Background It has been estimated that approximately 12% of women consume alcohol at some time during their pregnancy, and as many as 5% of children born in the United States are impacted by prenatal alcohol exposure (PAE). The range of physical, behavioral, emotional, and social dysfunctions that are associated with PAE are collectively termed fetal alcohol spectrum disorder (FASD). Methods Using a saccharin-sweetened ethanol solution, we developed a limited access model of PAE. C57BL/6J mice were provided access to a solution of either 10% (w/v) ethanol and 0.066% (w/v) saccharin or 0.066% (w/v) saccharin (control) for 4 h/d. After establishing consistent drinking, mice were mated and continued drinking during gestation. Following parturition, solutions were decreased to 0% in a stepwise fashion over a period of 6 days. Characterization of the model included measurements of maternal consumption patterns, blood ethanol levels, litter size, pup weight, maternal care, and the effects of PAE on fear-conditioned and spatial learning, and locomotor activity. Results Mothers had mean daily ethanol intake of 7.17 ± 0.17 g ethanol/kg body weight per day, with average blood ethanol concentrations of 68.5 ± 9.2 mg/dl after 2 hours of drinking and 88.3 ± 11.5 mg/dl after 4 hours of drinking. Food and water consumption, maternal weight gain, litter size, pup weight, pup retrieval times, and time on nest did not differ between the alcohol-exposed and control animals. Compared with control offspring, mice that were exposed to ethanol prenatally displayed no difference in spontaneous locomotor activity but demonstrated learning deficits in 3 hippocampal-dependent tasks: delay fear conditioning, trace fear conditioning, and the delay nonmatch to place radial-arm maze task. Conclusions These results indicate that this model appropriately mimics the human condition of PAE and will be a useful tool in studying the learning deficits seen in FASD.

Although it is well documented that heavy consumption of alcohol during pregnancy impairs brain development, it remains controversial whether moderate consumption causes significant damage. Using a limited access, voluntary consumption paradigm, we recently demonstrated that moderate prenatal alcohol exposure (MPAE) is associated with dentate gyrus-dependent learning and memory deficits that are manifested in adulthood. Here, we identified a novel mechanism that may underlie this effect of MPAE. We found that MPAE mice exhibit deficits in N-methyl-d-aspartate receptor (NMDAR)-dependent long-term potentiation (LTP) in the dentate gyrus. Further, using semi-quantitative immunoblotting techniques, we found that the levels of GluN2B subunits were decreased in the synaptic membrane, while levels of C2’-containing GluN1 and GluN3A subunits were increased, in the dentate gyrus of MPAE mice. These data suggest that MPAE alters the subunit composition of synaptic NMDARs, leading to impaired NMDAR-dependent LTP in the dentate gyrus.

GABAA receptor subunit composition is a critical determinant of receptor localization and physiology, with synaptic receptors generating phasic inhibition and extrasynaptic receptors producing tonic inhibition. Extrasynaptically localized α5 GABAA receptors are largely responsible for tonic inhibition in hippocampal neurons. However, we show here that inhibitory synapses also contain a constant level of α5 GABAA receptors throughout neuronal development, as measured by its colocalization with gephyrin, the inhibitory postsynaptic scaffolding protein. Immunoprecipitation of the α5 subunit from both cultured neurons and adult rat brain coimmunoprecipitated gephyrin, confirming this interaction in vivo. Furthermore, the α5 subunit can interact with gephyrin independent of other synaptically localized alpha subunits, as shown by immunoprecipitation experiments in HEK cells. By replacing the α5 predicted gephyrin binding domain (Residues 370–385) with either the high affinity gephyrin binding domain of the α2 subunit or homologous residues from the extrasynaptic α4 subunit that does not interact with gephyrin, α5 GABAA receptor localization shifted into or out of the synapse, respectively. These shifts in the ratio of synaptic/extrasynaptic α5 localization disrupted dendritic outgrowth and spine maturation. In contrast to the predominant view of α5 GABAA receptors being extrasynaptic and modulating tonic inhibition, we identify an intimate association of the α5 subunit with gephyrin, resulting in constant synaptic levels of α5 GABAAR throughout circuit formation that regulates neu ronal development.

γ-aminobutyric acid (GABA) begins as the key excitatory neurotransmitter in newly forming circuits, with chloride efflux from GABA type A receptors (GABARs) producing membrane depolarization, which promotes calcium entry, dendritic outgrowth and synaptogenesis. As development proceeds, GABAergic signaling switches to inhibitory hyperpolarizing neurotransmission. Despite the evidence of impaired GABAergic neurotransmission in neurodevelopmental disorders, little is understood on how agonist-dependent GABAR activation controls the formation and plasticity of GABAergic synapses. We have identified a weakly depolarizing and inhibitory GABAR response in cortical neurons that occurs during the transition period from GABAR depolarizing excitation to hyperpolarizing inhibitory activity. We show here that treatment with the GABAR agonist muscimol mediates structural changes that diminish GABAergic synapse strength through postsynaptic and presynaptic plasticity via intracellular Ca stores, ERK and BDNF/TrkB signaling. Muscimol decreases synaptic localization of surface γ2 GABARs and gephyrin postsynaptic scaffold while β2/3 non-γ2 GABARs accumulate in the synapse. Concurrent with this structural plasticity, muscimol treatment decreases synaptic currents while enhancing the γ2 containing benzodiazepine sensitive GABAR tonic current in an ERK dependent manner. We further demonstrate that GABAR activation leads to a decrease in presynaptic GAD65 levels via BDNF/TrkB signaling. Together these data reveal a novel mechanism for agonist induced GABAergic synapse plasticity that can occur on the timescale of minutes, contributing to rapid modification of synaptic and circuit function.

Age-dependent, MK801-induced, activated caspase-3 expression in the postnatal brain is generally not observed in neurons expressing calcium-binding proteins (CaBPs), suggesting that apoptosis and calcium buffering are inversely related. In regions such as the cingulate and retrosplenial cortex, injury peaks at postnatal Day 7 (P7) and rapidly diminishes thereafter, whereas expression of calbindin (CB) and calretinin (CR) was relatively low from P0 to P7 and steadily increased from P7 to P14. At ages thereafter, CB and CR expression either remained stable then declined or rapidly declined. Parvalbumin (PV) was generally low-absent prior to P7 but expression dramatically increased from P10 onwards, peaking at P21. These studies suggest calcium entry (through N-methyl-D-aspartate receptor (NMDARs)) and buffering (by CaBPs) are integral to normal CNS maturation. Because schizophrenia is associated with glutamate hypo-function, developmental injury, and aberrant CaBP expression, our data indicate that this postnatal brain injury model may offer important insights into the nature of this disorder.