Sex on the brain.

Release of testosterone during early development is necessary for masculinization of brain structures in rodents. Wu et al. (2009) now elucidate the neuronal changes caused by aromatase-mediated conversion of testosterone into estrogen in perinatal mice and the ensuing effects on adult behavior.

Maternal diabetes induces autism-like behavior by hyperglycemia-mediated persistent oxidative stress and suppression of superoxide dismutase 2.

Epidemiological studies show that maternal diabetes is associated with an increased risk of autism spectrum disorders (ASDs), although the detailed mechanisms remain unclear. The present study aims to investigate the potential effect of maternal diabetes on autism-like behavior in offspring. The results of in vitro study showed that transient hyperglycemia induces persistent reactive oxygen species (ROS) generation with suppressed superoxide dismutase 2 (SOD2) expression. Additionally, we found that SOD2 suppression is due to oxidative stress-mediated histone methylation and the subsequent dissociation of early growth response 1 (Egr1) on the SOD2 promoter. Furthermore, in vivo rat experiments showed that maternal diabetes induces SOD2 suppression in the amygdala, resulting in autism-like behavior in offspring. SOD2 overexpression restores, while SOD2 knockdown mimics, this effect, indicating that oxidative stress and SOD2 expression play important roles in maternal diabetes-induced autism-like behavior in offspring, while prenatal and postnatal treatment using antioxidants permeable to the blood-brain barrier partly ameliorated this effect. We conclude that maternal diabetes induces autism-like behavior through hyperglycemia-mediated persistent oxidative stress and SOD2 suppression. Here we report a potential mechanism for maternal diabetes-induced ASD.

Molecular profiling of reticular gigantocellularis neurons indicates that eNOS modulates environmentally dependent levels of arousal.

Neurons of the medullary reticular nucleus gigantocellularis (NGC) and their targets have recently been a focus of research on mechanisms supporting generalized CNS arousal (GA) required for proper cognitive functions. Using the retro-TRAP method, we characterized transcripts enriched in NGC neurons which have projections to the thalamus. The unique expression and activation of the endothelial nitric oxide (eNOS) signaling pathway in these cells and their intimate connections with blood vessels indicate that these neurons exert direct neurovascular coupling. Production of nitric oxide (NO) within eNOS-positive NGC neurons increases after environmental perturbations, indicating a role for eNOS/NO in modulating environmentally appropriate levels of GA. Inhibition of NO production causes dysregulated behavioral arousal after exposure to environmental perturbation. Further, our findings suggest interpretations for associations between psychiatric disorders and mutations in the eNOS locus.

Sex-specific gene-environment interactions underlying ASD-like behaviors.

The male bias in the incidence of autism spectrum disorders (ASDs) is one of the most notable characteristics of this group of neurodevelopmental disorders. The etiology of this sex bias is far from known, but pivotal for understanding the etiology of ASDs in general. Here we investigate whether a "three-hit" (genetic load × environmental factor × sex) theory of autism may help explain the male predominance. We found that LPS-induced maternal immune activation caused male-specific deficits in certain social responses in the contactin-associated protein-like 2 (Cntnap2) mouse model for ASD. The three "hits" had cumulative effects on ultrasonic vocalizations at postnatal day 3. Hits synergistically affected social recognition in adulthood: only mice exposed to all three hits showed deficits in this aspect of social behavior. In brains of the same mice we found a significant three-way interaction on corticotropin-releasing hormone receptor-1 (Crhr1) gene expression, in the left hippocampus specifically, which co-occurred with epigenetic alterations in histone H3 N-terminal lysine 4 trimethylation (H3K4me3) over the Crhr1 promoter. Although it is highly likely that multiple (synergistic) interactions may be at work, change in the expression of genes in the hypothalamic-pituitary-adrenal/stress system (e.g., Crhr1) is one of them. The data provide proof-of-principle that genetic and environmental factors interact to cause sex-specific effects that may help explain the male bias in ASD incidence.

Stress and corticosteroids regulate rat hippocampal mitochondrial DNA gene expression via the glucocorticoid receptor.

