Choline supplementation attenuates learning deficits associated with neonatal alcohol exposure in the rat: Effects of varying the timing of choline administration

Abstract

Despite the harmful effects of fetal alcohol exposure, some pregnant women continue to drink alcohol. Thus, it is imperative to pursue safe, effective treatments for children with fetal alcohol spectrum disorders. Using an animal model, our laboratory has demonstrated that choline, an essential nutrient, effectively reduces the severity of some fetal alcohol effects, even when administered after the ethanol insult is complete. The present study investigated whether there is a critical developmental period when choline is most effective in attenuating ethanol's teratogenic effects. Sprague–Dawley rats were exposed to 5.25 g/kg/day ethanol during the third trimester equivalent brain growth spurt (postnatal days (PD) 4–9) via intubation. A non-intubation control group and a sham intubation control group were included. Following ethanol exposure, pups received subcutaneous injections of saline vehicle or choline chloride (100 mg/kg/day) from PD 11–20, PD 21–30, or PD 11–30. Beginning on PD 45, subjects were tested on a Morris water maze spatial learning task. Performance of both the ethanol-exposed group that did not receive choline and the ethanol-exposed group treated with choline from PD 21–30 was significantly impaired compared to controls during acquisition of the Morris water maze task. Performance of ethanol-exposed groups treated with choline from PD 11–20 or PD 11–30 was intermediate, not differing significantly from any other groups. However, during the probe trial, ethanol exposure produced significant deficits in spatial memory which were mitigated by all choline treatments, regardless of the timing of administration. These findings suggest that choline's therapeutic window may be very large, or spans across the two developmental periods examined in this study. Importantly, these findings indicate that choline supplementation may effectively reduce some alcohol-related learning impairments, even when administered in later childhood.

Introduction

Fetal alcohol spectrum disorders (FASD) is a term used to describe the wide range of physical, neurological, and behavioral alterations associated with prenatal exposure to ethanol (NIAAA, 2000, Riley and McGee, 2005, Sokol et al., 2003). Alcohol-related disruptions in central nervous system (CNS) development constitute the most devastating consequences (see Spadoni et al., 2007 for review), with damage to areas such as the cerebellum (O'Hare et al., 2005, Sowell et al., 1996), corpus callosum (Bookstein et al., 2007, Riley et al., 1995, Sowell et al., 2008a, Sowell et al., 2001), caudate (Mattson et al., 1996), cortex (Sowell et al., 2008b) and hippocampus (Autti-Ramo et al., 2002, Riikonen et al., 1999). Ethanol-induced neuropathology leads to a variety of behavioral problems, including hyperactivity, attention deficits, motor dysfunction, impairments in language and social skills, and learning deficits (Coles et al., 1997, Kelly et al., 2000, Mattson et al., 2001, Riley and McGee, 2005, Roebuck et al., 1999). Although FASDs are preventable, many pregnant women still drink alcohol and FASDs continue to constitute a serious health concern throughout the world (Warren et al., 2001). In the U.S. alone, it is estimated that 1 in 100 live births exhibit at least some adverse effects of prenatal alcohol exposure (Sampson et al., 1997). Thus, it is imperative that potential interventions/treatments for FASD be identified.

Numerous animal studies have shown that pharmacological manipulations may effectively block ethanol's teratogenic effects (Bonthius et al., 2003, Chen et al., 2005, Endres et al., 2005, Ieraci and Herrera, 2006, Thomas et al., 2001, Wilkemeyer et al., 2003, Wilkemeyer et al., 2002). However, to be effective, such experimental therapeutics would have to administered during ethanol exposure and there is concern that such treatments could exert undesirable effects on nontargeted developmental processes. Moreover, the opportunity for treatment may not be available until a child is already born with FASD. Fortunately, both human and animal studies have shown that postnatal environmental manipulations can reduce the severity of some fetal alcohol effects. Animal studies have shown that enriched environment (Hannigan and Berman, 2000, Hannigan et al., 1993), exercise (Christie et al., 2005, Thomas et al., in press), and acrobatic motor training (Klintsova et al., 2000, Klintsova et al., 2002) can reduce alcohol's behavioral teratogenic effects. Similarly, clinical studies have shown that specialized training can improve social skills (O'Connor et al., 2006), behavioral problems (Kable et al., 2007) and math skills (Kable et al., 2007). These data suggest that the CNS can respond to environmental interventions even after a developmental alcohol insult.

