Ethanol-Induced Apoptotic Neurodegeneration and Fetal Alcohol Syndrome

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Science  11 Feb 2000:
Vol. 287, Issue 5455, pp. 1056-1060
DOI: 10.1126/science.287.5455.1056

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The deleterious effects of ethanol on the developing human brain are poorly understood. Here it is reported that ethanol, acting by a dual mechanism [blockade ofN-methyl-d-aspartate (NMDA) glutamate receptors and excessive activation of GABAA receptors], triggers widespread apoptotic neurodegeneration in the developing rat forebrain. Vulnerability coincides with the period of synaptogenesis, which in humans extends from the sixth month of gestation to several years after birth. During this period, transient ethanol exposure can delete millions of neurons from the developing brain. This can explain the reduced brain mass and neurobehavioral disturbances associated with human fetal alcohol syndrome.

Intrauterine exposure of the human fetus to ethanol causes a neurotoxic syndrome (1) termed fetal alcohol effects (FAE) or fetal alcohol syndrome (FAS), depending on severity. The most disabling features of FAE/FAS are neurobehavioral disturbances ranging from hyperactivity and learning disabilities to depression and psychosis (2, 3). It is thought that the brain is particularly sensitive to the neurotoxic effects of ethanol during the period of synaptogenesis, also known as the brain growth spurt period, which occurs postnatally in rats but prenatally (during the last trimester of gestation) in humans (4–6). Thus, ethanol treatment of neonatal rats causes reproducible effects relevant to FAE/FAS, including a generalized loss of brain mass and a specific loss of cerebellar and hippocampal neurons (7, 8). However, these circumscribed losses cannot account for the overall loss of brain mass, and the mechanism(s) underlying ethanol's injurious effects on the developing brain remain a mystery.

Having recently found (9) that transient blockade of NMDA glutamate receptors during the period of synaptogenesis causes widespread apoptotic neurodegeneration in the infant rat brain, we decided to evaluate ethanol, a known NMDA antagonist (10), for its ability to trigger apoptotic neurodegeneration in the developing rat brain. A 20% solution of ethanol in normal saline was administered to 7-day-old Sprague- Dawley rats in two separate treatments, 2 hours apart, each treatment delivering 2.5 g/kg subcutaneously (sc); control rats were treated with saline only. The brains were examined histologically 24 hours after the first treatment. In the brains of saline-treated rats, both silver staining and TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling) (11–13) revealed a very light pattern of neurodegeneration attributable to physiological cell death (PCD), the apoptotic process by which biologically redundant neurons are deleted from the developing brain. In the brains of ethanol-treated rats, these stains revealed a very dense and widely distributed pattern of neurodegeneration, a pattern that overlapped with but was more extensive than that (9) induced by other NMDA antagonists (Fig. 1, A to H). As assessed by electron microscopy, the ethanol-induced cell death process clearly met ultrastructural criteria (14, 15) for apoptosis (Fig. 1L).

Figure 1

(A to D) Low-magnification (25×) light microscopic overviews of silver-stained (11–13) transverse sections from the parietal and cingulate cortex of P8 rats treated 24 hours previously with saline, MK-801 (NMDA antagonist), phenobarbital, or ethanol. Degenerating neurons (small dark dots) are abundantly present in several brain regions after treatment with MK-801, phenobarbital, or ethanol, but are only sparsely present after saline treatment. Note that MK-801 and phenobarbital both affect neurons superficial to the cortical surface, whereas the middle cortical layers are affected very prominently by phenobarbital and are relatively spared by MK-801. The ethanol pattern resembles a combination of the MK-801 and phenobarbital patterns. (E toH) Light micrographs (55×) depicting the anterior thalamus at the level of the laterodorsal (LD), anterodorsal (AD), anteroventral (AV), and anteromedial (AM) nuclei, respectively. Note that MK-801 affects the LD, AV, and AM nuclei but not the AD nucleus, whereas phenobarbital affects the LD and AD nuclei very prominently but almost entirely spares the AV and AM nuclei. The ethanol pattern includes all four nuclei, as would be expected if it acts by a dual mechanism involving blockade of NMDA receptors plus activation of GABAA receptors. (I to L) Electron micrographs (1800×) illustrating that apoptotic neurodegeneration induced by MK801 (J), phenobarbital (K), or ethanol (L) has the same ultrastructural appearance as PCD (I), an apoptotic phenomenon that occurs spontaneously in the developing brain. As we have recently described (9, 14), in both spontaneous and induced apoptosis, the earliest signs are the formation of spherical chromatin masses and flocculent densities in the nucleus while the nuclear envelope remains intact and cytoplasmic organelles are relatively unaltered; this is followed in the middle and late stages by fragmentation of the nuclear envelope, intermixing of nucleoplasmic and cytoplasmic contents, and progressive condensation of the entire cell. All four examples shown here have a similar appearance, as they are all in the middle stage of apoptotic neurodegeneration.

