Clark W. Bird

Effects of moderate prenatal ethanol exposure and age on social behavior, spatial response perseveration errors and motor behavior ☆

Persistent deficits in social behavior are among the major negative consequences associated with exposure to ethanol during prenatal development. Prior work from our laboratory has linked deficits in social behavior following moderate prenatal alcohol exposure (PAE) in the rat to functional alterations in the ventrolateral frontal cortex [21]. In addition to social behaviors, the regions comprising the ventrolateral frontal cortex are critical for diverse processes ranging from orofacial motor movements to flexible alteration of behavior in the face of changing consequences. The broader behavioral implications of altered ventrolateral frontal cortex function following moderate PAE have, however, not been examined. In the present study we evaluated the consequences of moderate PAE on social behavior, tongue protrusion, and flexibility in a variant of the Morris water task that required modification of a well-established spatial response. PAE rats displayed deficits in tongue protrusion, reduced flexibility in the spatial domain, increased wrestling, and decreased investigation, indicating that several behaviors associated with ventrolateral frontal cortex function are impaired following moderate PAE. A linear discriminant analysis revealed that measures of wrestling and tongue protrusion provided the best discrimination of PAE rats from saccharin-exposed control rats. We also evaluated all behaviors in young adult (4-5 months) or older (10-11 months) rats to address the persistence of behavioral deficits in adulthood and possible interactions between early ethanol exposure and advancing age. Behavioral deficits in each domain persisted well into adulthood (10-11 months), however, there was no evidence that aging enhances the effects of moderate PAE within the age ranges that were studied.

HuD promotes BDNF expression in brain neurons via selective stabilization of the BDNF long 3'UTR mRNA.

Complex regulation of brain-derived neurotrophic factor (BDNF) governs its intricate functions in brain development and neuronal plasticity. Besides tight transcriptional control from multiple distinct promoters, alternative 3′end processing of the BDNF transcripts generates either a long or a short 3′untranslated region (3′UTR). Previous reports indicate that distinct RNA sequence in the BDNF 3′UTRs differentially regulates BDNF production in the brain to accommodate neuronal activity changes, conceivably through differential interactions with undefined trans-acting factors that regulate stability and translation of these BDNF mRNA isoforms. In this study, we report that the neuronal RNA-binding protein (RBP) HuD interacts with a highly conserved AU-rich element (ARE) specifically located in the BDNF long 3′UTR. Such interaction is necessary and sufficient for selective stabilization of mRNAs that contain the BDNF long 3′UTR in vitro and in vivo. Moreover, in a HuD transgenic mouse model, the BDNF long 3′UTR mRNA is increased in the hippocampal dentate granule cells (DGCs), leading to elevated expression of BDNF protein that is transported and stored in the mossy fiber (MF) terminals. Our results identify HuD as the first trans-acting factor that enhances BDNF expression specifically through the long 3′UTR and a novel mechanism that regulates BDNF protein production in selected neuronal populations by HuD abundance.

KSRP Modulation of GAP-43 mRNA Stability Restricts Axonal Outgrowth in Embryonic Hippocampal Neurons

The KH-type splicing regulatory protein (KSRP) promotes the decay of AU-rich element (ARE)-containing mRNAs. Although KSRP is expressed in the nervous system, very little is known about its role in neurons. In this study, we examined whether KSRP regulates the stability of the ARE-containing GAP-43 mRNA. We found that KSRP destabilizes this mRNA by binding to its ARE, a process that requires the presence of its fourth KH domain (KH4). Furthermore, KSRP competed with the stabilizing factor HuD for binding to these sequences. We also examined the functional consequences of KSRP overexpression and knockdown on the differentiation of primary hippocampal neurons in culture. Overexpression of full length KSRP or KSRP without its nuclear localization signal hindered axonal outgrowth in these cultures, while overexpression of a mutant protein without the KH4 domain that has less affinity for binding to GAP-43′s ARE had no effect. In contrast, depletion of KSRP led to a rise in GAP-43 mRNA levels and a dramatic increase in axonal length, both in KSRP shRNA transfected cells and neurons cultured from Ksrp+/− and Ksrp −/−embryos. Finally we found that overexpression of GAP-43 rescued the axonal outgrowth deficits seen with KSRP overexpression, but only when cells were transfected with GAP-43 constructs containing 3′ UTR sequences targeting the transport of this mRNA to axons. Together, our results suggest that KSRP is an important regulator of mRNA stability and axonal length that works in direct opposition to HuD to regulate the levels of GAP-43 and other ARE-containing neuronal mRNAs.

