0145-6008/95/1905-1359$03.00/0 ALCOHOLISM: CLINICAL AND EXPERIMENTAL RESEARCH

Vol. 19, No. 5 October 1995

RAPID COMMUNICATION

Effect of Pre- or Postnatal Exposure to Ethanol on the Total Number of Neurons in the Principal Sensory

Nucleus of the Trigeminal Nerve: Cell Proliferation and Neuronal Death

Michael W. Miller

Early exposure to ethanol reduces the number of neurons in many CNS structures in vivo. The present study determined whether such reductions are caused by the death of neurons. Three groups of ethanol-treated rats were prepared: those exposed to ethanol from gestational day (G) 11 to G19 (during the period of neuronal genera- tion and migration), from postnatal day (P) 4 to P12 (during the period of synaptogenesis), or from P31 to P39 [after the mature structure and function of neurons in the principal sensory nucleus (PSN) of the trigeminal nerve was established]. During these times, pregnant dams or pups were fed a liquid ethanol-containing diet that produced peak blood ethanol concentrations of 137-157 mg/dl. The number of PSN neurons in mature rats exposed to ethanol pre- or postnatally was determined using stereological procedures. The number of PSN neurons was also calculated for rats pair-fed an isocaloric liquid control diet or fed chow and water ad libitum. The volume of the PSN was not affected by pre- or postnatal ethanol exposure. The number of PSN neurons, however, was significantly affected by ethanol ex- posure in a time-dependent manner. Prenatal exposure lead to a 27.1 YO decrease in neuronal number. Early postnatal exposure led to a smaller decrease (-15.1Y0), and late postnatal exposure had no affect on the number of PSN neurons. These data show not only that ethanol directly depresses the proliferation of neuronal precursors, but also that ethanol causes the death of neurons during the period of synaptogenesis.

Key Words: Alcohol, Apoptosis, Critical Period, Fetal Alcohol Syn- drome, Neurogenesis.

ANY INVESTIGATORS have suggested that one of M the central mechanisms of fetal alcohol syndrome is the ethanol-induced neuronal death.'-' Although this hy- pothesis has been espoused widely and over many years, no in vivo studies have directly tested it. There are two main

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 April 28, 1995; accepted July 5, 1995 This study was supported by the Department of Veterans Affairs and the

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

M.E.B., University of Iowa College of Medicine, Iowa City, IA 52242-1000. Copyright 0 1995 by The Research Society on Alcoholism.

Alcohol Clin Exp Res, Vol19, No 5, 1995: pp 1359-1363

shortcomings to the studies on ethanol-induced neuronal death that have been conducted to date.

One shortcoming is methodological. Published studies only describe the effects of early exposure to ethanol on the density of neurons either in a single section or in a portion of the structure being examined. Such an approach is in- adequate, because ethanol-induced microcephaly has dif- ferential effects upon CNS structures. For example, the volume of CNS structures in ethanol-treated mature rats, that had peak blood ethanol concentrations of -140 mgidl, may be the same [the principal sensory nucleus (PSN) of the trigeminal nerve'), smaller (the somatosensory cor- tex"], or larger (the dentate gyrus") than in control rats. Such ethanol-induced changes in the volume of a structure can offset or amplify alterations in neuronal density.

Second, most previous studies on the effects of ethanol on neuronal survival have not accounted for the effects of ethanol on early ontogenetic processes (i.e., cell prolifera- tion and neuronal migration).12 Many CNS structures are heterogeneous, in which individual neurons develop on greatly different time scales than do neighboring neurons. Such overlapping developmental schedules make it virtu- ally impossible to tease out the effects of ethanol on neu- rons passing through specific developmental stages.

The present study examines the effect of limited ethanol exposure on the total number of neurons in the PSN, a small nucleus in the lateral pons. The PSN has various advantages. Chiefly, it is circumscribed by fiber bundles so that its volume and the total number of constituent neurons can be determined, it is composed of a mostly homoge- neous population of neurons, and the development of PSN neurons is relatively synchronous (i.e., relative to structures such as the cerebellum, hippocampus, and neocortex). Eth- anol exposure was restricted to three periods: (1) when cells were proliferating and migrating, (2) when neurons were forming synaptic connections, and (3) after the ma- ture structure was established.

