0145-6008/96/2001-Ol39$03.00/0 ALCOHOLISM: CLINICAL AND EXPERIMENTAL RESEARCH

Vol. 20, No. 1 February 1996

Limited Ethanol Exposure Selectively Alters Proliferation of Precursor Cells in the Cerebral

Michael W. Miller

The present in vivo study tests the hypothesis that limited (4-day) exposure to ethanol differentially affects the proliferation of cortical precursors in the two cortical germinal zones [the ventricular zone (VZ) and the subventricular zone (w] and their descendants in the mature brain. The offspring of pregnant rats fed a liquid diet contain- ing 6.7% (v/v) ethanol when prosencephalic stem cells [gestation day (0) 6-69], VZ cells (G12415), and St cells were proliferating (G18- 621) throughout much of gestation (064321). In addition, the off- spring of rats pair-fed a liquid control diet or fed chow were exam- ined. The pregnant dams were administered with bromodeoxyuridine (BrdU) on either 615 or 021. The ratio of the number of cells that incorporated BrdU to the total number (the labeling index) was de- termined l-hr postinjection (i.e., on G15 or G21) or on postnatal day 80. Ethanol treatment between 06 and 021 reduced the ratio of cells labeled by an injection of BrdU on G15 in the fetus and in the adult, and increased the ratio of cells labeled on G21. Regardless of when the injection was placed, ethanol treatment between 0 6 and 09 had no effect upon the ratio of BrdU-labeled cells in the fetus or mature cortex. Exposure from 012 to 615 decreased the number of VZ cells in the fetus and the number of immunolabeled cells in the adult cor- tex labeled by an injection on 015. This exposure had no effect on the incorporation by SZ cells. In contrast, ethanol exposure from G18 to 621 increased the labeling indices for fetal SZ cells and for cells in the adult, but it had no effect on the ratio of labeled VZ cells. Although ethanol had no apparent effect on the proli$eration of stem cells, it did alter the proliferation of cells in the VZ and SZ. These effects are time-dependent and underlie the ethanol-induced chap !% in the number of cells in the adult.

Key Words: Cell Proliferation, Cerebral Cortex, Critical Period, Subventricular Zone, Ventricular Zone.

HE TERATOGENIC effects of ethanol are temporally T delimited. That is, there are critical periods when the developing nervous system is particularly susceptible to ethanol toxicity. For example, the craniofacial malforma- tions characteristic of fetal alcohol syndrome can be repli- cated by exposing a rat to ethanol only on gestational days (G) 11 and G12.l.' Exposures at later times do not cause craniofacial malformations.

Developing neurons have periods of increased vulnera- bility to ethanol. For example, the number of hypothalamic neurons that express luteinizing hormone-releasing factor

From the Research Service, Veterans Affairs Medical Center; and the Departments of Psychiatry and Pharmacology, University of Iowa College of Medicine, Iowa City, Iowa.

Received for publication June 2, 1995; accepted August 14, 1995 This research was frrnded by the DepaHment of Veterans Affairs and the

National Institutes of Health (Grants AA06916, AA07568, and DE07734). Reprint requests: Michael W. Miller, M.D., Department of Psychiatry

M.E.B., University of Iowa College of Medicine, Iowa City, L4 52242-1000. Copyright 0 1996 by The Research Society on Alcoholism.

Alcohol Clin Exp Res, Vol 20, No 1, 1996: pp 139-144

the Cortex

is reduced by ethanol exposure on G10 or G11, but not on G7.3 On the other hand, exposures to ethanol on G7 or G8 elicited severe effects on the development of other midline forebrain structures, such as the septa1 nuclei and the ol- factory bulbs.4 The hippocampal formation provides an- other good example for the time-dependent effects of eth- anol. Prenatal exposure to ethanol does not affect the number of granule cells in the dentate gyrus, whereas post- natal ethanol exposure leads to an increase in neuronal number?-' In contrast, the number of hippocampal neu- rons is affected by prenatal, but not by postnatal ethanol exposure. Thus, the effects of ethanol are defined by re- gional and temporal differences.

