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
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.
References (97)
- et al.
Early hypergravity exposure effects calbindin-D28k and inositol-3-phosphate expression in Purkinje cells
Neurosci. Lett.
(2005) - et al.
Behavioural consequences of hypergravity in developing rats
Brain Res. Dev. Brain Res.
(2004) - et al.
Extrasynaptic GABA(A) receptors: their function in the CNS and implications for disease
Neuron
(2012) - et al.
Fetal alcohol syndrome: historical perspectives
Neurosci. Biobehav. Rev.
(2007) - et al.
Impaired cerebellar learning in children with prenatal alcohol exposure: a comparative study of eyeblink conditioning in children with ADHD and dyslexia
Cortex
(2005) - et al.
Dynamics of fast and slow inhibition from cerebellar golgi cells allow flexible control of synaptic integration
Neuron
(2009) - et al.
Binge ethanol exposure delays development of GABAergic miniature postsynaptic currents in septal neurons
Brain Res. Dev. Brain Res.
(2004) - et al.
GABAergic miniature postsynaptic currents in septal neurons show differential allosteric sensitivity after binge-like ethanol exposure
Brain Res.
(2006) - et al.
Actions and interactions of extracellular potassium and kainate on expression of 13 gamma-aminobutyric acid type A receptor subunits in cultured mouse cerebellar granule neurons
J. Biol. Chem.
(2003) - et al.
Effects of third trimester-equivalent ethanol exposure on Cl(-) co-transporter expression, network activity, and GABAergic transmission in the CA3 hippocampal region of neonatal rats
Alcohol
(2012)
Immature hippocampal neuronal networks do not develop tolerance to the excitatory actions of ethanol
Alcohol
Binge neonatal alcohol intubations induce dose-dependent loss of Purkinje cells
Neurotoxicol. Teratol.
Tonic and spillover inhibition of granule cells control information flow through cerebellar cortex
Neuron
Development of GABAA receptors on medial septum/diagonal band (MS/DB) neurons after postnatal ethanol exposure
Brain Res.
Postnatal ethanol exposure blunts upregulation of GABAA receptor currents in Purkinje neurons
Brain Res.
Maternal voluntary drinking in C57BL/6J mice: advancing a model for fetal alcohol spectrum disorders
Behav. Brain Res.
Developing rat Purkinje cells are more vulnerable to alcohol-induced depletion during differentiation than during neurogenesis
Alcohol
Neurodevelopment milestone abnormalities in rats exposed to stress in early life
Neuroscience
Resting intracellular calcium concentration, depolarizing gamma-aminobutyric acid and possible role of local estradiol synthesis in the developing male and female hippocampus
Neuroscience
Visual modulation of vestibularly-triggered air-righting in the rat
Behav. Brain Res.
Visual modulation of vestibularly-triggered air-righting in rats involves the superior colliculus
Behav. Brain Res.
GABA(A) receptors: immunocytochemical distribution of 13 subunits in the adult rat brain
Neuroscience
Maternal care alterations induced by repeated ethanol leads to heightened consumption of the drug and motor impairment during adolescence: a dose-response analysis
Physiol. Behav.
Decrease in levels and rates of synthesis of tubulin and actin in developing rat brain
Brain Res.
Postnatal development of rat pups is altered by prenatal methamphetamine exposure
Progr. Neuro-psychopharmacol. Biol. Psychiatry
Gestational stress induces post-partum depression-like behaviour and alters maternal care in rats
Psychoneuroendocrinology
Morphological development and maturation of the GABAergic synapses in the mouse cerebellar granular layer
Brain Res. Dev. Brain Res.
Developmental localization of potassium chloride co-transporter 2 in granule cells of the early postnatal mouse cerebellum with special reference to the synapse formation
Neuroscience
The effect of acute, chronic, and prenatal ethanol exposure on insulin sensitivity
Pharmacol. Therap.
Targeted disruption of the GABA(A) receptor delta subunit gene leads to an up-regulation of gamma 2 subunit-containing receptors in cerebellar granule cells
J. Biol. Chem.
Effects of prenatal ethanol exposure on postnatal growth and the insulin-like growth factor axis
Horm. Res. Paediat.
MRI findings in children with school problems who had been exposed prenatally to alcohol
Dev. Med. Child Neurol.
Electrophysiological characterization of cerebellar neurons from adult rats exposed to ethanol during development
Alcohol. Clin. Exp. Res.
Alcohol excites cerebellar Golgi cells by inhibiting the Na+/K+ ATPase
Neuropsychopharmacology
Ethanol sensitivity of GABAergic currents in cerebellar granule neurons is not increased by a single amino acid change (R100Q) in the alpha6 GABAA receptor subunit
J. Pharmacol. Exp. Therap.
Excitation of rat cerebellar golgi cells by ethanol: further characterization of the mechanism
Alcohol. Clin. Exp. Res.
An electrophysiological and anatomical study of afferents reaching the cerebellar uvula in the rabbit
Exp. Physiol.
Development of a tonic form of synaptic inhibition in rat cerebellar granule cells resulting from persistent activation of GABAA receptors
J. Physiol.
Multimodal integration in rostral fastigial nucleus provides an estimate of body movement
J. Neurosci.: Off. J. Soc. Neurosci.
Prenatal alcohol exposure, blood alcohol concentrations and alcohol elimination rates for the mother, fetus and newborn
J. Perinatol.: Off. J. Cal. Perinatal Assoc.
Alcohol enhances GABAergic transmission to cerebellar granule cells via an increase in Golgi cell excitability
J. Neurosci.: Off. J. Soc. Neurosci.
Motor coordination and balance in rodents
Acute effects of ethanol and the first suckling episode in the newborn rat
Alcohol. Clin. Exp. Res.
GABA increases Ca2+ in cerebellar granule cell precursors via depolarization: implications for proliferation
IUBMB Life
Prenatal alcohol use and offspring size at 10 years of age
Alcohol. Clin. Exp. Res.
si-RNA inhibition of brain insulin or insulin-like growth factor receptors causes developmental cerebellar abnormalities: relevance to fetal alcohol spectrum disorder
Mol. Brain
Prostaglandin E2 stimulates estradiol synthesis in the cerebellum postnatally with associated effects on Purkinje neuron dendritic arbor and electrophysiological properties
Endocrinology
Bestrophin1 channels are Insensitive to ethanol and do not mediate tonic GABAergic currents in cerebellar granule cells
Front. Neurosci.
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