adult rodents is associated with various neuroendocrine (16,23,38),

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FIG. 5. Histogram showing the size distribution of ChAT-unmunoreactive neurons in the septum of wine-treated (black bars) or sucrose-treated rats (hatched bars). The )'-axis indicates the area and the x-axis the number of ChAT-positive neurons. Note the higher number of small size ChAT-positive neurons and a decrease of medium- and large-size neurons after intragastric administration of wine as compared to control rats.

behavioral (6,351, immunological (17,32), and neuroanatomicat (12,20,21,25,30,44,47) alterations. It has been reported that alcohol consumption induces morphological deficits of FCN, both in humans (12,431 and in animal models (3,4, 20,21). The mechanism(s) of this deficit, however, have not yet been elucidated. More recent studies have shown that continuous alcohol consumption induces transient loss of dentritic spines from hippocampal pyramidal neurons. These studies showed that normal conditions are re-established after 2 months of ethanol withdrawal (21,26). This observation, although providing further evidence that neuronal plasticity occurs in the CNS, suggested the possibility that brain and NGF-target cells might be affected by chronic alcohol intake. Since FCN express NGF-R and are receptive to the action of NGF (27,45,48), the question was also raised as to whether a link exists between the deficit induced by alcohol intake and brain NGF levels. This hypothesis was further strengthened by the observation, reported in a previous in vitro study, that alcohol intake results in a dose-dependent inhibition of NGFinduced neurite outgrowth (10), and that in vivo alcohol expo-

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COMPARISON OF CHAT AND NGF-R IMMUNOREACTIVITY IN T H E F O R E B R A 1 N C H O L 1 N E R G I C N E U R O N S O F A G E D RATS T R E A T E D A S I N D I C A T E D

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191 177 206 194

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All densely stained neurons with a well-defined neurite profile in four representative coded sections from each experimental group and selected brain regions were counted using a Zeiss standard microscope equipped with × 40 objective. All values are means ± SEM (n = 5). *p < 0.001 and ~p < 0.01 versus corresponding control rats (sucrose-treated).

ALCOHOL EFFECT ON BRAIN-NGF TARGET CELLS

303

sure induces deficits on young and adult Purkinje cells (36,37) which are known to express N G F - R . Although several studies on alcohol-induced deficits have been reported, no experimental evidence has been provided regarding the effect of chronic wine consumption on the CNS (8,9,11,15). Therefore, it was considered worthwhile to study the effect of wine on FCN in order to provide further insight into the neuropathology o f ethilism. Our studies demonstrated that 16 weeks o f intragastric wine administration cause brain cholinergic deficits which are, to some extent, comparable to those reported by others using other forms o f alcohol administration (3,4). They also showed that wine intake causes a concomitant decrease of N G F - R distribution on FCN. These findings suggest that the impairment of cholinergic enzyme and N G F - R expression following wine intake might be the result of a reduced brain N G F level. The fact that the decrease of C h A T activity in the ST and the NB is restored after icv injection o f N G F supports this hypothesis. Furthermore, because both chronic alcohol consumption and aging result in a decrease o f brain N G F level and C h A T activity, it would be of interest to investigate whether similar pathogenic mechanisms are involved. The present studies failed, however, to detect significant differences in N G F activity in the cortex. The possibility that this negative result is due to a limitation o f the sensitivity of our bioassay a n d / o r the fact that N G F assays were carried

out on the entire cortex, rather than in a more restricted NBprojecting field of innervation, cannot be excluded. It could also be hypothesized that the unconventional alcohol administration utilized in our study does not affect the N G F level in the cortex. We are currently studying the effect o f alcohol intake on NGF-target cells in the peripheral and CNS o f developing rodents to verify this hypothesis. In conclusion, although both our results and those o f others suggest that chronic alcohol ingestion causes specific deficits in the CNS, it should be taken into consideration that a high level of circulating alcohol is associated with a broad spectrum o f actions affecting the nervous, endocrine, and immune systems. Most probably, there is no single mechanism which can account for such a complex physiopathological alteration, and more systematic studies are required for a thorough understanding of the dynamic destructive and compensatory changes that are produced in the hippocampus during and following alcohol intake. ACKNOWLEDGEMENTS We are very grateful to Professor R. Levi-Montalcini for continuous advice and intellectual contributions during these studies. This work was supported in part through the Progetto Finalizzato Invecchiamento, Sottoprogetto: Sistema, Neuroendocrino/Gerontobiologia Grant no. 922107, to Rita Levi-Montalcini and Luigi Aloe.

