Elsevier

Neuropharmacology

Volume 79, April 2014, Pages 262-274
Neuropharmacology

Repeated intermittent alcohol exposure during the third trimester-equivalent increases expression of the GABAA receptor δ subunit in cerebellar granule neurons and delays motor development in rats

https://doi.org/10.1016/j.neuropharm.2013.11.020Get rights and content

Highlights

  • Impact of 3rd trimester-equivalent ethanol exposure on the cerebellum was studied.

  • Rats were exposed to ethanol vapor for 4 h/day between postnatal days 2–12.

  • Basal GABA transmission in developing granule cells was unaffected by ethanol.

  • Ethanol exposure increased GABAA receptor δ-subunit expression at postnatal day 28.

  • Behaviorally, ethanol exposure delayed the acquisition of the air righting reflex.

Abstract

Exposure to ethanol (EtOH) during fetal development can lead to long-lasting alterations, including deficits in fine motor skills and motor learning. Studies suggest that these are, in part, a consequence of cerebellar damage. Cerebellar granule neurons (CGNs) are the gateway of information into the cerebellar cortex. Functionally, CGNs are heavily regulated by phasic and tonic GABAergic inhibition from Golgi cell interneurons; however, the effect of EtOH exposure on the development of GABAergic transmission in immature CGNs has not been investigated. To model EtOH exposure during the 3rd trimester-equivalent of human pregnancy, neonatal pups were exposed intermittently to high levels of vaporized EtOH from postnatal day (P) 2 to P12. This exposure gradually increased pup serum EtOH concentrations (SECs) to ∼60 mM (∼0.28 g/dl) during the 4 h of exposure. EtOH levels gradually decreased to baseline 8 h after the end of exposure. Surprisingly, basal tonic and phasic GABAergic currents in CGNs were not significantly affected by postnatal alcohol exposure (PAE). However, PAE increased δ subunit expression at P28 as detected by immunohistochemical and western blot analyses. Also, electrophysiological studies with an agonist that is highly selective for δ-containing GABAA receptors, 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridine-3-ol (THIP), showed an increase in THIP-induced tonic current. Behavioral studies of PAE rats did not reveal any deficits in motor coordination, except for a delay in the acquisition of the mid-air righting reflex that was apparent at P15 to P18. These findings demonstrate that repeated intermittent exposure to high levels of EtOH during the equivalent of the last trimester of human pregnancy has significant but relatively subtle effects on motor coordination and GABAergic transmission in CGNs in rats.

Introduction

The cerebellum is best known for its role in motor coordination and motor learning; however, it also critically regulates numerous cognitive processes including attention, the speed of information processing, visuo-spatial functions, and language (O'Halloran et al., 2012). Alterations in cerebellar functions have been demonstrated in neurodevelopmental disorders such as autism, Down's syndrome, and fetal alcohol spectrum disorders (FASDs) [reviewed in O'Halloran et al., 2012, Steinlin, 2008]. FASDs are among the most common neurodevelopmental disorders, affecting up to 5% of the population in the United States and Western Europe (May et al., 2009). Individuals with FASDs often exhibit impairments in balance, delays in gross and fine motor skills (Jaatinen and Rintala, 2008, Mattson and Riley, 1998, Nguyen et al., 2012), and deficits in motor learning (i.e., cerebellar-mediated eyeblink conditioning (Jacobson et al., 2011, Jacobson et al., 2008)), and additional cognitive deficits that could also be related to cerebellar damage, such as: impaired math skills (Lebel et al., 2010) and dyslexia (Coffin et al., 2005). A reduction in cerebellar volume and white matter tracts have been detected in patients with FASDs (Autti-Ramo et al., 2002, O'Hare et al., 2005, Roebuck et al., 1998, Sowell et al., 1996, Wozniak et al., 2006, Wozniak and Muetzel, 2011). However, the relationship between the pattern of alcohol exposure and the extent of the cerebellar alterations is not fully understood. Furthermore, the precise molecular mechanisms underlying the cerebellar deficits associated with FASDs have yet to be elucidated.