Quantitative evaluation (16, 17) revealed that the densities of degenerating neurons in saline-treated rats varied from 0.13 to 1.55% of the total neuronal density in brain regions examined (Table 1). This represents the rate of spontaneous apoptosis (PCD) that occurs naturally in the rat brain at postnatal day 8 (P8). In contrast, the densities of degenerating neurons in ethanol-treated pups ranged from 5 to 30% of the total neuronal densities in the same brain regions. The mean number (±SEM) of degenerating neurons in selected regions of the forebrain (16) was 12,567,726 ± 1,008,195 for the ethanol-treated rats (n = 6), compared to 835,360 ± 101,079 for the saline-treated group (n = 6).

Table 1

Rate of apoptotic neurodegeneration in 15 brain regions of P8 rats 24 hours after treatment with saline, ethanol, diazepam, or MK801.

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In additional experiments with P7 rats, we administered ethanol by various dosing regimens and compared the severity of the apoptotic response (16) with the blood ethanol curves produced by each regimen. We found that the apoptotic response induced by ethanol cannot be predicted by the dose, but rather depends on how rapidly the dose is administered and on how long the blood ethanol levels are elevated above a toxic threshold in the range of 180 to 200 mg/dl (Fig. 2). Maintaining blood ethanol concentrations at or above 200 mg/dl for four consecutive hours was the minimum condition for triggering neurodegeneration. If ethanol concentrations remained above 200 mg/dl for more than 4 hours, the degenerative response became progressively more severe and more widespread in proportion to the length of time that the concentrations remained above this level.

Figure 2

Ethanol was administered to P7 rats by several dosing regimens. The total dose ranged from 0 to 5 g/kg sc and was administered either in a single injection or in multiple injections spaced 2 hours apart. (A) Blood ethanol curves associated with each of several dosing regimens. (B) Severity of apoptotic neurodegeneration associated with each dose–blood ethanol curve. The histographic values in (B) represent total numbers of apoptotic neurons (means ± SEM, n = 6 per group) in the forebrains of saline- and ethanol-treated rats. Severity of degeneration was established using silver-stained sections and by counting argyrophilic profiles in 13 brain regions, as described (16). Many blood ethanol curves not shown were generated; those shown were selected because each is representative of a type of curve required to trigger a certain amount of apoptotic neurodegeneration. The data show that the severity of apoptotic degeneration does not correlate with total dose, but rather with the rate at which the dose is given and the length of time the blood ethanol level remains elevated above a toxic threshold in the range of 200 mg/dl. Dosing regimens that produced blood ethanol concentrations that did not exceed 200 mg/dl for more than 2 hours did not increase the rate of apoptotic neurodegeneration significantly above the spontaneous rate in saline-treated rats. If blood ethanol concentrations exceeded 200 mg/dl for 4 hours, apoptotic neurodegeneration was significantly increased, and if concentrations exceeded 200 mg/dl for more than 4 hours, the degenerative response became progressively more severe in proportion to the length of time the concentrations exceeded 200 mg/dl.

To determine how the apoptotic response to ethanol might differ as a function of developmental age, we administered either saline or ethanol (2.5 g/kg sc at 0 and 2 hours; total dose 5 g/kg) to pregnant rats on embryonic day 17 (E17) or E19 or to their offspring on P0, P3, P14, or P21; after 24 hours, we compared the neurodegenerative response in the fetal or infant brains with the response in P7 rats. To assess the degenerative response to ethanol, we compared the density of degenerating neurons in various brain regions of saline-treated rats with the density of degenerating neurons in the same brain regions of ethanol-treated rats. We found that there is a time window from E19 to P14 when various neurons in the forebrain show transient sensitivity to ethanol-induced neurodegeneration, and within this period, which coincides with the synaptogenesis period, different neuronal populations display transient sensitivity at different times. Three response patterns were observed (Fig. 3).

Figure 3

Age dependency of ethanol-induced apoptosis in the brains of developing rats. Immature rats were exposed at different developmental ages (E17 to P21) to saline or ethanol (2.5 g/kg sc at 0 and 2 hours; total dose 5 g/kg), and 24 hours later we used the stereological disector method (16, 17) to assess the numerical densities of degenerating neurons in DeOlmos silver-stained sections of various brain regions. To determine the apoptotic degeneration that could be attributed to ethanol, we subtracted the mean numerical density count for the saline-treated rats (n = 6) in a given brain region from the mean numerical density count for the ethanol-treated rats (n = 6) in the same brain region. In each brain region there was a time window during which neurons showed vulnerability to ethanol-induced apoptosis, and the timing of this vulnerability period was different for different brain regions. However, each region displayed a temporal profile that fit into an early-, middle-, or late-stage category. Neuronal populations showing the early-stage profile (ventromedial hypothalamus, mediodorsal and ventral thalamus) began to display a significant response to ethanol on day E19, which reached a peak at P0 and rapidly declined thereafter. Neurons showing the middle-stage profile (subiculum, hippocampus, caudate, and laterodorsal and anteroventral thalamus) began to show a response on E19, which reached a peak at P3 and gradually declined to zero by P14. Those showing the late-stage profile (frontal, parietal, temporal, cingulate, and retrosplenial cortices) exhibited a degenerative response that began on day P3, peaked at P7, was markedly diminished at P14, and was absent at P21. Each of the curves presented here pertains to a single brain region that is representative of a response pattern: ventromedial hypothalamus, early stage; laterodorsal thalamus, middle stage; and frontal cortex, layer II, late stage.