The period of vulnerability to ethanol for a particular structure coincides with the period of cell proliferation. Previous in vivo studies show that ethanol does affect the proliferative activity of neuronal precursors throughout the central nervous system.' Ethanol-induced changes in cell proliferation have been demonstrated in the neocorte~?-'~ the hippocampal formation: the cerebel l~m, '~~ '~ and the trigeminal brainstem complex.1417

The present study tests the hypothesis that the period of vulnerability to ethanol neurotoxicity is defined by the pe- riod of cell proliferation. The developing cerebral cortex of the rat was examined because it contains two germinal zones that have peak proliferative activities at different

The ventricular zone (VZ) is most active during the first half of the period of neuronal generation, whereas the subventricular zone (SZ) is more prominent during the latter part of neuronogenesis. These proliferative zones are affected in opposite fashions by blood ethanol concentra- tions of -150 mg/dl; proliferation in the VZ is depressed, whereas proliferation in the SZ is stimulated. Thus, focus- ing the present study on the cerebral cortex permits a temporal dissection of the effects of ethanol on proliferat- ing populations.

METHODS

Animals, Feeding, and Care

Long-Evans rats were mated by placing a male with 4 nulligravid females for 2 hr (17:OO-19:OO). Positive concepti were identified the next day, G1, by the presence of a sperm-positive vaginal smear. Pregnant dams were fed a high-protein liquid diet containing 6.7% ethanol (Et; Research Diets, New Brunswick, NJ)I8 ad libitum during a 4-day interval (Fig. 1). These intervals, between G6 and G9, between G12 and G15, or between 018 and G21, were periods when stem cells, VZ cells, or SZ cells,

139

140 MILLER

insemination GO.

sperm positive GI.

0 6 '

stem cell proliferation

0 9 .

012.

ventricular cell proliferation

015 '

G l 8 '

subventricular cell proliferation

021 '

birth 022'

Fig. 1. Experimental paradigm. Pregnant rats were fed a liquid diet (Et or Ct) for a 4-day period between G6 and G21 or fed a liquid diet for the entire time between G6 and G21. Each rat was given a single injection of BrdU on G15 or G21. Solid lines identify the periods when rats were fed the 6.7% Ettontaining diet. Discontinuous line denotes the period during which the rats chronically fed an ethanol-containing diet were acclimatized to the diet with 6.7% ET.

respectively, were the predominant proliferative population. Other rats were fed the Et throughout much of gestation (between G6 and G21).93" When rats were not being fed the Et, they were provided chow and water ad libitum.

A second set of rats was matched by weight with the Et-fed rats. These rats were fed a control liquid diet (Ct) that was isocaloric to the Et, with maltose and dextrins substituted for the ethanol. As with the Et-fed rats, these rats were fed the Ct either between G6 and G21 or for a specific 4-day period. The Ct-fed rats were pair-fed the amount of food consumed by a weight-matched Et-fed rat or fed chow and water (Ch) ad libitum.

A third set of rats was fed Ch ad libitum throughout gestation. These rats served as a control for the possible malnutrition imposed on the Ct-fed rats by the pair-feeding regimen.

Within 4 hr of birth, each litter was culled to 10 and surrogate-fostered to a nursing Ch-fed dam. The day of birth was designated as postnatal day (P) 0. During the next 3 wk, nursing mothers were fed chow and water. Offspring were weaned on P21, and then fed chow and water until P60. Fetal and adult rats used in the study were randomly sampled (i.e., with respect to size and gender).

Blood Ethanol Concenfration (BEC)

The BEC of each rat fed Et was determined. Samples of venous blood were removed from the tails of pregnant dams fed Et between G6 and G9, between G12 and G15, or between G18 and G21 on G8, G14, or G20, respectively. All samples were taken at 1000 PM and assessed with a diagnostics kit (#332UV; Sigma, St. Louis, MO). Accordingly, the BECs were 132 t 11, 148 2 18, and 135 2 22 mg/dl for the three groups of rats.

Immunohistochernishy

Each pregnant rat was given a single intraperitoneal injection of bro- modeoxyuridine (BrdU; 25 m a g ; Sigma) at 1490 on G15 or G21. Two complementary studies were conducted. In one study, the long-term ef-

fects of limited exposure to ethanol was assessed in mature rats. Sixty- day-old rats from each of the treatment groups were killed by transcardial perfusion with 70% ethanol. In the second study, the incorporation of BrdU by cells in the VZ and SZ was determined in Et-, Ct-, and Ch- treated fetuses. These fetuses were delivered by Cesarean section 1-hour postinjection (i.e., on G15 or G21) and perfused with alcohol.

