Elsevier

Brain Research

Volume 911, Issue 1, 17 August 2001, Pages 71-81
Brain Research

Research report
Effects of prenatal exposure to ethanol on the expression of bcl-2, bax and caspase 3 in the developing rat cerebral cortex and thalamus

https://doi.org/10.1016/S0006-8993(01)02718-4Get rights and content

Abstract

Prenatal exposure to ethanol causes neuronal death in somatosensory cortex, but apparently not in the ventrobasal nucleus of the thalamus. Effectors such as bcl-2, bax, and caspase 3 can determine whether a neuron survives or dies. We hypothesize that ethanol differentially affects the expression of these proteins in the cortex and thalamus during the periods of naturally occurring and ethanol-induced neuronal death. Pregnant rats were fed ad libitum with an ethanol-containing liquid diet (Et) or pair-fed an isocaloric non-alcoholic diet (Ct). Samples were collected from fetuses (gestational day (G) 16 and G19) and pups (postnatal day (P) 0 through P30) and examined for bcl-2, bax, or caspase 3 expression using a quantitative immunoblotting procedure. Prenatal exposure to ethanol reduced cortical bcl-2 expression, but not bax expression on P6. Hence, the bcl-2/bax ratio was lower in Et-treated rats than in controls. In contrast, thalamic expression of neither bcl-2 nor bax was significantly different in the two groups of rats. Thus, the thalamic bcl-2/bax ratio was unaffected by exposure to ethanol. During the period of naturally occurring neuronal death, the expression of the active (20 kDa) and inactive isoforms (32 kDa) of caspase 3 was altered in the cortices of Et-treated rats, but not in their thalami. Thus, prenatal exposure to ethanol affected the early postnatal expression of death-related proteins in the cortex, but not in the thalamus. These biochemical changes concur with anatomical data on the spatial and temporal selectivity of ethanol toxicity in the developing CNS.

Introduction

Prenatal ethanol exposure can alter the number of neurons in the mature central nervous system (CNS). Such damage varies among CNS structures; indeed, variations can even exist within the same motor or sensory system. For example, in the trigeminal-somatosensory system, the number of neurons in the primary somatosensory cortex [40] and in the principal sensory nucleus of the trigeminal nerve (PSN) [33], [35], [38] of ethanol-treated rats is one-third fewer than in controls. Neuronal number in the ventrobasal nucleus of the thalamus (VB), however, is unaffected by ethanol [44].

The number of neurons within a structure is the sum of two opposing processes: the additive effect of cell acquisition (proliferation and neuronal migration) and the subtractive effect of neuronal death. Ethanol affects both processes in vivo. Studies show that exposure to ethanol during early development can reduce the proliferation of neuronal precursors [15], [35], [36], [39] and alter the timing of neuronal migration [28], [29], [31], [34]. A recent longitudinal study of neuronal number in the developing PSN shows that ethanol reduces neuronal acquisition and increases neuronal death [35]. This dual effect of ethanol on cell proliferation and neuronal death is also evident in vitro. Ethanol causes the death of cultured cortical neurons and inhibits the neuronal proliferation seen in primary neuronal cultures [13], [55]. It is noteworthy that the balance of the effects of ethanol on cell proliferation and death in vitro is similar to the effects seen in the PSN in vivo.

Various features of ethanol-induced cell death echo those associated with naturally occurring apoptosis [5], [7], [53]. These include morphological changes (e.g., in the cytoskeleton and nucleus) and biochemical changes [16]. For example, chronic exposure to ethanol can increase the incidence of DNA damage in vitro [2], [13], [21], [27] and in vivo [5], [53]. Furthermore, prenatal exposure to ethanol causes transient increases in the cortical expression of a protein recognized by the antibody ALZ-50 [19], a protein associated with naturally occurring neuronal death (NOND) [1], [37], [58]. Likewise, p53 is upregulated during NOND [19], [37], [52], [59]. p53 can act as a transcription factor for bax [42] and directly affects the relative expression of pro- and anti-apoptotic bcl proteins. The balanced expression of bcl proteins is considered to be a critical determinant in cell survival [18], [50].

Effectors that are downstream of the bcl proteins in the apoptotic cascade include the caspases [6], [41]. Activation of caspases results in the cleavage of crucial cytoskeletal and regulatory proteins and, ultimately, in cell death [24]. Ethanol causes the death of primary cultured cerebellar granule neurons [23], [51] and neocortical neurons [55]. This is associated with the upregulation of caspase activity [48] which can be offset by treatment with the anti-apoptotic agent N-methyl-d-aspartate [3] or with ganglioside treatment [54].

The temporal expression of bcl-2, bax and caspase 3 [45], [56], [57] shows changes during normal development. These changes coincide with the period of NOND in the cerebral cortex [45], [57], thalamus and midbrain/pons [45]. For example, the rise in expression of an active (20 kDa) isoform of caspase 3 temporally correlates with an increase in bax expression, and with a decrease in the ratio of bcl-2 to bax expression [45], suggesting an interaction between these proteins.

Ethanol-induced neuronal death is evident in select regions of the CNS, e.g. in the somatosensory cortex, but not in the VB. This death may be apoptotic, and may result from a direct toxic effect of ethanol on the cell, or it may occur indirectly by the ability of ethanol to exacerbate processes associated with NOND. We hypothesize that prenatal exposure to ethanol differentially alters the relative expression of proteins involved in naturally occurring neuronal apoptosis, e.g. bcl-2, bax and caspase 3. These effects should be evident in cortex where ethanol causes neuronal death, but not in the thalamus. Therefore, a quantitative immunoblotting technique was used to examine the temporal expression of bcl-2, bax and caspase 3 proteins in the cerebral cortex and the thalamus in ethanol-treated rats.

Section snippets

Animals, feeding, and care

Pregnant Long-Evans rats were obtained from Harlan-Sprague-Dawley (Indianapolis, IN, USA) on gestational day (G) 4. The day on which a dam was first identified as having a sperm-positive vaginal plug was designated as G1. Animals were maintained in a temperature/humidity controlled facility in which the light/dark cycle was 12 h/12 h.

Rats were arbitrarily assigned to one of two treatment groups [28], [30]. One group was fed ad libitum a liquid diet containing 6.7% (v/v) ethanol (Et) from G11

Bcl proteins

The bcl-2-positive protein (25 kDa) was expressed in the offspring of Ct-fed dams throughout development (Fig. 1). Densitometric analyses showed that the relative amount of bcl-2 expression did not significantly change over development (Fig. 2). The temporal changes of bcl-2 expression in Et-treated rats followed the same pattern as that described for Ct-treated rats. Even so, there was a notable ethanol-induced difference. Prenatal exposure to ethanol significantly decreased bcl-2 expression

Protein expression in the developing prosencephalon

While interpreting the present data, it must be kept in mind that they represent the summed changes for a neuronal population(s). Neuronal development within a population is asynchronous. Thus, whereas the expression of death-related protein may be intense, but short-lived for an individual cell, alterations for a population may be dilute but evident over a broader period of time. This situation may be exacerbated by the removal of apoptotic cells that transiently altered bcl proteins or

Acknowledgements

We thank Dustin Hays for his technical assistance in the immunoblotting studies. This study was supported by grants from the Department of Veterans Affairs and from the National Institutes of Health (AA06916, AA07568, AA09611, and DE07734).

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