Evolution of brain-wide activity in the awake behaving mouse after acute fear by longitudinal manganese-enhanced MRI.

Life threatening fear after a single exposure evolves in a subset of vulnerable individuals to anxiety, which may persist for their lifetime. Yet neither the whole brain's response to innate acute fear nor how brain activity evolves over time is known. Sustained neuronal activity may be a factor in the development of a persistent fear response. We couple two experimental protocols to provoke acute fear leading to prolonged fear: Predator stress (PS), a naturalistic approach to induce fear in rodents; and Serotonin transporter knockout mouse (SERT-KO) that responds to PS with sustained defensive behavior. Behavior was monitored before, during and at short and long times after PS in wild type (WT) and SERT-KO mice. Both genotypes responded to PS with defensive behavior. SERT-KO retained defensive behavior for 23 days, while WT mice returned to baseline exploratory behavior by 9 days. Thus, differences in neural activity between WT and SERT-KO 9 days after PS identifies neural correlates of persistent defensive behavior, in mice. We used longitudinal manganese-enhanced magnetic resonance imaging (MEMRI) to identify brain-wide neural activity associated with different behaviors. Mn2+ accumulation in active neurons occurs in awake, behaving mice and is retrospectively imaged. Following the same two cohorts of mice, WT and SERT-KO, longitudinally allowed unbiased quantitative comparisons of brain-wide activity by statistical parametric mapping (SPM). During natural behavior in WT, only low levels of activity-induced Mn2+-accumulation were detected, while much more accumulation appeared immediately after PS in both WT and SERT-KO, and evolved at 9 days to a new activity pattern (p < 0.0001, uncorr., T = 5.4). Patterns of accumulation differed between genotypes, with more regions of the brain and larger volumes within regions involved in SERT-KO than WT. A new computational segmentation analysis, using our InVivo Atlas based on a manganese-enhanced MR image of a living mouse, revealed dynamic changes in the volume of significantly enhanced voxels within each segment that differed between genotypes across 45 of 87 segmented regions. At Day 9 after PS, the striatum and ventral pallidum were active in both genotypes but more so in the SERT-KO. SERT-KO also displayed sustained or increased volume of Mn2+ accumulations between Post-Fear and Day 9 in eight segments where activity was decreased or silenced in WT. C-fos staining, an alternative neural activity marker, of brains from the same mice fixed at conclusion of imaging sessions confirmed that MEMRI detected active neurons. Intensity measurements in 12 regions of interest (ROIs) supported the SPM results. Between group comparisons by SPM and of ROI measurements identified specific regions differing between time points and genotypes. We report brain-wide activity in response to a single exposure of acute fear, and, for the first time, its evolution to new activity patterns over time in individuals vulnerable to persistent fear. Our results show multiple regions with dynamic changes in neural activity and that the balance of activity between segments is disordered in the SERT-KO. Thus, longitudinal MEMRI represents a powerful approach to discover how brain-wide activity evolves from the natural state either after an experience or during a disease process.

Decoupling the Effects of the Amyloid Precursor Protein From Amyloid-ß Plaques on Axonal Transport Dynamics in the Living Brain.

Amyloid precursor protein (APP) is the precursor to Aß plaques. The cytoplasmic domain of APP mediates attachment of vesicles to molecular motors for axonal transport. In APP-KO mice, transport of Mn2+ is decreased. In old transgenic mice expressing mutated human (APPSwInd) linked to Familial Alzheimer's Disease, with both expression of APPSwInd and plaques, the rate and destination of Mn2+ axonal transport is altered, as detected by time-lapse manganese-enhanced magnetic resonance imaging (MEMRI) of the brain in living mice. To determine the relative contribution of expression of APPSwInd versus plaque on transport dynamics, we developed a Tet-off system to decouple expression of APPSwInd from plaque, and then studied hippocampal to forebrain transport by MEMRI. Three groups of mice were compared to wild-type (WT): Mice with plaque and APPSwInd expression; mice with plaque but suppression of APPSwInd expression; and mice with APPSwInd suppressed from mating until 2 weeks before imaging with no plaque. MR images were captured before at successive time points after stereotactic injection of Mn2+ (3-5 nL) into CA3 of the hippocampus. Mice were returned to their home cage between imaging sessions so that transport would occur in the awake freely moving animal. Images of multiple mice from the three groups (suppressed or expressed) together with C57/B6J WT were aligned and processed with our automated computational pipeline, and voxel-wise statistical parametric mapping (SPM) performed. At the conclusion of MR imaging, brains were harvested for biochemistry or histopathology. Paired T-tests within-group between time points (p = 0.01 FDR corrected) support the impression that both plaque alone and APPSwInd expression alone alter transport rates and destination of Mn2+ accumulation. Expression of APPSwInd in the absence of plaque or detectable Aß also resulted in transport defects as well as pathology of hippocampus and medial septum, suggesting two sources of pathology occur in familial Alzheimer's disease, from toxic mutant protein as well as plaque. Alternatively mice with plaque without APPSwInd expression resemble the human condition of sporadic Alzheimer's, and had better transport. Thus, these mice with APPSwInd expression suppressed after plaque formation will be most useful in preclinical trials.

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.

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.

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

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 24h before ifenprodil or vehicle was infused into AIC, and after 15min 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.

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.

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.

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.