The brains of the fetuses and 60-day-old offspring were prepared immunohistochemically. Each brain was removed from the skull, dehy- drated, cleared, and embedded in paraffin. A complete set of 8.0-pm coronal sections through cortex (between the levels of the rostrum of the corpus callosum and the dorsal extent of the hippocampus) was cut. One series of alternate sections was stained with cresyl violet. These sections were used for orientation with respect to cortical depth, layer, and cyto- architectonic area, and for determining the total number of cells in a defined segment of cortex. The other series was processed for BrdU immunohi~tochemistry.'~ Briefly, the DNA in these sections was dena- tured with 0.07 N NaOH. The sections were incubated in a 5.0% solution of an anti-BrdU antibody (Becton-Dickenson, San Jose, CA) in phosphate buffer (0.15 M for the fetuses and 0.10 M for the mature offspring). The distribution of bound antibody was determined by conjugating the primary antibody with an avidin-biotin-peroxidase complex.20

In the adults, the number of BrdU-positive cells through the full depth of dorsal primary somatosensory cortex was determined by counting the number of labeled cells in a 250-pm-wide segment of cortex. This analysis was performed without regard to laminar distribution or whether a cell was a neuron or glia. The counts of the labeled cells were corrected for biases using the formulae described by FloderusZ1 and modified by Smolen et al.= The immunohistochemical procedures did not permit the discrim- ination of intensely BrdU-positive cells (first generation cells) from weakly labeled cells (second or later generations). Primary somatosensory cortex was identified by a characteristic densely packed layer IV?3

"he labeling indices for the VZ and SZ of the fetuses was determined using the same stereological procedures applied to the adult tissue (see herein). The VZ and SZ were discriminated using previously described criteria." The VZ and SZ produced neurons that generally terminated their migrations in the deep and superficial laminae of the mature cortex, respectively?

The sample size was four fetuses or adult rats per treatment group. To elimin'ak Mralitter variations, each fetus or adult was taken from a different litter. Statistically significant differences between the Et- and Ct-treated rats and between the Ct-and Ch-treated rats were assessed with t tests for independent samples.

RESULTS

BrdU Labeling in the Mature Brain Injections of BrdU on G15 labeled cells in the deep

cortex of 60-day-old rats, chiefly layer Vla. The number of cells labeled by such an injection was significantly ( p < 0.05) lower in rats that were exposed to the Et between G6 and G22 than in the Ct-treated rats (Fig. 2). Limited expo- sure to ethanol also significantly ( p < 0.05) affected the number of cells labeled by an injeclion on G15; however, only when the exposure was between G12 and G15. Expo- sures to ethanol before or after this time had little impact on the number of cells labeled.

In mature Ch- and Ct-treated rats, an injection of BrdU on G21 labeled cells in supragranular cortex. On the other hand, a similar injection in an Et-treated rat labeled cells throughout the depth of cortex. These findings were similar to those previously shown with [3H]thymidine autoradiog- r a ~ h y . ~ ? ~ Chronic ethanol exposure through much of gesta- tion produced a significant ( p < 0.01) increase in the

LIMITED ETHANOL EXPOSURE AFFECTS CORTICAL CELL PROLIFERATION 141

150 *0° 1 50

0

3 30 z 40!

O i

= Chow

INJECTION ON G15 VENTRICULAR ZONE Injection on G I 5 Injection on G21

** ** 0.50

* *

X 040 n z

(3 z 1 020 LLI

0 30

-

010 4 0.00

INJECTION ON G21 **

r'! **

G6-G9 G6-G15 GGG9 G18-G21 G6-G22 G12-Gl5 G I 2-G 15 = Chow

ZB Control [~=l Ethanol TIMING OF ETHANOL EXPOSURE Fig. 3. Labeling indices for BrdU-labeled Fells in the fetal VZ. Effects of limited

exposure to ethanol on the ratio of VZ cells that incorporated BrdU after an injection on G15 (left) or on G21 (right) is shown. Data are expressed as the ratio of the density of BrdU-labeled cells to the density of cresyl violet-stained cells (i.e., the labeling index). Notations as in Fig. 2.