1359

1360 MILLER

METHODS

Animals, Feeding, and Care

Pregnant Long-Evans rats were obtained from Harlan-Sprague-Dawley (Altamount, NY). The day a sperm-positive vaginal plug was first identi- fied was designated as gestational day (G) 1. Pregnant rats or their offspring were fed ethanol for 9 days either during gestation or during one of two postnatal periods.

A group of pregnant rats was fed ad libitum with a high-protein, liquid diet containing 6.7% (v/v) ethanol (Et-G) from G11 to G19.13-15 During this period, the precursors of PSN neurons p r ~ l i f e r a t e d ~ ~ ’ ~ and the post- mitotic neurons migrated to the PSN.” Two sets of weight-matched control rats were used. One set was pair-fed a nutritionally matched, isocaloric liquid diet that did not contain ethanol (Ct-G). The second set was fed chow and water ad libitum (Ch-G). After birth, the litters were culled to 10, and the pups were surrogate-fostered by Ch-G mothers until postnatal day (P) 21 when they were weaned.

One set of rats was exposed to ethanol during the early postnatal period (from P4 to P12). During this 9-day period, PSN neurons were differen- tiating and forming synaptic connections,’8-21 and some were dying nat- urally.22 These rats were reared by the “pup-in-a-cup’’ m e t h ~ d . ~ ~ . ~ ~ Pups of Ch-G-treated mothers (that were not used in the prenatal feeding study) were fed a diet of artificial milk containing 2.8% (v/v) ethanol (Et-PE) in six feedings between 19:OO and 7:OO (i.e., during the dark cycle) on P4-Pl2. During the light cycle, the pups were fed a diet of artificial milk containing carbohydrates that were isocaloric with the ethanol con- tent of the Et-PE. A control group of rats was pair-fed this isocaloric diet (Ct-PE) from P4 to P12. On P12, the Et-PE- and Ct-PE-treated rats were removed from the feeding apparatus and surrogate-fostered by a suckling dam. In addition, a second control group of pups was kept with a nursing mother (Ch-PE) between P4 and P12.

Ch-PE-treated weanlings that had not been used in any other studies were fed and used to examine the effects of ethanol exposure from P31 to P39. This late ethanol exposure occurred after the period of neuronal differentiation”-” and naturally occurring neuronal death was com- plete22 (i.e., after the mature structure and function of the PSN neurons was established). These rats were fed ad libitum with the same 6.7% ethanol diet (Et-PL) as that provided to the Et-G-treated rats. Two control groups, rats pair-fed the isocaloric, isonutritional liquid control diet (Ct-PL) and rats ad libitum fed chow and water (Ch-PL), were run.

In summary, there were nine groups of rats. Pregnant rats exposed to ethanol during gestation (Et-G) and pups were exposed to ethanol either early (Et-PE) or late (Et-PL) in the postnatal period. Each of these groups of ethanol-treated rats was matched with two control groups: those that were pair-fed an isocaloric diet that did not contain ethanol (Ct-G-, Ct-PE-, and Ct-PL-treated rats) and those fed chow (Ch-G-, Ch-PE-, and Ch-PL-treated rats).

Blood Ethanol Concentrations

Blood ethanol concentrations were determined at 10:OO PM on 1 day during the period of ethanol exposure using a Sigma Diagnostics Kit #332UV. Only one representative day was chosen, because a similar peak blood ethanol concentration was achieved daily.’4,’5.Z4 The blood ethanol concentration for the pregnant dams exposed to ethanol was 149 ir 17 mg/dl on G17, and the blood ethanol concentration for the Et-PE- and Et-PL-treated pups was 137 2 14 on P10 and 157 ? 24 mg/dl on P37, respectively.