We have shown that choline supplementation may serve as an effective dietary intervention to reduce the severity of alcohol's adverse effects on behavioral development. Choline is an essential nutrient that is critical for brain development and function (Zeisel, 2006a). A growing literature illustrates that pre- and/or early postnatal choline supplementation in otherwise typically developing rats leads to long-lasting morphological (Li et al., 2004, Loy et al., 1991, Williams et al., 1998), electrophysiological (Jones et al., 1999, Pyapali et al., 1998), and neurochemical (Blusztajn et al., 1998, Cermak et al., 1999, Cermak et al., 1998, Coutcher et al., 1992, Meck et al., 1989, Montoya et al., 2000) changes in the CNS that contribute to long-lasting cognitive enhancements (McCann et al., 2006, Meck and Williams, 1997, Meck et al., 1988, Meck and Williams, 2003). Perinatal choline supplementation can also enhance cognitive improvements to later environmental manipulations, such as enriched environment (Tees, 1999). Using a rat model of fetal alcohol exposure, we found that choline supplementation not only reduces the severity of prenatal alcohol effects when administered concurrently with the alcohol (Thomas et al., submitted for publication), but is also effective when administered postnatally. For example, we first reported that choline supplementation during postnatal days (PD) 2–21 reduced the severity of working memory deficits in adult rats following prenatal alcohol exposure (Thomas et al., 2000). Importantly, these findings illustrated that choline is effective even when administered after ethanol exposure is complete. More recently, it's been shown that choline supplementation from either PD 4–30 or 10–30 reduces overactivity in the open field (Thomas et al., 2007, Thomas et al., 2004), spatial reversal learning deficits (Thomas et al., 2004), trace eyeblink conditioning deficits (Tran and Thomas, 2007), trace fear conditioning deficits (Wagner and Hunt, 2006), and spatial learning deficits (Thomas et al., 2007) associated with alcohol exposure during the 3rd trimester equivalent (PD 4–9). In all of these studies, behavioral testing occurred after choline treatment had ceased, indicating that early postnatal choline supplementation leads to relatively long-lasting changes in behavior.

One important question is whether there are critical periods during which choline is most effective in mitigating ethanol's adverse effects on behavioral development. Indeed, the effects of choline supplementation among rats not exposed to alcohol have been shown to depend on the timing of administration. Choline supplementation during gestation produces more robust long-lasting improvements in visuospatial memory compared to postnatal choline supplementation (from PD 1–24), although the combination of pre- and postnatal choline supplementation is more effective than either period alone (Meck et al., 1989). Meck et al. (2008) further investigated critical periods of choline sensitivity among otherwise typically developing rats, administering choline for various periods throughout gestation and up to PD 75. They found that choline led to long-lasting improvements in spatial memory, as well as increased hippocampal dendritic spine densities, only when administered from gestational days (GD) 12–17 or PD 16–30. However, to date, no studies have examined the effects of varying developmental timing of choline administration among subjects exposed to alcohol during development.

In the present experiment, rat pups were exposed to ethanol during a period of brain development equivalent to the third trimester brain growth spurt. This period of brain development occurs postnatally in rats, from PD 4–9 (Dobbing and Sands, 1979). Subjects were subsequently treated with choline (choline chloride 100 mg/kg/day via s.c. injection) during one of two developmental phases following alcohol exposure: PD 11–20 (EtOH c/s) or PD 21–30 (EtOH s/c), or a combination of both periods (PD 11–30) (EtOH c/c). Ethanol-treated subjects that did not receive choline (EtOH s/s), sham intubated (SHAM s/s), and non-intubated controls (NC s/s) were included and injected with saline from PD 11–30. From PD 45–50, subjects were tested on a standard spatial learning version of the Morris maze, where the location of a hidden escape platform within a tank of opaque water remained constant throughout training for each subject and had to be found using extra-maze spatial cues. Twenty-four hours after training, subjects were tested on a probe trial to measure spatial memory. Finally, performance on a visible platform was measured to determine if other variables (i.e. motivation, motor, sensory) influenced spatial learning performance.