Seico Benner

Developmental origin of abnormal dendritic growth in the mouse brain induced by in utero disruption of aryl hydrocarbon receptor signaling

Increased prevalence of mental disorders cannot be solely attributed to genetic factors and is considered at least partly attributable to chemical exposure. Among various environmental chemicals, in utero and lactational dioxin exposure has been extensively studied and is known to induce higher brain function abnormalities in both humans and laboratory animals. However, how the perinatal dioxin exposure affects neuromorphological alterations has remained largely unknown. Therefore, in this study, we initially studied whether and how the over-expression of aryl hydrocarbon receptor (AhR), a dioxin receptor, would affect the dendritic growth in the hippocampus of the developing brain. Transfecting a constitutively active AhR plasmid into the hippocampus via in utero electroporation on gestational day (GD) 14 induced abnormal dendritic branch growth. Further, we observed that 14-day-old mice born to dams administered with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dose: 0, 0.6, or 3.0 μg/kg) on GD 12.5 exhibited disrupted dendritic branch growth in both the hippocampus and amygdala. Finally, we observed that 16-month-old mice born to dams exposed to perinatal TCDD as described above exhibited significantly reduced spine densities. These results indicated that abnormal micromorphology observed in the developing brain may persist until adulthood and may induce abnormal higher brain function later in life.

Neuronal Heterotopias Affect the Activities of Distant Brain Areas and Lead to Behavioral Deficits

Neuronal heterotopia refers to brain malformations resulting from deficits of neuronal migration. Individuals with heterotopias show a high incidence of neurological deficits, such as epilepsy. More recently, it has come to be recognized that focal heterotopias may also show a range of psychiatric problems, including cognitive and behavioral impairments. However, because focal heterotopias are not always located in the brain areas responsible for the symptoms, the causal relationship between the symptoms and heterotopias remains elusive. In this study, we showed that mice with focal heterotopias in the somatosensory cortex generated by in utero electroporation exhibited spatial working memory deficit and low competitive dominance behavior, which have been shown to be closely associated with the activity of the medial prefrontal cortex (mPFC) in rodents. Analysis of the mPFC activity revealed that the immediate-early gene expression was decreased and the local field potentials of the mPFC were altered in the mice with heterotopias compared with the control mice. Moreover, activation of these ectopic and overlying sister neurons using the DREADD (designer receptor exclusively activated by designer drug) system improved the working memory deficits. These findings suggest that cortical regions containing focal heterotopias can affect distant brain regions and give rise to behavioral abnormalities. SIGNIFICANCE STATEMENT Recent studies reported that patients with heterotopias have a variety of clinical symptoms, such as cognitive disturbance, psychiatric symptoms, and autistic behavior. However, the causal relationship between the symptoms and heterotopias remains elusive. Here we showed that mice with focal heterotopias in the somatosensory cortex generated by in utero electroporation exhibited behavioral deficits that have been shown to be associated with the mPFC activity in rodents. The existence of heterotopias indeed altered the neural activities of the mPFC, and direct manipulation of the neural activity of the ectopic neurons and their sister neurons in the overlying cortex improved the behavioral deficit. Thus, our results indicate that focal heterotopias could affect the activities of distant brain areas and cause behavioral abnormalities.

Environmental insults in early life and submissiveness later in life in mouse models

Dominant and subordinate dispositions are not only determined genetically but also nurtured by environmental stimuli during neuroendocrine development. However, the relationship between early life environment and dominance behavior remains elusive. Using the IntelliCage-based competition task for group-housed mice, we have previously described two cases in which environmental insults during the developmental period altered the outcome of dominance behavior later in life. First, mice that were repeatedly isolated from their mother and their littermates (early deprivation; ED), and second, mice perinatally exposed to an environmental pollutant, dioxin, both exhibited subordinate phenotypes, defined by decreased occupancy of limited resource sites under highly competitive circumstances. Similar alterations found in the cortex and limbic area of these two models are suggestive of the presence of neural systems shared across generalized dominance behavior.

Association of impaired neuronal migration with cognitive deficits in extremely preterm infants

Many extremely preterm infants (born before 28 gestational weeks [GWs]) develop cognitive impairment in later life, although the underlying pathogenesis is not yet completely understood. Our examinations of the developing human neocortex confirmed that neuronal migration continues beyond 23 GWs, the gestational week at which extremely preterm infants have live births. We observed larger numbers of ectopic neurons in the white matter of the neocortex in human extremely preterm infants with brain injury and hypothesized that altered neuronal migration may be associated with cognitive impairment in later life. To confirm whether preterm brain injury affects neuronal migration, we produced brain damage in mouse embryos by occluding the maternal uterine arteries. The mice showed delayed neuronal migration, ectopic neurons in the white matter, altered neuronal alignment, and abnormal corticocortical axonal wiring. Similar to human extremely preterm infants with brain injury, the surviving mice exhibited cognitive deficits. Activation of the affected medial prefrontal cortices of the surviving mice improved working memory deficits, indicating that decreased neuronal activity caused the cognitive deficits. These findings suggest that altered neuronal migration altered by brain injury might contribute to the subsequent development of cognitive impairment in extremely preterm infants.

Clinical potential of oxytocin in autism spectrum disorder: current issues and future perspectives

The effects of oxytocin on social cognition and behavior have recently attracted considerable attention. In particular, oxytocin has been proposed as a novel therapeutic for psychiatric disorders with social deficits such as autism spectrum disorders. This review provides a brief overview of behavioral and neural responses to oxytocin manipulations in humans and animal models. Although the differences in findings between human and animal studies should be interpreted carefully, shared behavioral phenotypes have been recognized, such as social bonding, social responses, and recognition and usage of social cues. Previous literature suggests that the neural effects of oxytocin in humans and animals overlap in the prefrontal, limbic, and paralimbic cortices. Oxytocin-induced alterations in these regions may indicate a fundamental basis for how oxytocin modulates social behaviors and facilitate the discovery of new pharmaceutical targets for treating social deficits.