Anatomical Studies

All pregnant rats were injected on G13 with [3H]thymidine ([jH]dT; New England Nuclear, Boston MA; specific activity 78.9 Ci/mmol; 5.0 pCi/g body weight). [3H]dT is incorporated into proliferating cells as they pass through the S-phase of the cell cycle, and G13 is the middle of the period of neuronogenesis in the PSN.9,’6 Ninety-day-old male rats from each of the nine treatment groups were killed by transcardial perfusion

z v) a

= Chow Control 0 Ethanol Exposure period

Fig. 1. Volume of the PSN. The size of the PSN was determined from three- dimensional reconstructions of the pons of chow-fed (solid bars), pair-fed control (striped bars), and ethanol-treated (open bars) rats. Each bar represents the means of four rats, and bars signify the SEMs.

with 4.0% paraformaldehyde in 0.10 M phosphate buffer. The brains were removed, and the brainstems were embedded in paraffin. The paraffin block was cut into a complete series of 10-pm sections. The sections were processed by standard autoradiographic procedures,14 stained with cresyl violet, and coverslipped.

The number of neurons in the PSN was determined using a rigorous procedure? The neuronal density was estimated using the stereological procedure described by Smolen et al?’ The total number of neurons in a section of the PSN was counted. This number was corrected for overes- timation because of counting cell fragments as whole cells. The volume of the PSN was calculated by generating a three-dimensional reconstruction of the PSN. The product of the neuronal density and the volume of the PSN was the total number of PSN neurons. In addition, the number of neurons that was heavily labeled by the injection of [3H]dT was deter- mined by the same procedure. Heavily labeled neurons were considered to be the cells with more than half the number of silver grain over their nuclei as the number of grains over the nuclei of the maximally labeled neurons. Presumably, these are the cells that passed through only one mitotic division after [3H]dT was incorporated.

Mean data for four animals per treatment group were calculated; each of these four rats was taken from a different litter. Statistical differences between the means were tested using a t test for independent samples. Only comparisons between the Et-G, Et-PE, and Et-PL, and the paired Ct-G, Ct-PE, and Ct-PL treatment groups, respectively, and between the pairs of the control groups (Ct-G, Ct-PE, and Ct-PL- versus the Ch-G, Ch-PE, and Ch-PL treatment groups, respectively) were examined. Thus, the effects of ethanol exposure and the malnutrition imposed by the pair-feeding paradigm were determined.

RESULTS

Prenatal exposure to ethanol had no affect on the vol- ume of the PSN (Fig. 1). These data concur with those of a previous study on the effects of gestational ethanol expo- sure on the PSN.9 Postnatal exposure, be it either early (from P4 to P12) or late (from P31 to P39), also did not have a significant affect on the size of the PSN.

The packing density for the total population of PSN neurons was affected by ethanol exposure in a time-depen- dent manner. The neuronal packing density in the Et-G- treated rats was -24.3% that in the Ct-G-treated rats (Fig.

EFFECTS OF ETHANOL ON CELL PROLIFERATION AND NEURONAL DEATH 1361

= Chow

0 Control Ethanol Exposure period Fig. 2. Neuronal packing density. The number of neurons per unit volume was

determined stereologically. The top graph shows the effects of ethanol on the total population of neurons, and the bottom graph shows the density of neurons that were heavily labeled by an injection of fH]dT on G13. Asterisks denote statistically significant differences between the ethanol- and control-treated rats: *p < 0.05; **p < 0.01.

2). This difference was statistically significant ( p < 0.01). Early postnatal exposure to ethanol also significantly ( p < 0.05) affected the neuronal packing density. It was 14.0% less in the Et-PE-treated rats than in the Ct-PE-treated rats. On the other hand, late postnatal ethanol exposure had no affect on neuronal packing density.

The density of neurons heavily labeled by an injection of [3H]dT on G13 was greatly affected by prenatal ethanol exposure and to a lesser extent by ethanol exposure from P4 to P12. Exposure to ethanol from P31 to P39 had no impact on the density of neurons born on G13.

The net effect of the lack of an ethanol-induced change in the PSN volume and the decrease in neuronal packing density was that the total number of neurons in the PSN was lower in rats exposed to ethanol than in controls (Fig. 3). The greatest effect (-27.1%; Et-G- versus Ct-G-treated rats) occurred when the exposure to ethanol was confined to the prenatal period. This difference was statistically significant ( p < 0.01). A significant ( p < 0.05) difference (-15.1%) was also observed between the total number of PSN neurons in Et-PE- and in Ct-PE-treated rats.