To assess whether the apoptotic response to ethanol is associated with a loss of brain mass, we administered saline or ethanol (2.5 g/kg sc at 0 and 2 hours; total dose 5 g/kg) to infant rats on P7 and found, when the experiment was terminated at P12, that the brain weights (whole brain or forebrain and cerebellum weighed separately) of the ethanol-treated rats were significantly lower than those of the saline-treated rats (Fig. 4).

Figure 4

On day P12, the brains of rats that were treated with saline (n = 6) or ethanol (n = 6) on day P7 were weighed to obtain a whole brain weight, then were dissected at the level of the pons into two portions, one including the forebrain (FB) and midbrain (MB) and the other including the cerebellum (CB) and brainstem (BS). Sampling and weighing of the brains were performed in a blinded manner. The weights for the ethanol-treated brains (whole or in parts) were significantly lower than those for the saline-treated brains (*P < 0.05, **P < 0.01; Student's t test).

Because ethanol triggered apoptosis in some brain regions that are not typically affected by NMDA antagonists, we attempted to identify other possible mechanisms to explain ethanol's effects in these brain regions. In a series of experiments (18), we were unable to demonstrate an appreciable apoptotic response to agents that act as either agonists or antagonists at dopamine receptors, block kainic acid or muscarinic cholinergic receptors, or block voltage-gated ion channels. However, a robust apoptotic response was triggered by benzodiazepines and barbiturates, “GABAergic” agents that either mimic or potentiate the action of GABA at GABAA receptors. The agents tested were diazepam [10 to 30 mg/kg intraperitoneally (ip) at 0 hours, n = 6], clonazepam (0.5 to 4 mg/kg ip at 0 hours, n = 6), pentobarbital (10 mg/kg ip at 0 and 4 hours, n = 6), and phenobarbital (50 to 75 mg/kg ip at 0 hours, n = 6). These agents, in a dose-dependent manner, triggered widespread cell death in the infant rat brain, which was apoptotic as assessed by ultrastructural analysis (Fig. 1K). The pattern of degeneration was similar for each GABAergic agent, but this pattern differed in several major respects from that induced by NMDA antagonists (Fig. 1, A to H). However, superimposing one pattern on the other resulted in a composite pattern closely resembling that induced by ethanol. We also studied the window of vulnerability to the proapoptotic actions of diazepam and phenobarbital, and we determined that it coincides with the period of synaptogenesis.

Our results show that exposure of the developing rat brain to ethanol for a period of hours during a specific developmental stage (synaptogenesis) predictably induces an apoptotic neurodegenerative reaction that deletes large numbers of neurons from several major regions of the developing brain. Of ethanol's many actions in the brain, it appears that two—its blocking action at NMDA glutamate receptors and its positive modulatory action at GABAAreceptors—are primarily responsible for its proapoptotic effects. In addition, the developmental period during which the immature brain is vulnerable to the proapoptotic action of NMDA antagonists, GABAergic agents, and ethanol is the same: For all three, it coincides with the synaptogenesis period.

In humans, as noted earlier, the period of synaptogenesis occurs prenatally, during the last 3 months of gestation (6). If a pregnant mother imbibes ethanolic beverages for several hours in a single drinking episode, she could expose her third-trimester fetus to blood ethanol levels equivalent to those required to trigger apoptotic neurodegeneration in the immature rat brain (200 mg/dl lasting 4 hours or more).

From a clinical perspective, it is important to recognize that both NMDA antagonists and GABAA agonists are frequently used as sedatives, tranquilizers, anticonvulsants, or anesthetics in pediatric and/or obstetric medicine. These agents also are drugs of abuse. Because the human brain growth spurt spans not only the last trimester of pregnancy but several years after birth (6), the developing human brain may be exposed to these agents by medical professionals or by drug-abusing pregnant mothers. Also relevant is our observation that within the synaptogenesis period, different neuronal populations have different temporal patterns of response to the apoptosis-inducing effects of these drugs. Thus, depending on the timing of exposure, different combinations of neuronal groups will be deleted, which signifies that this is a neurodevelopmental mechanism that can contribute to a wide spectrum of neuropsychiatric disturbances.


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