Application of NeuroTrace staining in the fresh frozen brain samples to laser microdissection combined with quantitative RT-PCR analysis

BackgroundThe heterogeneity of the brain requires appropriate molecular biological approaches to account for its morphological complexity. Laser-assisted microdissection followed by transcript profiling by quantitative determination has been reported to be an optimal methodology. Nevertheless, not all brain regions can be identified easily without staining, restricting the accuracy and efficiency in sampling. The aim of the present study was to validate whether fixation and staining treatments are suitable for quantitative transcript expression analysis in laser microdissection (LMD) samples. Quantitative RT-PCR was used to determine the absolute transcript expression levels and profiles of samples obtained from the hippocampal dentate gyrus from fresh frozen mice brain sections that had been fixed with ethanol and stained with NeuroTrace. The results were compared with those obtained from unfixed and unstained samples.ResultsWe found that the quantitative relationship of transcript expression levels between various housekeeping genes and immediate early genes was preserved, although the preparation compromised the yield of the transcripts. In addition, histological and molecular integrities of the fixed and stained specimens were preserved for at least a week at room temperature. Based on the lobe specific profiles of transcripts in the anterior and posterior lobes of the pituitary, we confirmed that no cross-contamination on transcription expressions occurred as a result of the fixation and staining.ConclusionsWe have provided detailed information of the procedures on ethanol fixation followed by NeuroTrace staining on the absolute quantitative RT-PCR analysis using microdissected fresh frozen mouse brain tissues. The present study demonstrated that quantitative transcript expression analysis can be conducted reliably on stained tissues. This method is suitable for applications in basic and clinical studies on particular transcript expressions in various regions of the brain.

Effect of intranasal oxytocin on the core social symptoms of autism spectrum disorder: a randomized clinical trial

Although small-scale studies have described the effects of oxytocin on social deficits in autism spectrum disorder (ASD), no large-scale study has been conducted. In this randomized, parallel-group, multicenter, placebo-controlled, double-blind trial in Japan, 106 ASD individuals (18–48 y.o.) were enrolled between Jan 2015 and March 2016. Participants were randomly assigned to a 6-week intranasal oxytocin (48IU/day, n = 53) or placebo (n = 53) group. One-hundred-three participants were analyzed. Since oxytocin reduced the primary endpoint, Autism Diagnostic Observation Schedule (ADOS) reciprocity, (from 8.5 to 7.7; P < .001) but placebo also reduced the score (8.3 to 7.2; P < .001), no between-group difference was found (effect size −0.08; 95% CI, −0.46 to 0.31; P = .69); however, plasma oxytocin was only elevated from baseline to endpoint in the oxytocin-group compared with the placebo-group (effect size −1.12; −1.53 to −0.70; P < .0001). Among the secondary endpoints, oxytocin reduced ADOS repetitive behavior (2.0 to 1.5; P < .0001) compared with placebo (2.0 to 1.8; P = .43) (effect size 0.44; 0.05 to 0.83; P = .026). In addition, the duration of gaze fixation on socially relevant regions, another secondary endpoint, was increased by oxytocin (41.2 to 52.3; P = .03) compared with placebo (45.7 to 40.4; P = .25) (effect size 0.55; 0.10 to 1.0; P = .018). No significant effects were observed for the other secondary endpoints. No significant difference in the prevalence of adverse events was observed between groups, although one participant experienced temporary gynecomastia during oxytocin administration. Based on the present findings, we cannot recommend continuous intranasal oxytocin treatment alone at the current dose and duration for treatment of the core social symptoms of high-functioning ASD in adult men, although this large-scale trial suggests oxytocin’s possibility to treat ASD repetitive behavior.

Neurochemical evidence for differential effects of acute and repeated oxytocin administration

A discrepancy in oxytocin’s behavioral effects between acute and repeated administrations indicates distinct underlying neurobiological mechanisms. The current study employed a combination of human clinical trial and animal study to compare neurochemical changes induced by acute and repeated oxytocin administrations. Human study analyzed medial prefrontal metabolite levels by using 1H-magnetic resonance spectroscopy, a secondary outcome in our randomized, double-blind, placebo-controlled crossover trial of 6 weeks intranasal administrations of oxytocin (48 IU/day) and placebo within-subject design in 17 psychotropic-free high-functioning men with autism spectrum disorder. Medial prefrontal transcript expression levels were analyzed in adult male C57BL/6J mice after intraperitoneal injection of oxytocin or saline either once (200 ng/100 μL/mouse, n = 12) or for 14 consecutive days (200 ng/100 μL/mouse/day, n = 16). As the results, repeated administration of oxytocin significantly decreased the medial prefrontal N-acetylaspartate (NAA; p = 0.043) and glutamate–glutamine levels (Glx; p = 0.001), unlike the acute oxytocin. The decreases were inversely and specifically associated (r = 0.680, p = 0.004 for NAA; r = 0.491, p = 0.053 for Glx) with oxytocin-induced improvements of medial prefrontal functional MRI activity during a social judgment task not with changes during placebo administrations. In wild-type mice, we found that repeated oxytocin administration reduced medial frontal transcript expression of N-methyl-d-aspartate receptor type 2B (p = 0.018), unlike the acute oxytocin, which instead changed the transcript expression associated with oxytocin (p = 0.0004) and neural activity (p = 0.0002). The present findings suggest that the unique sensitivity of the glutamatergic system to repeated oxytocin administration may explain the differential behavioral effects of oxytocin between acute and repeated administration.