The number of neurons that incorporated [3H]dT on G13 was affected by ethanol exposure. The number was 32.0% lower in Et-G-treated rats than it was in Ct-G- treated rats. Early postnatal exposure also significantly ( p < 0.05) affected the number of neurons born on G13,

Chow

0 Control Ethanol Exposure period Fig. 3. Number of PSN neurons. An estimate for the total number of neurons

(top graph) and the number of neurons born on GI3 (bottom graph) was calcu- lated as the product of the volume of the PSN and the neuronal packing density. Asterisks denote statistically significant differences between the ethanol- and control-treated rats: ' p < 0.05; **p < 0.01.

but to a lesser extent (-19.9%) than did the gestational exposure. Later ethanol exposure did not affect the number of surviving neurons that were born on G13. Thus, the effect of ethanol on the number of neurons born on G13 was similar to that for the total population of PSN neurons.

No differences (in neuronal packing density or number) were detected between the pairs of control groups for the prenatal, early postnatal, or late postnatal exposure.

DISCUSSION

The effects of ethanol on the developing nervous system in vivo are time-dependent. The number of neurons in the PSN is adversely affected only when the exposure occurs during the period of cell proliferation and neuronal migra- tion or during the period of neuronal synaptogenesis. This pattern is evident for the overall population of PSN neu- rons and for the neurons born on G13. Three conclusions can be drawn from these data. (1) Prenatal exposure to ethanol has direct effects on the population of proliferating neuronal precursors. Prenatal exposure to ethanol can lead to a reduction in neuronal number by altering the cell cycle kinetic^^^^^-*^ or by inducing the death of proliferating cells? (2) PSN neurons born at a particular time are not selectively affected by pre- or postnatal ethanol exposure. This conclusion is supported by data from a previous study showing that, although ethanol exposure does delay the

1362 MILLER

period of neurogenesis, prenatal exposure to ethanol does not have a selective effect on PSN neurons with a particular time of origin.’ (3) Exposure to ethanol during the period of synaptogenesis induces neuronal death. Early postnatal exposure can only affect postmitotic neurons because the generation of PSN neurons is complete by G16.9,16

Exposure to ethanol during the period of cell prolifera- tion and neuronal migration has a greater negative effect on neuronal number than does exposure during the period of synaptogenesis; however, it is unclear which ontogenetic stage is most affected by ethanol. Exposure during the period of cell proliferation can produce not only to a direct effect, but a cascade of “latent” secondary defects that appear well after the ethanol exposure ended. Suffice it to say, that the effects of ethanol toxicity are more devastat- ing, because they include both primary and secondary ef- fects. Similar effects have been described for the effects of ethanol on the number of PC12 cells in proliferating and nonproliferating cultures.29

Justifying such apparently antagonistic effects of ethanol on developmental stages has confounded alcohol research- ers for many years (for reviews, see Refs. 12 and 30). How is it possible that a single toxin can affect proliferating and differentiating cells? In fact, these ethanol-induced alter- ations on disparate developmental events may result from a singular effect on a class of substances that have dual roles [i.e., trophic factor(s) and cell cycle regulators]. These sub- stances affect the development of cells at two crucial points in their development: they determine when cells exit from the cell cycle, which for neurons is a permanent decision, and they promote the growth and survival of differentiating n e u r o n ~ . ~ l - ~ ~ This thesis is supported by data from in vitro studies that show that ethanol affects growth factor-medi- ated cell proliferation and neurotrophin-regulated neurite outgrowth and cell Thus, whereas early ex- posure to ethanol may affect cells in many developmental stages, the primary chemical targets of ethanol may be few. If indeed the chief target(s) of ethanol neuroteratogenicity is a trophic factor(s), then it may be conceivable that the neural and possibly craniofacial abnormalities associated with fetal alcohol syndrome may be offset by the timely application of exogenous neurotrophins.

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