REFERENCES 1. Alderson, R. F.; Alterman, A. L.; Barde, Y. A.; Lindsay, R. M. Brain-derived neurotrophic factor increase survival and differentiated functions of rat septal cholinergic neurons in culture. Neurons 5:297-306; 1990. 2. Aloe, L. Adrenalectomy decreases nerve growth factor in young adult rat hippocampus. Proc. Natl. Acad. Sci. USA 88:56365640; 1989. 3. Arendt, T.; Allen, Y.; Marchbanks, R. M.; Schugens, M. M.; Sinden, J.; Lantos, P. L.; Grey, J. A. Cholinergic system and memory in the rat: Effects of chronic ethanol, embryonic basal forebrain brain transplants and excitotoxic lesions of cholinergic basal forebrain projection system. Neuroscience 33:435-462; 1989. 4. Arendt, T.; Hennig, D.; Gray, J. A.; Marchbanks, R. M. Loss of neurons in the rat basal forebrain cholinergic projection system after a prolonged intake of ethanol. Brain Res. Bull. 21:563-570; 1988. 5. Bartus, R. T.; Dean, R. L.; Beer, B.; Lippa, A. S. The cholinergic hypothesis of geriatric memory dysfunction. Science 217:408417; 1982. 6. B~racoch~a, D.; Jaffard, R. Memory deficits subsequent to chronic consumption of alcohol in mice: An analysis based on spontaneous alternation behavior. Behav. Brain Res. 15:15-26; 1985. 7. Bocchini, G.; Angeletti, P. U. The nerve growth factor: purification as a 30,000-molecular-weight protein. Proc. Natl. Acad. Sci. USA 64;787-794; 1969. 8. Carlen, P. L.; Wilkinson, D. A. Alcoholic brain damage and reversible deficits. Acta Psychiatry Scand. 286:(Suppl.), 103-118; 1980. 9. Dally, S.; Girre, C.; Hispard, E.; Thomas, G.; Fournnier, L. High blood lead level in alcoholics: Wine vs. beer. Drug Alc. Depend. 23:45-49; 1989. 10. Dow, E. K.; Riopelle, R. J. Ethanol neurotoxicity. Effects on neurite formation and neurotrophic factor production in vitro. Science 228:591-593; 1985. I1. Elton, M. Do brain damage and liver disease interrelate in advanced abusers of alcohol? Acta Psychiatry Scand. 68:437-444; 1983. 12. Ferrer, i.; Fabregues, I.; Pineda, M.; Gracia, I.; Ribalta, T. A.

13. 14. 15. 16. 17.

18. 19.

20.

21. 22.

23.

24.

Golgi. Study of cerebellar atrophy in human chronic alcoholism. Neuropathol. Appl. Neurobiol. 10;245-253; 1984. Fischer, W.; Gage, F. H.; Bjorklund, A. Degenerative changes in forebrain cholinergic nuclei correlate with cognitive impairments in aged rats. Eur. J. Neurosci. 1:34-45; 1989. Fonnum, F. A rapid radiocbemical method for the determination of choline acetyltransferase. J. Neurochem. 24:407--409; 1975. Giesmann, G. Migraine nach rotwein. Deutsche Medizinische Wochenschrift ! 13:1941-1942; 1988. Gottesfeld, Z.; Silverman, P. B. Developmental delays associated with prenatal alcohol exposure are reversed by thyroid hormone treatment. Neurosci. Lett. 109:42-47; 1990. Gottesfeld, Z.; Christie, R.; Felten, D. L.; LeGrue, S. J. Prenatal ethanol exposure alters immune capacity and noradrenergic synaptic transmission in lymphoid organs of the adult mouse. Neuroscience 35:185-194; 1990. Grant, K. A.; Lovinger, D. M.; White, G.; Berret, J. E. Blockade of the discriminative stimulus properties of ethanol by 5-HT receptors antagonist. FASEB J. 4:A989; 1990. Hefti, F.; Dravid, A.; Hartikka, J. Chronic intraventricular injections of nerve growth factor elevate hippocampal choline acetyltransferase activity in adult rats with partial septohippocampal lesions. Brain Res. 293:305-311; 1984. lrle, E.; Markowitch, H. J. Widespread neuroanatomical damage and learning deficits following chronic alcohol consumption or vitamin-Bl (thiamin) deficiency in rats. Behav. Brain Res. 9:277294; 1983. King, M. A.; Hunter, B. E.; Walker, D. W. Alteration and recovery of dentritic spines in rat hippocampus following long-term ethanol ingestion. Brain Res. 459:381-385; 1985. Koh, S.; Chang, P.; Collier, T. J.; Loy, R. Loss of NGF receptor immunoreactivity in basal forebraln neurons of aged rats: Correlation with spatial memory impairment. Brain Res. 498:397-404; 1989. Kornguth, D. J.; Rutledge, J. J.; Sunderland, E.; Segel, F.; Carlson, I.; Smallens, J.; Juhl, U.; Young, D. Impeded cerebellar development and reduced serum thyroxine levels associated with fetal alcohol intoxication. Brain Res. 177:347-360; 1979. Larkfors, L.; Ebendal, T.; Whittemore, S. R.; Persson, H.; Hoffer, J.; Olson, L. Decreased level of nerve growth factor (NGF)

304

25.

26.

27. 28. 29. 30. 31. 32.

33. 34. 35. 36.

37.

