Peptidergic Agonists of Activity-Dependent Neurotrophic Factor Protect Against Prenatal Alcohol-Induced Neural Tube Defects and Serotonin Neuron Loss

Fetal alcohol syndrome (FAS) is the leading nongenetic cause of developmental disabilities in the Western world, with an estimated incidence in the United States between 0.5 and 2.0 cases per 1000 live births (Bertrand et al., 2005). FAS is diagnosed by the presence of craniofacial dysmorphology, growth retardation, and evidence of central nervous system dysfunction (Jones and Smith, 1973; Jones et al., 1973). However, it is estimated that there may be up to 10 times as many individuals with confirmed prenatal alcohol exposure who have significant brain damage and cognitive/behavioral deficits, but who do not have the facial dysmorphology or growth retardation needed to fulfill the diagnostic criteria for FAS (Aase et al., 1995; Barr and Streissguth, 2001; Mattson et al., 1998; Roebuck et al., 1998; Sampson et al., 1997; Stratton et al., 1996). The term fetal alcohol spectrum disorders (FASD) has been adopted as an umbrella classification to convey the wide range of phenotypic effects resulting from prenatal alcohol exposure, ranging from subtle effects on growth or neurobehavioral function to full-blown FAS at the severe end of the spectrum (Bertrand et al., 2005; Manning and Hoyme, 2007). Abnormal development of midline structures of the face and brain is a hallmark of FAS. These include the characteristic features of facial dysmorphology (indistinct philtrum, narrow palpebral fissures, short nose, and flattened mid-face) and the disproportionate reductions in midline brain structures such as the corpus callosum, the anterior vermis of the cerebellum, and the caudate nucleus (Bookstein et al., 2002a,b; Sowell et al., 2001; Swayze et al., 1997; Zimmerberg and Scalzi, 1989). Profound structural abnormalities in midline brain regions have also been modeled by heavy-binge alcohol exposure on gestational day 7 in mice (Sulik and Johnston, 1983; Sulik et al., 1984). These outcomes suggest that alcohol may alter cellular processes associated with neural tube fusion and early morphogenesis of the midline neuroaxis. Under very high exposure circumstances, total dysraphia may result, i.e., the failure of the neural tube to close or rupture of a closed neural tube (Gardener, 1973), as has been reported in a mouse embryo culture model (Chen et al., 2005) and in human autopsy cases of fetuses from the first trimester that failed to survive heavy prenatal alcohol exposure (Kovetskii, 1991). Using a mouse model of prenatal alcohol exposure in which inbred C57BL/6 (B6) dams drink alcohol in a chocolate-flavored liquid diet as their sole source of calories beginning on gestational day 7 (just before the onset of neurulation), we have identified a midline neural tube deficit that is evident microscopically in the fetal brain in either (or both) the floor or roof plates (Zhou et al., 2003). In the brainstem, a delayed occlusion of ventral canal (fusion of 2 sides of the brainstem) was evident. This effect on embryonic brain development may best be characterized as an incomplete dysraphia, because the neural tube and ventral canal eventually did close with this alcohol exposure paradigm. Yet, adverse consequences are likely to result from this midline abnormality, including mistimed formation and differentiation of midline neurons and altered growth of fiber processes that cross the midline. Consistent with that, we have shown a delayed formation of the rostral raphe nuclei in the brainstem and reduced numbers and dystrophic morphogenesis of serotonin (5-HT) neurons in the dorsal and median raphe in this model (Zhou et al., 2001, 2002). Deficits in development of the 5-HT system have also been shown in rat models of prenatal exposure using liquid diets (Druse et al., 1991). Development of effective strategies of interventions (prevention and treatment) is an important goal of current fetal alcohol research. Prevention of FASD would be assured by abstinence from alcohol drinking during pregnancy. However, interventions intended to reduce at-risk drinking during pregnancy are often unsuccessful in alcohol-dependent individuals or are difficult to implement for individuals at greatest risk for an FASD outcome. Moreover, effective treatment of children with FASD has not yet been identified (Kalberg and Buckley, 2007). One potential approach to reduce the incidence and life-long adverse consequences of FASD is to develop a molecular intervention that could limit or prevent the effects of prenatal alcohol exposure on the developing brain. Towards that goal, several agents targeting various putative mechanisms of pathogenesis have been demonstrated to have some success in different experimental animal models [(Chen et al., 2001; Ieraci and Herrera, 2006; Kim and Druse, 1996; Thomas et al., 2000, 1997; Wilkemeyer et al., 2002; for review see Goodlett et al. (2005)]. One promising molecular intervention for alcohol-induced teratogenesis is a class of neurotrophic factors that are regulated by vasoactive intestinal peptide in an activity-dependent fashion. These trophic molecules known as activity-dependent neurotrophic factor (ADNF) (Gozes and Brenneman, 1996; Gressens et al., 1999; Guo et al., 1999) and the structural and immunological homologue, activity-dependent neuroprotective protein (ADNP) (Bassan et al., 1999; Gozes et al., 1999) have been shown to prevent alcohol-induced growth deficits and embryonic teratogenesis (Spong et al., 2001), including the total dysraphia produced in cultured mouse embryos by high alcohol concentrations (Chen et al., 2005). ADNF and ADNP have extremely potent neurotrophic (Brenneman and Gozes, 1996; Brenneman et al., 1997) or neuroprotective actions (Gressens et al., 1997), with an efficacy in the femtomolar range (Gozes and Brenneman, 1996). A functional peptide fragment of ADNF, SALLRSIPA (SAL), a potent neurotrophic agent (Brenneman et al., 1998), ameliorated microencephaly in B6 fetal mice induced by prenatal alcohol exposure via maternal liquid diet consumption (Zhou et al., 2004). A functional peptide fragment of ADNP, NAPVSIPQ (NAP), which shares many protective effects of SAL, has also been identified as a potent neuroprotectant (Gozes and Brenneman, 2000; Zamostiano et al., 1999). The goal of the current study was to test the hypothesis that the NAP and SAL peptides would prevent alcohol-induced deficits in embryonic midline development in the brainstem (dysraphia) and would prevent the alcohol-induced deficits in early development of 5-HT neurons in the rostral and caudal raphe. Notably, the current study is the first to test whether there is an alcohol-induced effect on development of 5-HT neurons in the caudal raphe.