While it has long been recognized that acute high dose ethanol exposure at early gastrulation stages in mice selectively impacts the forebrain [82
], there is only one previous study employing this FASD model in which a detailed examination of ethanol-induced insult to specific forebrain populations is reported [82
]. In this previous report, a reduction in choline acetyltransferase (ChAT)-expressing neurons in the forebrain of fetal mice following GD 7 ethanol exposure was shown. Within the past fifteen years, the fact that the ventromedian forebrain serves as a source for tangentially-migrating interneurons and oligodendrocytes has been firmly established [5
]. More recently, transcription factors that regulate the differentiation of interneurons have been identified, and mechanisms controlling tangential migration have been defined [12
]. This new information is critical for more fully understanding the impact of ethanol-induced ventromedian forebrain deficiency. In the current study, the region or cell type-specific markers, Nkx2.1, Fzd8, Olig2, and GABA, were employed to further examine ethanol's teratogenic effect. Reductions in MGE and POA tissues and effects on oligodendrocyte and GABAergic interneuron progenitor populations have been shown.
The Nkx2.1 and Fzd8 expression pattern alterations identified in this study are remarkably similar to that in the Nkx2.1 mutant mouse described by Sussel and colleagues [93
]. This study was the first to show that Nkx2.1 is required for normal development of the pallidum-related ventral telencephalon. The mutant mice lacked a morphological and molecular MGE, although the dorsal telencephalon was relatively normal. The globus pallidus (pallidum), a derivative of the MGE, was absent at later stages of development, and instead, was replaced by striatal-like tissue. Also, there was an overall reduction in an isoform of glutamic acid decarboxylase (Gad67), a GABA synthesizing enzyme. Similar to the results of the ethanol-teratogenesis studies of Schambra et al [82
], cholinergic neurons, another subset of progenitor cells derived from the MGE/POA region, were also absent in the Nkx2.1 mutant [93
Nkx2.1 expression is dependent on Shh signaling. Shh is a ventralizing morphogen that is necessary for cell proliferation, differentiation, and embryonic patterning. At gastrulation stages, Shh is expressed in the notochord, prechordal plate, and floor plate of the neural tube. In mouse embryos having approximately 8 somites (approximately GD 8.5), its expression can be localized to the median aspect of the anterior neural plate. By about GD 9.5, Shh is expressed in the mantle zone of the MGEs [83
]. Importantly, mutations in Shh result in HPE, one of the severe manifestations of early prenatal alcohol exposure [28
Recognizing that early gastrulation stages represent a critical exposure time for FAS and that Shh is a key signaling pathway at this point in time, a number of investigations have explored insult to this pathway as a primary mechanism of ethanol's teratogenesis [2
]. The results of studies employing a variety of animal models, including zebrafish, chicks, and mice, have provided evidence indicating that ethanol exposure occurring during gastrulation does decrease Shh expression, with resulting craniofacial and CNS abnormalities being consistent with HPE. Strongly supporting a role for diminished Shh signaling is that the ethanol-induced defects can be rescued by Shh [2
] or by other molecules involved in Shh signaling [i.e. cholesterol [55
]]. Despite a considerable amount of work, however, it remains unclear whether the ethanol-mediated affects on Shh signaling are direct or indirect.
The results of the current work highlight the potential of acute, early ethanol insult to have a protracted effect on the structure and function of the developing brain. A reduction in the tissues from which GABAergic populations are derived is expected to have functional consequences that might include disruption in the balance of excitation and inhibition in the CNS. In this regard it is notable that ADHD is the highest comorbidity in FASD [35
]; an outcome that may, at least in part, result from reduced GABAergic neurotransmission [81
]. Another result of inhibitory interneuron loss is seizure activity [26
]. Importantly, seizures are estimated to occur in 3-21% of children with FASD [16
]. The developmental basis for this remains unknown, though Bonthius and colleagues [19
] have suggested that ethanol-induced hippocampal dysfunction may be one cause. Additionally, Godin et al [38
] have proposed that seizure activity might be the result of an error in cell migration leading to the development of cerebral cortical heterotopias. Also notable are that seizures are common in patients with HPE [30
]; a decrease in interneurons in individuals with HPE has been reported [32
]; and individuals with Shh mutations that have microcephaly, but lack overt HPE features, present with hyperactivity and seizures. Also, in the Nkx2.1 mutant mouse, there are reduced populations of interneurons [93
]. In fact, Nkx2.1 has been suggested to regulate the balance of excitation and inhibition in the postnatal cerebral cortex [41
]. Although the mutant mice do not survive postnatally because of lung and thyroid problems, a conditional knockout of the gene on either GD 9.5 or 10.5 is consistent with viability. These conditional knockouts develop spontaneous seizures as juveniles, a finding thought to be related to decreased GABA activity [25
]. Clearly, studies directed toward examining seizure thresholds in the FASD mouse model employed for the current study are needed.
In addition to hyperactivity and seizures, another potential consequence of abnormal GABA levels is altered sensitivity to GABA-modulating drugs, including ethanol. Ethanol is a GABA-agonist, an action that yields relaxing, anti-anxiety effects. That most individuals with FASD have an increased risk for developing both alcohol and drug abuse problems may, in part, be related to their need to drink more in order to achieve a desired effect [3
]. Interestingly, a reduced ataxic and hypothermic response to an acute ethanol challenge has been demonstrated among mice that were exposed to ethanol on GD 7 (personal communication, SOM).
In addition to the consequences of damage to GABAergic populations, additional consideration of early ethanol-mediated insult involving oligodendrocyte populations is warranted. A marked reduction in the tissue from which oligodendrocyte progenitor cell populations originated was shown in this study. Importantly, in human FASD, fiber tracts, including the corpus callosum, are affected. Defects in the corpus callosum have been shown to range from complete agenesis to decreased white matter organization/integrity [10
]. While the existence of functionally redundant oligodendrocyte progenitor cells that may compensate if another population is affected has been suggested [47
], it remains possible that loss of ventromedian derived-oligodendrocyte progenitor cells may contribute to the white matter integrity changes that follow prenatal ethanol exposure. Additional investigations directed toward examining early ethanol exposure mediated effects on myelination in later prenatal and postnatal stages are indicated.
Imaging studies assessing human FASD have shown that individuals prenatally exposed to ethanol have basal ganglia that are reduced in size [9
]. Importantly, Sowell and colleagues have recently identified size reductions in the pallidum that are associated with the upper median facial dysmorphology that characterizes FAS (personal communication). This result is consistent with an early insult comparable to that in the model employed for this study. In the mice, it would be of interest to explore ethanol-induced functional changes in movement as might be expected to follow insult to the pallidum [40
In conclusion, the results of this study show that in mice, acute ethanol exposure occurring at a time in development equivalent to that in the third week of pregnancy in humans adversely affects the ventromedian forebrain, reducing tissues that are a source of interneuron and oligodendrocyte populations and that form the septal region and components of basal ganglia, especially the pallidum. The functional consequences of this insult are expected to include an increased potential for hyperactivity, seizures and susceptibility to drug and alcohol abuse. This work highlights the need for additional pre- and postnatal studies directed toward a more comprehensive examination of ethanol-induced ventromedian forebrain deficiency and resulting structural and functional changes.