Glucocorticoids (GCs) are involved in stress and circadian regulation, and produce many actions via the GC receptor (GR), which is classically understood to function as a nuclear transcription factor. However, the nuclear genome is not the only genome in eukaryotic cells. The mitochondria also contain a small circular genome, the mitochondrial DNA (mtDNA), that encodes 13 polypeptides. Recent work has established that, in the brain and other systems, the GR is translocated from the cytosol to the mitochondria and that stress and corticosteroids have a direct influence on mtDNA transcription and mitochondrial physiology. To determine if stress affects mitochondrially transcribed mRNA (mtRNA) expression, we exposed adult male rats to both acute and chronic immobilization stress and examined mtRNA expression using quantitative RT-PCR. We found that acute stress had a main effect on mtRNA expression and that expression of NADH dehydrogenase 1, 3, and 6 (ND-1, ND-3, ND-6) and ATP synthase 6 (ATP-6) genes was significantly down-regulated. Chronic stress induced a significant up-regulation of ND-6 expression. Adrenalectomy abolished acute stress-induced mtRNA regulation, demonstrating GC dependence. ChIP sequencing of GR showed that corticosterone treatment induced a dose-dependent association of the GR with the control region of the mitochondrial genome. These findings demonstrate GR and stress-dependent transcriptional regulation of the mitochondrial genome in vivo and are consistent with previous work linking stress and GCs with changes in the function of brain mitochondria.

Can distinctly different rapid estrogen actions share a common mechanistic step?

Contribution to Special Issue on Fast effects of steroids. This paper reviews early evidence for the existence of rapid, non-genomic effects of estrogens on neurons, and, further, proposes that these rapid effects are often synergistic with later, genomic effects. Finally, suggestions about potential molecular mechanisms underlying the rapid effects of estrogens are offered. A mechanistic step we propose to be common among rapid estrogenic actions includes membrane ER's binding to histamine, and NMDA receptors and subsequent dimerization, and clustering (respectively) in a manner that enhances histamine and NMDA actions.

Stress and the dynamic genome: Steroids, epigenetics, and the transposome.

Stress plays a substantial role in shaping behavior and brain function, often with lasting effects. How these lasting effects occur in the context of a fixed postmitotic neuronal genome has been an enduring question for the field. Synaptic plasticity and neurogenesis have provided some of the answers to this question, and more recently epigenetic mechanisms have come to the fore. The exploration of epigenetic mechanisms recently led us to discover that a single acute stress can regulate the expression of retrotransposons in the rat hippocampus via an epigenetic mechanism. We propose that this response may represent a genomic stress response aimed at maintaining genomic and transcriptional stability in vulnerable brain regions such as the hippocampus. This finding and those of other researchers have made clear that retrotransposons and the genomic plasticity they permit play a significant role in brain function during stress and disease. These observations also raise the possibility that the transposome might have adaptive functions at the level of both evolution and the individual organism.

Rapid increases in immature synapses parallel estrogen-induced hippocampal learning enhancements.

Dramatic increases in hippocampal spine synapse density are known to occur within minutes of estrogen exposure. Until now, it has been assumed that enhanced spinogenesis increased excitatory input received by the CA1 pyramidal neurons, but how this facilitated learning and memory was unclear. Delivery of 17ß-estradiol or an estrogen receptor (ER)-α (but not ER-ß) agonist into the dorsal hippocampus rapidly improved general discrimination learning in female mice. The same treatments increased CA1 dendritic spines in hippocampal sections over a time course consistent with the learning acquisition phase. Surprisingly, estrogen-activated spinogenesis was associated with a decrease in CA1 hippocampal excitatory input, rapidly and transiently reducing CA1 AMPA activity via a mechanism likely reflecting AMPA receptor internalization and creation of silent or immature synapses. We propose that estrogens promote hippocampally mediated learning via a mechanism resembling some of the broad features of normal development, an initial overproduction of functionally immature connections being subsequently "pruned" by experience.

Recovery of consciousness is mediated by a network of discrete metastable activity states.

It is not clear how, after a large perturbation, the brain explores the vast space of potential neuronal activity states to recover those compatible with consciousness. Here, we analyze recovery from pharmacologically induced coma to show that neuronal activity en route to consciousness is confined to a low-dimensional subspace. In this subspace, neuronal activity forms discrete metastable states persistent on the scale of minutes. The network of transitions that links these metastable states is structured such that some states form hubs that connect groups of otherwise disconnected states. Although many paths through the network are possible, to ultimately enter the activity state compatible with consciousness, the brain must first pass through these hubs in an orderly fashion. This organization of metastable states, along with dramatic dimensionality reduction, significantly simplifies the task of sampling the parameter space to recover the state consistent with wakefulness on a physiologically relevant timescale.