Moderate Prenatal Alcohol Exposure Enhances GluN2B Containing NMDA Receptor Binding and Ifenprodil Sensitivity in Rat Agranular Insular Cortex

Prenatal exposure to alcohol affects the expression and function of glutamatergic neurotransmitter receptors in diverse brain regions. The present study was undertaken to fill a current gap in knowledge regarding the regional specificity of ethanol-related alterations in glutamatergic receptors in the frontal cortex. We quantified subregional expression and function of glutamatergic neurotransmitter receptors (AMPARs, NMDARs, GluN2B-containing NMDARs, mGluR1s, and mGluR5s) by radioligand binding in the agranular insular cortex (AID), lateral orbital area (LO), prelimbic cortex (PrL) and primary motor cortex (M1) of adult rats exposed to moderate levels of ethanol during prenatal development. Increased expression of GluN2B-containing NMDARs was observed in AID of ethanol-exposed rats compared to modest reductions in other regions. We subsequently performed slice electrophysiology measurements in a whole-cell patch-clamp preparation to quantify the sensitivity of evoked NMDAR-mediated excitatory postsynaptic currents (EPSCs) in layer II/III pyramidal neurons of AID to the GluN2B negative allosteric modulator ifenprodil. Consistent with increased GluN2B expression, ifenprodil caused a greater reduction in NMDAR-mediated EPSCs from prenatal alcohol-exposed rats than saccharin-exposed control animals. No alterations in AMPAR-mediated EPSCs or the ratio of AMPARs/NMDARs were observed. Together, these data indicate that moderate prenatal alcohol exposure has a significant and lasting impact on GluN2B-containing receptors in AID, which could help to explain ethanol-related alterations in learning and behaviors that depend on this region.

Effects of sex and housing on social, spatial, and motor behavior in adult rats exposed to moderate levels of alcohol during prenatal development.

Persistent deficits in social behavior, motor behavior, and behavioral flexibility are among the major negative consequences associated with exposure to ethanol during prenatal development. Prior work from our laboratory has linked moderate prenatal alcohol exposure (PAE) in the rat to deficits in these behavioral domains, which depend upon the ventrolateral frontal cortex (Hamilton et al., 2014) [20]. Manipulations of the social environment cause modifications of dendritic morphology and experience-dependent immediate early gene expression in ventrolateral frontal cortex (Hamilton et al., 2010) [19], and may yield positive behavioral outcomes following PAE. In the present study we evaluated the effects of housing PAE rats with non-exposed control rats on adult behavior. Rats of both sexes were either paired with a partner from the same prenatal treatment condition (ethanol or saccharin) or from the opposite condition (mixed housing condition). At four months of age (∼3 months after the housing manipulation commenced), social behavior, tongue protrusion, and behavioral flexibility in the Morris water task were measured as in (Hamilton et al., 2014) [20]. The behavioral effects of moderate PAE were primarily limited to males and were not ameliorated by housing with a non-ethanol exposed partner. Unexpectedly, social behavior, motor behavior, and spatial flexibility were adversely affected in control rats housed with a PAE rat (i.e., in mixed housing), indicating that housing with a PAE rat has broad behavioral consequences beyond the social domain. These observations provide further evidence that moderate PAE negatively affects social behavior, and underscore the importance of considering potential negative effects of housing with PAE animals on the behavior of critical comparison groups.

The Simple Video Coder: A free tool for efficiently coding social video data.

Videotaping of experimental sessions is a common practice across many disciplines of psychology, ranging from clinical therapy, to developmental science, to animal research. Audio–visual data are a rich source of information that can be easily recorded; however, analysis of the recordings presents a major obstacle to project completion. Coding behavior is time-consuming and often requires ad-hoc training of a student coder. In addition, existing software is either prohibitively expensive or cumbersome, which leaves researchers with inadequate tools to quickly process video data. We offer the Simple Video Coder—free, open-source software for behavior coding that is flexible in accommodating different experimental designs, is intuitive for students to use, and produces outcome measures of event timing, frequency, and duration. Finally, the software also offers extraction tools to splice video into coded segments suitable for training future human coders or for use as input for pattern classification algorithms.

Moderate Prenatal Alcohol Exposure and Quantification of Social Behavior in Adult Rats

Alterations in social behavior are among the major negative consequences observed in children with Fetal Alcohol Spectrum Disorders (FASDs). Several independent laboratories have demonstrated robust alterations in the social behavior of rodents exposed to alcohol during brain development across a wide range of exposure durations, timing, doses, and ages at the time of behavioral quantification. Prior work from this laboratory has identified reliable alterations in specific forms of social interaction following moderate prenatal alcohol exposure (PAE) in the rat that persist well into adulthood, including increased wrestling and decreased investigation. These behavioral alterations have been useful in identifying neural circuits altered by moderate PAE(1), and may hold importance for progressing toward a more complete understanding of the neural bases of PAE-related alterations in social behavior. This paper describes procedures for performing moderate PAE in which rat dams voluntarily consume ethanol or saccharin (control) throughout gestation, and measurement of social behaviors in adult offspring.