1 1

SUBVENTRICULAR ZONE Injection on GI5 Injection on G21

0 1 5 r

G6-G9 G12-GI5 G18-G21 G6-G22

EZ3 Control D Ethanol TIMING OF ETHANOL EXPOSURE

Fig. 2. Number of BrdU-labeled cells in the adult. The number of BrdU-labeled cells through the full depth of cortex was determined for rats injected with BrdU on G15 or G21. Solid, striped, and open bars signify the mean values for the Ch-, Ct-, and Et-treated rats, respectively. T-bars denote SEMs. Single and double asterisks identify statistically significant differences (p < 0.05 and p < 0.01, respectively) between the Ct- and Et-treated rats. No differences between the controls were detected.

number of BrdU-labeled cells, relative t? the Ct-treated rats. Moreover, exposure to ethanol between G12 and G15 or between G18 and G21 led to a significant ( p < 0.01) increase in labeling.

Note that the number of cells in the mature brain that was labeled by an injection of BrdU on either G15 or G21 was not affected by the pair-feeding paradigm. That is, no significant differences were detected between the Ch- and Ct-treated rats.

3rdU Labeling in the Prolifeative Zones Cells in the VZ were labeled by an injection of BrdU on

G15 or on G21; however, many more VZ cells were labeled by injections on G15 (Fig. 3). Rats exposed to ethanol from G6 to G15 had one-third fewer cells labeled by an injection on G15 than did the Ct-treated rats. Likewise, the number of cells labeled by an injection on G15 was one-third of that in Ct-treated rats when the ethanol exposure was limited to G12-G15. These differences were statistically significant ( p < 0.01). Exposure to ethanol between G6 and G9, however, had no effect on the density of cells labeled by an injection of G15.

k! U

0.10 z (3 Z ** ** -

005

m 4

0 00 . ~- G6-G9 G6-Gl5 G6-G9 GIB-GZI G6-G22

G I 2-G 15 G12-GI5 - Chow E Z l Control u Ethanol

Fig. 4. Labeling indices for BrdU-labeled cells in the fetal SZ. Labeling index for SZ cells that were labeled by an injection of BrdU was affected by the limited exposure during gestation. Notations as in Fig. 2.

TIMING OF ETHANOL EXPOSURE

The number of VZ cells labeled by an injection on G21 was significantly ( p < 0.01) lower in rats chronically ex- posed to ethanol. There was also a significant ( p < 0.01) effect when the ethanol exposure was confined to between G12 and G15.

The ratio of SZ cells labeled by an injection of BrdU on G15 was not affected by ethanol exposure independent of when that exposure occurred (Fig. 4). On the other hand, the number of SZ cells labeled by an injection on G21 was affected by ethanol exposure. The ratio of labeled cells was significantly ( p < 0.01) higher in rats that were exposed to ethanol between G6 and G22 than in the Ct-treated rats. Limited ethanol exposures (between G12 and G15 or be- tween G18 and G21) also significantly ( p < 0.01) increased (>15%) the ratio of cells labeled by an injection of BrdU on G21.

MILLER 142

DISCUSSION

Effects of Ethanol on Mature Cortex The number of cells in mature cortex is significantly af-

fected by ethanol exposure throughout gestation. This is evi- denced by a decrease in the number of early-generated cells (those born on G15) and an increase in the number of late- generated cells (those born on G21). The effects of the long- term exposure to ethanol can be replicated by a short (4-day) exposure to ethanol, as long as the exposure includes the cell's time of generation. Exposure to ethanol during the first half of cortical neuronogenesis decreases the number of early-gener- ated cells, whereas later exposure affects the number of late- generated cells. Because most early-generated cells are VZ derivatives and late-generated neurons are largely SZ deriva- tives,8-1' the present data imply that the ethanol exposure differentially affects the two proliferative zones.

When interpreting data from the adult, it is important to keep in mind that it is not possible, with BrdU immunohis- tochemistry, to discriminate intensely labeled cells (first- generation cells) from weakly labeled cells (later genera- tions of cells). Such a discrimination is important because ethanol affects the number of times that proliferating cells pass through the cell cycle before permanently leaving the proliferative population and beginning their Nevertheless, data from the adult brain are consistent with the hypothesis that the effects of ethanol are time- and proliferation site-dependent.

Eflects on Proliferative Zones At the time of sacrifice (1-hr postinjection), all of the

cells that incorporated the BrdU are trapped in a premi- totic stage of the cell cycle (predominantly in the S-phase). Thus, in these preparations, the effects of ethanol on the activity of the VZ and SZ cells could be examined directly. Chronic exposure to ethanol sufficient to produce peak BECs of 150 mg/dl depresses the activity of VZ cells, but stimulates the proliferative activity in the SZ. Limited ex- posure to ethanol also affects cell proliferation in the VZ and SZ, if exposure occurs when the particular zone is prominent. Thus, the incorporation data from the fetus support the notion that the period of vulnerability to eth- anol is defined by the timing and site of origin for a particular cell. VZ cells are affected by early exposure to ethanol and late exposure to ethanol affect SZ cells. The short ethanol exposures apparently have a maximal effect on cells in a particular proliferative zone. This conclusion is supported by data from the acute study of fetuses exposed to ethanol for only 4 days.