Development of Electrophysiological Properties of Nucleus Gigantocellularis Neurons Correlated with Increased CNS Arousal.

Many types of data have suggested that neurons in the nucleus gigantocellularis (NGC) in the medullary reticular formation are critically important for CNS arousal and behavioral responsiveness. To extend this topic to a developmental framework, whole-cell patch-recorded characteristics of NGC neurons in brainstem slices and measures of arousal-dependent locomotion of postnatal day 3 (P3) to P6 mouse pups were measured and compared. These neuronal characteristics developed in an orderly, statistically significant monotonic manner over the course of P3-P6: (1) proportion of neurons capable of firing action potential (AP) trains, (2) AP amplitude, (3) AP threshold, (4) amplitude of inward and outward currents, (5) amplitude of negative peak currents, and (6) steady state currents (in I-V plot). These measurements reflect the maturation of sodium and certain potassium channels. Similarly, all measures of locomotion, latency to first movement, total locomotion duration, net locomotion distance, and total quiescence time also developed monotonically over P3-P6. Most importantly, electrophysiological and behavioral measures were significantly correlated. Interestingly, the behavioral measures were not correlated with frequency of excitatory postsynaptic currents or the proportion of neurons showing these currents, responses to a battery of neurotransmitter agents, or rapid activating potassium currents (including IA). Considering the results here in the context of a large body of literature on NGC, we hypothesize that the developmental increase in NGC neuronal excitability participates in causing the increased behavioral responsivity during the postnatal period from P3 to P6.

Stochastic modeling of mouse motor activity under deep brain stimulation: the extraction of arousal information.

In the present paper, we quantify, with a rigorous approach, the nature of motor activity in response to Deep Brain Stimulation (DBS), in the mouse. DBS is currently being used in the treatment of a broad range of diseases, but its underlying principles are still unclear. Because mouse movement involves rapidly repeated starting and stopping, one must statistically verify that the movement at a given stimulation time was not just coincidental, endogenously-driven movement. Moreover, the amount of activity changes significantly over the circadian rhythm, and hence the means, variances and autocorrelations are all time varying. A new methodology is presented. For example, to discern what is and what is not impacted by stimulation, velocity is classified (in a time-evolving manner) as being zero-, one- and two-dimensional movement. The most important conclusions of the paper are: (1) (DBS) stimulation is proven to be truly effective; (2) it is two-dimensional (2-D) movement that strongly differs between light and dark and responds to stimulation; and, (3) stimulation in the light initiates a manner of movement, 2-D movement, that is more commonly seen in the (non-stimulated) dark. Based upon these conclusions, it is conjectured that the above patterns of 2-D movement could be a straightforward, easy to calculate correlate of arousal. The above conclusions will aid in the systematic evaluation and understanding of how DBS in CNS arousal pathways leads to the activation of behavior.

Etiologies underlying sex differences in Autism Spectrum Disorders.

The male predominance of Autism Spectrum Disorders (ASD) is one of the best-known, and at the same time, one of the least understood characteristics of these disorders. In this paper we review genetic, epigenetic, hormonal, and environmental mechanisms underlying this male preponderance. Sex-specific effects of Y-linked genes (including SRY expression leading to testicular development), balanced and skewed X-inactivation, genes that escape X-inactivation, parent-of-origin allelic imprinting, and the hypothetical heterochromatin sink are reviewed. These mechanisms likely contribute to etiology, instead of being simply causative to ASD. Environments, both internal and external, also play important roles in ASD's etiology. Early exposure to androgenic hormones and early maternal immune activation comprise environmental factors affecting sex-specific susceptibility to ASD. The gene-environment interactions underlying ASD, suggested here, implicate early prenatal stress as being especially detrimental to boys with a vulnerable genotype.

Impact of Thyroid Hormones on Estrogen Receptor α-Dependent Transcriptional Mechanisms in Ventromedial Hypothalamus and Preoptic Area.