Ifenprodil infusion in agranular insular cortex alters social behavior and vocalizations in rats exposed to moderate levels of ethanol during prenatal development.

HighlightsIfenprodil infusion into agranular insular cortex alters social behaviors in rats.Many effects of ifenprodil infusion on behaviors are specific to PAE animals.Ifenprodil alters social communication, with many effects specific to PAE rats. ABSTRACT Moderate exposure to alcohol during development leads to subtle neurobiological and behavioral effects classified under the umbrella term fetal alcohol spectrum disorders (FASDs). Alterations in social behaviors are a frequently observed consequence of maternal drinking, as children with FASDs display inappropriate aggressive behaviors and altered responses to social cues. Rodent models of FASDs mimic the behavioral alterations seen in humans, with rats exposed to ethanol during development displaying increased aggressive behaviors, decreased social investigation, and altered play behavior. Work from our laboratory has observed increased wrestling behavior in adult male rats following prenatal alcohol exposure (PAE), and increased expression of GluN2B‐containing NMDA receptors in the agranular insular cortex (AIC). This study was undertaken to determine if ifenprodil, a GluN2B preferring negative allosteric modulator, has a significant effect on social behaviors in PAE rats. Using a voluntary ethanol exposure paradigm, rat dams were allowed to drink a saccharin‐sweetened solution of either 0% or 5% ethanol throughout gestation. Offspring at 6–8 months of age were implanted with cannulae into AIC. Animals were isolated for 24 h before ifenprodil or vehicle was infused into AIC, and after 15 min they were recorded in a social interaction chamber. Ifenprodil treatment altered aspects of wrestling, social investigatory behaviors, and ultrasonic vocalizations in rats exposed to ethanol during development that were not observed in control animals. These data indicate that GluN2B‐containing NMDA receptors in AIC play a role in social behaviors and may underlie alterations in behavior and vocalizations observed in PAE animals.

Social order: Using the sequential structure of social interaction to discriminate abnormal social behavior in the rat

Social interactions form the basis of a broad range of functions related to survival and mating. The complexity of social behaviors and the flexibility required for normal social interactions make social behavior particularly susceptible to disruption. The consequences of developmental insults in the social domain and the associated neurobiological factors are commonly studied in rodents. Though methods for investigating social interactions in the laboratory are diverse, animals are typically placed together in an apparatus for a brief period (under 30 min) and allowed to interact freely while behavior is recorded for subsequent analysis. A standard approach to the analysis of social behavior involves quantification of the frequency and duration of individual social behaviors. This approach provides information about the allocation of time to particular behaviors within a session, which is typically sufficient for detection of robust alterations in behavior. Virtually all social species, however, display complex sequences of social behavior that are not captured in the quantification of individual behaviors. Sequences of behavior may provide more sensitive indicators of disruptions in social behavior. Sophisticated analysis systems for quantification of behavior sequences have been available for many years; however, the required training and time to complete these analyses represent significant barriers to high-throughput assessments. We present a simple approach to the quantification of behavioral sequences that requires minimal additional analytical steps after individual behaviors are coded. We implement this approach to identify altered social behavior in rats exposed to alcohol during prenatal development, and show that the frequency of several pairwise sequences of behavior discriminate controls from ethanol-exposed rats when the frequency of individual behaviors involved in those sequences does not. Thus, the approach described here may be useful in detecting subtle deficits in the social domain and identifying neural circuits involved in the organization of social behavior.

Long-term Reductions in the Population of GABAergic Interneurons in the Mouse Hippocampus following Developmental Ethanol Exposure

Developmental exposure to ethanol leads to a constellation of cognitive and behavioral abnormalities known as Fetal Alcohol Spectrum Disorders (FASDs). Many cell types throughout the central nervous system are negatively impacted by gestational alcohol exposure, including inhibitory, GABAergic interneurons. Little evidence exists, however, describing the long-term impact of fetal alcohol exposure on survival of interneurons within the hippocampal formation, which is critical for learning and memory processes that are impaired in individuals with FASDs. Mice expressing Venus yellow fluorescent protein in inhibitory interneurons were exposed to vaporized ethanol during the third trimester equivalent of human gestation (postnatal days 2-9), and the long-term effects on interneuron numbers were measured using unbiased stereology at P90. In adulthood, interneuron populations were reduced in every hippocampal region examined. Moreover, we found that a single exposure to ethanol at P7 caused robust activation of apoptotic neurodegeneration of interneurons in the hilus, granule cell layer, CA1 and CA3 regions of the hippocampus. These studies demonstrate that developmental ethanol exposure has a long-term impact on hippocampal interneuron survivability, and may provide a mechanism partially explaining deficits in hippocampal function and hippocampus-dependent behaviors in those afflicted with FASDs.