Differential effects of ethanol on the two proliferative zones are perplexing. Although it has been replicated in a number of ~ t u d i e s ~ - ' ~ ~ ~ ~ the underlying mechanism of the differential effects remains elusive. There are two appealing hypothe- S ~ S . ' , ~ First, the effective ethanol concentration may differ in the two zones. Ethanol has a concentration-dependent effect on proliferating cells. Both in vitro and in vivo studies show

that cell proliferation is stimulated at low concentrations and inhibited by high concentration^.^'^'^ Thus, the effective eth- anol concentration may be higher in the VZ than it is in the SZ. This situation may arise from a differential bathing of cells in the two zones. After all, VZ cells are in contact with two bathing fluids that contain ethanol, the cerebrospinal fluid and, blood, whereas SZ cells are perfused only with blood. Second, the two proliferative populations express different receptors. The appropriate ligand and/or receptor may be selectively affected by ethanol. Two possibilities are the opiate receptor, that is expressed by subventricular and growth factor receptors, which are elaborated by ventricular cells (unpublished results).

Note that exposure to ethanol during the period of stem cell proliferation has no effect on the number of cells born on G15 or G21. Presumably this is because the effects of ethanol on the stem cell population dissipate by the third postnatal week. Thus, the proliferative activity of later- generated cells compensates for earlier ethanol-induced damage. Compensatory responses may explain the lack of a circadian rhythm for VZ cells12 and for the stimulation of proliferation in the SZ after a depression of proliferative activity in the VZ.7,10911913

Windows of Vulnerability Limited exposure to ethanol can have specific effects on an

ontogenetic stage. In fact, two stages seem to be most suscep- tible to ethanol teratogenicity. One stage is cell proliferation. In addition to neocortex (as described herein'), proliferating neuronal precursors in other central nervous system sites seem to be especially vulnerable to ethanol. These include the c e r e b e l ! ~ , ' ~ ~ ~ ~ the principal sensory nucleus of the trigemi- nal nerve,16717 the hypothalamus,3 and the hippocampal for- mation.' Although the neuronogenetic period differs for each of these structures, moderate amounts of ethanol are toxic when the exposure occurs during the period of cell prolifera- tion. For example, prenatal exposure to ' ethhol has little effect on the number of dentate granule cells, whereas post- natal exposure significantly increases the number of these neurons? The generation of most dentate granule cells occurs

In contrast, the number of pyramidal neu- rons in the neighboring hippocampal segment CA1 are af- fected by prenatal ethanol exposure, but not postnatal expo- sure.7 All CA1 pyramidal neurons are born by the day of

This differential ethanol effect is particularly strik- ing because the dentate gyrus and the CA1 hippocampal region are adjacent to one another.

The second window of ethanol toxicity occurs early in the period of neuronal differentiation when the survival of maturing neurons is in the b a l a n ~ e . ' ~ , ~ ' - ~ ~ During this win- dow, neurons are actively forming synapses and the matur- ing neurons are acutely responsive to neurotrophic factors. If differentiating neurons are unsuccessful in forming syn- apses and/or incorporating sufficient amounts of a neuro- trophic factor(s), they die.

In summary, there are critical periods for ethanol teratoge-

143 LlMlTED ETHANOL EXPOSURE AFFECTS CORTICAL CELL PROLIFERATION

nicity. The primary one occurs during the period of cell pro- liferation. It has even been suggested that exposure to ethanol during the period of cell proliferation can influence the phe- notypic expression of a neuron.33 After the early insult, the developmental timeline is sufficiently disrupted that a cascade of defects ensues,34 including the death of many neurons.17 Despite the permanence of these defects, some data show that various agents, such as neurotrophins and signal transduction blockers, may protect against ethanol-induced damage?5-37 Thus, the sequelae of defects in cell proliferation may be ameliorated by a timely chemical manipulation with one or more growth-influencing substances.

ACKNOWLEDGMENTS

I thank Mein Sun, Guy Potter, and Jeffrey Stadler for their assistance in generating data for this study.

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