ALOE AND TIRASSA and its messenger RNA in aged rat brain. Mol. Brain Res. 3:5560; 1987. Lescaudron, L.; Verna, A. Effect of chronic ethanol consumption on pyramidal neurons of the mouse dorsal and ventral hippocampus: A quantitative histological analysis. Exp. Brain Res. 58: 362-367; 1985. Lescaudron, L.; Jaffard, R.; Verna, A. Modifications in number and morphology of dendritic spines resulting from chronic ethanol consumption and withdrawal: A golgi study in the mouse anterior and posterior hippocampns. Exp. Neurol. 106:i56-163; 1989. Levi-Montalcini, R. The nerve growth factor 35 years later. Science 237:1154-1162; 1987. Levi-Montalcini, R.; Aloe, L.; Alleva, E. A role for nerve growth factor in nervous, endocrine and immune systems. PNEI 3: I-10; 1990. Levi-Montalcini, R.; Angeletti, P. U. The nerve growth factor. Physiol. Rev. 48:534-569; 1968. Lovinger, D. M.; White, G.; Weigth, F. F. Ethanol inhibits NMDA-activated ion current in hippocampal neurons. Science 243: 1721-1724; 1990. Miller, M. W. Effects of prenatal exposure to ethanol on neocortical development: II. Cell proliferation in the ventricular and subventricular zones of the rat. J. Comp. Neuroi. 287:362-338; 1989. Norman, D. C.; Chang, M. P.; Castle, S. C.; Van Zuylen, J. E.; Taylor, A. N. Diminished proliferative response of Con A blast cells to interleukin 2 in rats exposed to ethanol in utero. Alcohol.: Clin. Exp. Res. 13:69-72; 1989. Often, U. Nerve factor and the peptidergic sensory neurons. Trends Pharmacol. 7:307-310; 1984. Paxinos, G.; Watson, C. The rat brain in stereotaxic coordinates. 2nd ed. London: Academic Press; 1986. Peterson, J. T.; Pohorecky, L. A. Effect of chronic ethanol administration on intermale aggression in rats. Aggr. Behav. 15: 201-215; 1989. Philips, S. C.; Cragg, B. G. A change in susceptibility of rat cerebellar Purkinje ceils to damage by alcohol during fetal, neonatal and adult life. Neuropath. Appl. Neurobiol. 8:441-454; 1982. Philips, S. C.; Cragg, B. G.; Singh, S. C. The short-term toxicity

38.

39. 40. 41.

42. 43. 44. 45.

46. 47.

48.

of ethanol to neurons in rat cerebellar cortex tested by topical application in vivo, and a note on a proble estimating ethanol concentrations in tissue. J. Nenrol. Sci. 49:353-361; 1981. Rachamin, G.; Luttge, W. G.; Hunter, B. E.; Walker, D. W. Neither chronic exposure to ethanol nor aging affects type I or II conicosteroid receptors in rat hippocampns. Exp. Neurol. 106: 164-171; 1989. Riley, J. N.; Walker, D. W. Morphological alterations in hippocampus after long-term alcohol consumption in mice. Science 201:646--648; 1978. Sapolsky, R. M.; Krey, L. C.; McEwen, B. S. Prolonged glucocorticoid exposure reduces hippocampal neuron number. Implication for aging. J. Neurosci. 5:1222-1227; 1985. Stephani, U.; SuRer, A.; Zimmermann, A. Nerve growth factor (NGF) in serum. Evaluation of serum NGF levels with a sensitive bioassay employing embryonic sensory neurons. J. Neurosci. Res. 17:25-35; 1987. Stockel, K.; Paravicini, U.; Thoeneu, H. Specificity of the retograde axonal transport of nerve growth factors. Brain Res. 76: 413--421; 1974. Streissguth, A. P.; Landesman-Dwyer, S.; Martin, J. C.; Smith, D. W. Teratogenic effect of alcohol in humans and laboratory animals. Science 209:353-361; 1980. Takada, R.; Saito, K.; Matsuura, H.; Inoki, R. Effect of ethanol on hippocampal GABA receptors in the rat brain. Alcohol 6:115i 19; 1989. Thoenen, H.; Barde, Y. A.; Edgar, D.; Hatanaka, H.; Otten, U.; Schwab, M. Mechani~m of action and possible sites of synthesis of nerve growth factor. Progr. Brain Res. 51:95-107; 1979. Wafford, K. A.; Burnett0 D. M.; Dwiddie, T. V.; Harris, R. A. Genetic differences in ethanol sensitivity of GABA receptors expressed in xenopns oocytes. Science 249:291-293; 1990. Walker, D. W.; Hunter, B. E.; Abraham, W. C. Neuroanatomical and functional def'~its subsequent to chronic ethanol administration in animals. Alcohol.: Clin. Exp. Res. 5:267-282; 1981. Whittemore, S. R.; Seiger, A. The expression, localiTation and functional significance of a nerve growth factor in the central nervous system. Brain Res. Rev. 12:439-464; 1987.