Elevated levels of thyroid hormones (TH) reduce estradiol (E2)-dependent female sexual behavior. E2 stimulates progesterone receptor (Pgr) and oxytocin receptor (Oxtr) within the ventromedial hypothalamus and preoptic area, critical hypothalamic nuclei for sexual and maternal behavior, respectively. Here, we investigated the impact of TH on E2-dependent transcriptional mechanisms in female mice. First, we observed that triiodothyronine (T3) inhibited the E2 induction of Pgr and Oxtr. We hypothesized that differences in histone modifications and receptor recruitment could explain the influence of TH on E2-responsive Pgr and Oxtr expression. We observed that histone H3 acetylation (H3Ac) and methylation (H3K4me3) was gene and brain-region specific. We then analyzed the recruitment of estrogen receptor α (ERα) and TH receptor α (TRα) on the putative regulatory sequences of Pgr and Oxtr. Interestingly, T3 inhibited E2-induced ERα binding to a specific Pgr enhancer site, whereas TRα binding was not affected, corroborating our theory that the competitive binding of TRα to an ERα binding site can inhibit ERα transactivation and the subsequent E2-responsive gene expression. On the Oxtr promoter, E2 and T3 worked together to modulate ERα and TRα binding. Finally, the E2-dependent induction of cofactors was reduced by hypothyroidism and T3. Thus, we determined that the Pgr and Oxtr promoter regions are responsive to E2 and that T3 interferes with the E2 regulation of Pgr and Oxtr expression by altering the recruitment of receptors to DNA and changing the availability of cofactors. Collectively, our findings provide insights into molecular mechanisms of response to E2 and TH interactions controlling sex behavior in the hypothalamus.

Scale invariance in the dynamics of spontaneous behavior.

Typically one expects that the intervals between consecutive occurrences of a particular behavior will have a characteristic time scale around which most observations are centered. Surprisingly, the timing of many diverse behaviors from human communication to animal foraging form complex self-similar temporal patterns reproduced on multiple time scales. We present a general framework for understanding how such scale invariance may arise in nonequilibrium systems, including those that regulate mammalian behaviors. We then demonstrate that the predictions of this framework are in agreement with detailed analysis of spontaneous mouse behavior observed in a simple unchanging environment. Neural systems operate on a broad range of time scales, from milliseconds to hours. We analytically show that such a separation between time scales could lead to scale-invariant dynamics without any fine tuning of parameters or other model-specific constraints. Our analyses reveal that the specifics of the distribution of resources or competition among several tasks are not essential for the expression of scale-free dynamics. Rather, we show that scale invariance observed in the dynamics of behavior can arise from the dynamics intrinsic to the brain.

siRNA silencing of estrogen receptor-α expression specifically in medial preoptic area neurons abolishes maternal care in female mice.

The medial preoptic area has been shown to be intricately involved in many behaviors, including locomotion, sexual behavior, maternal care, and aggression. The gene encoding estrogen receptor-α (ERα) protein is expressed in preoptic area neurons, and a very dense immunoreactive field of ERα is found in the preoptic region. ERα knockout animals show deficits in maternal care and sexual behavior and fail to exhibit increases in these behaviors in response to systemic estradiol treatment. In the present study, we used viral-vector mediated RNA interference to silence ERα expression specifically in the preoptic area of female mice and measured a variety of behaviors, including social and sexual aggression, maternal care, and arousal activity. Suppression of ERα in the preoptic area almost completely abolished maternal care, significantly increasing the latency to pup retrieval and significantly reducing the time the moms spent nursing and licking the pups. Strikingly, maternal aggression toward a male intruder was not different between control and preoptic ERα-silenced mice, demonstrating the remarkably specific role of ERα in these neurons. Reduction of ERα expression in preoptic neurons significantly decreased sexual behavior in female mice and increased aggression toward both sexual partners and male intruders in a seminatural environment. Estrogen-dependent increases in arousal, measured by home cage activity, were not mediated by ERα expression in the preoptic neurons we targeted, as ERα-suppressed mice had increases similar to control mice. Thus, we have established that a specific gene in a specific group of neurons is required for a crucially important natural behavior.

Acute stress and hippocampal histone H3 lysine 9 trimethylation, a retrotransposon silencing response.

The hippocampus is a highly plastic brain region particularly susceptible to the effects of environmental stress; it also shows dynamic changes in epigenetic marks in response to stress and learning. We have previously shown that, in the rat, acute (30 min) restraint stress induces a substantial, regionally specific, increase in hippocampal levels of the repressive histone H3 lysine 9 trimethylation (H3K9me3). Because of the large magnitude of this effect and the fact that stress can induce the expression of endogenous retroviruses and transposable elements in many systems, we hypothesized that the H3K9me3 response was targeted to these elements as a means of containing potential genomic instability. We used ChIP coupled with next generation sequencing (ChIP-Seq) to determine the genomic localization of the H3K9me3 response. Although there was a general increase in this response across the genome, our results validated this hypothesis by demonstrating that stress increases H3K9me3 enrichment at transposable element loci and, using RT-PCR, we demonstrate that this effect represses expression of intracisternal-A particle endogenous retrovirus elements and B2 short interspersed elements, but it does not appear to have a repressive effect on long interspersed element RNA. In addition, we present data showing that the histone H3K9-specific methyltransferases Suv39h2 is up-regulated by acute stress in the hippocampus, and that this may explain the hippocampal specificity we observe. These results are a unique demonstration of the regulatory effect of environmental stress, via an epigenetic mark, on the vast genomic terra incognita represented by transposable elements.