Cerebellar granule neurons (CGNs) are considered to be the gateway of information to the cerebellar cortex. CGNs receive afferent input from the brain stem and spinal cord via glutamatergic mossy fibers (MFs). The glutamatergic axons from CGNs relay MF input to Purkinje cells (PCs), which are the sole output of the cerebellar cortex. PC activity is also regulated by glutamatergic climbing fibers, GABAergic molecular layer interneurons, and collateral inputs from neighboring PCs (Watt et al., 2009). Numerous animal models of FASDs have shown that binge-like ethanol (EtOH) exposure during development induces apoptosis of PCs [reviewed in Green, 2004, Luo, 2010]. Functionally, prenatal EtOH exposure during the equivalent to the 1st and 2nd trimester of human pregnancy altered spontaneous firing, synaptic transmission, and plasticity in PCs in mice (Servais et al., 2007), whereas exposure during the equivalent of the 3rd trimester decreased the proportion of PCs that exhibit complex spikes in anesthetized rats (Backman et al., 1998). CGNs have also been shown to be vulnerable to developmental EtOH exposure. Binge-like exposure to low-to-high doses of EtOH during the 1st, 2nd, or 3rd trimester-equivalents disrupts the migration of CGNs and triggers apoptosis of these neurons in mice [reviewed in Luo (2010)]. However, the effects of EtOH exposure during the equivalent of the human 3rd trimester on synaptic transmission at CGNs have not been characterized using animal models of FASDs. It is critically important to determine the effects of EtOH during the 3rd trimester-equivalent because one of the most common patterns of alcohol abuse in pregnant women is abstinence during the first two trimesters of pregnancy followed by consumption during the last trimester (Ethen et al., 2009).

CGNs receive two distinct forms of GABAergic transmission: phasic and tonic. Phasic inhibition is mediated by conventional vesicular GABA release from specialized interneurons, referred to as Golgi cells (GoCs), which activate synaptic GABAA receptors (GABAARs). Tonic inhibition is mediated by extrasynaptic GABAARs that are activated by ambient levels of GABA whose source remains controversial (Brickley and Mody, 2012, Diaz et al., 2011, Lee et al., 2010). While GABAergic transmission at CGNs has been shown to be an important target for acute EtOH in the mature cerebellar cortex (Botta et al., 2010, Botta et al., 2007, Botta et al., 2011, Carta et al., 2004, Hanchar et al., 2005), the impact of EtOH exposure during development has not been explored. Importantly, the development of GABAergic transmission in other brain regions has been shown to be impaired in various models of 3rd trimester-equivalent EtOH exposure (Everett et al., 2012, Hsiao et al., 1998, Zucca and Valenzuela, 2010). In this study, we tested the hypothesis that repeated intermittent exposure to EtOH during the 3rd trimester-equivalent also impairs GABAergic transmission at CGNs.

Section snippets

Animals

All animal procedures were approved by the UNM-Health Sciences Center Institutional Animal Care and Use Committee and conformed to National Institutes of Health Guidelines. Timed-pregnant Sprague–Dawley rats from Harlan (Indianapolis, IN) were received at the UNM-Health Science Center at embryonic day (E) 12–16. Dams were individually housed, received food and water ad libitum, and were housed in cages with a plastic hut to reduce stress (lights on at 6 am and off at 6 pm). Animals were

Characterization of a 3rd trimester equivalent repeated intermittent EtOH vapor exposure model

In rodents, the equivalent developmental time period to the human 3rd trimester occurs during the first two postnatal weeks in rodents. To model exposure of the human fetus that mimics heavy drinking during the 3rd trimester of pregnancy (Burd et al., 2012), postnatal pups housed with their respective dams were repeatedly exposed to EtOH via vapor inhalation. Analysis of the time course of SECs during the 4th day of exposure on P6 (Fig. 2A) demonstrated that peak SECs of ∼60 mM (U.S.A. legal

Summary of findings

This study characterized the impact of EtOH exposure during the 3rd trimester-equivalent on the developing cerebellum. The major findings of this study can be summarized as follows. Repeated intermittent exposure to a high dose of EtOH during P2 to P12, using a paradigm where EtOH levels gradually increase and decrease, caused mild growth delay without any significant changes in the indices of maternal care, except for a decrease in maternal grooming time. PAE caused growth retardation that was

Conclusion

This study illustrates that repeated intermittent exposure to high levels of EtOH during the 3rd trimester-equivalent does alter the development of phasic and tonic GABAergic transmission in immature CGNs. However, this EtOH exposure increases the expression of the GABAAR δ subunit in mature CGNs, suggesting that EtOH exposure alters the programming of these neurons. Furthermore, this EtOH exposure delayed the acquisition of the air-righting reflex. In contrast to our findings, other studies

Conflict of interest

The authors declare no competing financial interests.

Acknowledgments

Funding provided by NIH grant R01-AA014973 (CFV) and minority supplement AA014973-S1 (MRD). The confocal microscopy facility at the UNM Cancer Center is supported by grant NCI P30 CA118100. We would like to thank Dr. L. Don Partridge for critically reading the manuscript.

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