Self-assessment of anatomy, sexual sensitivity, and function of the labia and vagina.

Patient perceptions of genital esthetics are motivating requests for plastic surgeries that could change sexual sensitivity. There is little information about the sensitivities of labial and introital sites. The aim of this study is to assess the relationship between sexual sensitivity and self-reported sizes of labial and introital sites. Sixty-two healthy, sexually active, adult women (mean age 37.9, range 21-60) with no history of genital or vaginal surgery gave written consent to participate in this study. A modified version of Self-Assessment of Genital Anatomy and Sexual Function (L-SAGASF-F) was used to assess labial and introital size. Site-specific sensation was rated on Likert scales of 1-5. Anatomical locations were compared for ratings. Of 62 responders, 84% (52) described their labia as "average-sized," 11% (7) described their labia minora and 13% (8) their labia majora as "large", and 3% (2) and 5% (3) as "small". Sexual pleasure ratings were "moderate" (median value: 3.0 for external genitalia and vaginal lumen) or "strong" (median value: 4.0 for the interior vagina). Significantly higher rankings related to the vaginal opening (P=0.007). Orgasm intensity for stimulation of the external genitalia progressively increased toward the vaginal opening, from 1.0 to 3.0 (P=0.001); vaginal ratings showed a similar progression, from 2.0 at the external luminal margin to 3.0 in the deep interior (P<0.0001). Orgasm effort scores were intermediate (median: 3.0), uniform throughout the external and internal areas (P=0.626). Ratings for labial and introital sensitivity, regardless of self-reported size, were very similar to those at other genital sites for sexual pleasure. Surgical excision of labial and introital structures could modify sexual sensation.

Estradiol regulates responsiveness of the dorsal premammillary nucleus of the hypothalamus and affects fear- and anxiety-like behaviors in female rats.

Research suggests a causal link between estrogens and mood. Here, we began by examining the effects of estradiol (E2 ) on rat innate and conditioned defensive behaviors in response to cat odor. Second, we utilized whole-cell patch clamp electrophysiological techniques to assess noradrenergic effects on neurons within the dorsal premammillary nucleus of the hypothalamus (PMd), a nucleus implicated in fear reactivity, and their regulation by E2 . Our results show that E2 increased general arousal and modified innate defensive reactivity to cat odor. When ovariectomized females treated with E2 as opposed to oil were exposed to cat odor, they showed elevations in risk assessment and reductions in freezing, indicating a shift from passive to active coping. In addition, animals previously exposed to cat odor showed clear cue + context conditioning 24 h later. However, although E2 persisted in its effects on general arousal in the conditioning task, its effects on fear disappeared. In the patch clamp experiments noradrenergic compounds that typically induce fear clearly excited PMd neurons, producing depolarizations and action potentials. E2 treatment shifted some excitatory effects of noradrenergic agonists to inhibitory, possibly by differentially affecting α- and ß-adrenoreceptors. In summary, our results implicate E2 in general arousal and fear reactivity, and suggest these may be governed by changes in noradrenergic responsivity in the PMd. These effects of E2 may have ethological relevance, serving to promote mate seeking even in contexts of ambiguous threat and shed light on the involvement of estrogen in mood and its associated disorders.

Quantitative descriptions of generalized arousal, an elementary function of the vertebrate brain.

We review a concept of the most primitive, fundamental function of the vertebrate CNS, generalized arousal (GA). Three independent lines of evidence indicate the existence of GA: statistical, genetic, and mechanistic. Here we ask, is this concept amenable to quantitative analysis? Answering in the affirmative, four quantitative approaches have proven useful: (i) factor analysis, (ii) information theory, (iii) deterministic chaos, and (iv) application of a Gaussian equation. It strikes us that, to date, not just one but at least four different quantitative approaches seem necessary for describing different aspects of scientific work on GA.