AMPA receptors control fear extinction through an Arc-dependent mechanism.

Activity-regulated cytoskeleton-associated protein (Arc) supports fear memory through synaptic plasticity events requiring actin cytoskeleton rearrangements. We have previously shown that reducing hippocampal Arc levels through antisense knockdown leads to the premature extinction of contextual fear. Here we show that the AMPA receptor antagonist CNQX elevates hippocampal Arc levels during extinction and blocks extinction that can be rescued by reducing Arc. Increasing Arc levels with CNQX also overcomes the actin-destabilizing properties of cytochalasin D and promotes extinction. Therefore, extinction is dependent on AMPA-mediated reductions of Arc via a mechanism consistent with a role for Arc in stabilizing the actin cytoskeleton to constrain extinction.

Hippocampal Regulation of Postsynaptic Density Homer1 by Associative Learning.

Genes involved in synaptic plasticity, particularly genes encoding postsynaptic density proteins, have been recurrently linked to psychiatric disorders including schizophrenia and autism. Postsynaptic density Homer1 proteins contribute to synaptic plasticity through the competing actions of short and long isoforms. The activity-induced expression of short Homer1 isoforms, Homer1a and Ania-3, is thought to be related to processes of learning and memory. However, the precise regulation of Homer1a and Ania-3 with different components of learning has not been investigated. Here, we used in situ hybridization to quantify short and long Homer1 expression in the hippocampus following consolidation, retrieval, and extinction of associative fear memory, using contextual fear conditioning in rats. Homer1a and Ania-3, but not long Homer1, were regulated by contextual fear learning or novelty detection, although their precise patterns of expression in hippocampal subregions were dependent on the isoform. We also show for the first time that the two short Homer1 isoforms are regulated after the retrieval and extinction of contextual fear memory, albeit with distinct temporal and spatial profiles. These findings support a role of activity-induced Homer1 isoforms in learning and memory processes in discrete hippocampal subregions and suggest that Homer1a and Ania-3 may play separable roles in synaptic plasticity.

FMRP and CYFIP1 at the Synapse and Their Role in Psychiatric Vulnerability.

There is increasing awareness of the role genetic risk variants have in mediating vulnerability to psychiatric disorders such as schizophrenia and autism. Many of these risk variants encode synaptic proteins, influencing biological pathways of the postsynaptic density and, ultimately, synaptic plasticity. Fragile-X mental retardation 1 (FMR1) and cytoplasmic fragile-X mental retardation protein (FMRP)-interacting protein 1 (CYFIP1) contain 2 such examples of highly penetrant risk variants and encode synaptic proteins with shared functional significance. In this review, we discuss the biological actions of FMRP and CYFIP1, including their regulation of (i) protein synthesis and specifically FMRP targets, (ii) dendritic and spine morphology, and (iii) forms of synaptic plasticity such as long-term depression. We draw upon a range of preclinical studies that have used genetic dosage models of FMR1 and CYFIP1 to determine their biological function. In parallel, we discuss how clinical studies of fragile X syndrome or 15q11.2 deletion patients have informed our understanding of FMRP and CYFIP1, and highlight the latest psychiatric genomic findings that continue to implicate FMRP and CYFIP1. Lastly, we assess the current limitations in our understanding of FMRP and CYFIP1 biology and how they must be addressed before mechanism-led therapeutic strategies can be developed for psychiatric disorders.

Cyfip1 Haploinsufficiency Does Not Alter GABAA Receptor δ-Subunit Expression and Tonic Inhibition in Dentate Gyrus PV+ Interneurons and Granule Cells.

Copy number variation (CNV) at chromosomal region 15q11.2 is linked to increased risk of neurodevelopmental disorders including autism and schizophrenia. A significant gene at this locus is cytoplasmic fragile X mental retardation protein (FMRP) interacting protein 1 (CYFIP1). CYFIP1 protein interacts with FMRP, whose monogenic absence causes fragile X syndrome (FXS). Fmrp knock-out has been shown to reduce tonic GABAergic inhibition by interacting with the δ-subunit of the GABAA receptor (GABAAR). Using in situ hybridization (ISH), qPCR, Western blotting techniques, and patch clamp electrophysiology in brain slices from a Cyfip1 haploinsufficient mouse, we examined δ-subunit mediated tonic inhibition in the dentate gyrus (DG). In wild-type (WT) mice, DG granule cells (DGGCs) responded to the δ-subunit-selective agonist THIP with significantly increased tonic currents. In heterozygous mice, no significant difference was observed in THIP-evoked currents in DGGCs. Phasic GABAergic inhibition in DGGC was also unaltered with no difference in properties of spontaneous IPSCs (sIPSCs). Additionally, we demonstrate that DG granule cell layer (GCL) parvalbumin-positive interneurons (PV+-INs) have functional δ-subunit-mediated tonic GABAergic currents which, unlike DGGC, are also modulated by the α1-selective drug zolpidem. Similar to DGGC, both IPSCs and THIP-evoked currents in PV+-INs were not different between Cyfip1 heterozygous and WT mice. Supporting our electrophysiological data, we found no significant change in hippocampal δ-subunit mRNA expression or protein level and no change in α1/α4-subunit mRNA expression. Thus, Cyfip1 haploinsufficiency, mimicking human 15q11.2 microdeletion syndrome, does not alter hippocampal phasic or tonic GABAergic inhibition, substantially differing from the Fmrp knock-out mouse model.

Regulation and Function of Activity-Dependent Homer in Synaptic Plasticity.

Alterations in synaptic signaling and plasticity occur during the refinement of neural circuits over the course of development and the adult processes of learning and memory. Synaptic plasticity requires the rearrangement of protein complexes in the postsynaptic density (PSD), trafficking of receptors and ion channels and the synthesis of new proteins. Activity-induced short Homer proteins, Homer1a and Ania-3, are recruited to active excitatory synapses, where they act as dominant negative regulators of constitutively expressed, longer Homer isoforms. The expression of Homer1a and Ania-3 initiates critical processes of PSD remodeling, the modulation of glutamate receptor-mediated functions, and the regulation of calcium signaling. Together, available data support the view that Homer1a and Ania-3 are responsible for the selective, transient destabilization of postsynaptic signaling complexes to facilitate plasticity of the excitatory synapse. The interruption of activity-dependent Homer proteins disrupts disease-relevant processes and leads to memory impairments, reflecting their likely contribution to neurological disorders.

Cyfip1 haploinsufficient rats show white matter changes, myelin thinning, abnormal oligodendrocytes and behavioural inflexibility.

The biological basis of the increased risk for psychiatric disorders seen in 15q11.2 copy number deletion is unknown. Previous work has shown disturbances in white matter tracts in human carriers of the deletion. Here, in a novel rat model, we recapitulated low dosage of the candidate risk gene CYFIP1 present within the 15q11.2 interval. Using diffusion tensor imaging, we first showed extensive white matter changes in Cyfip1 mutant rats, which were most pronounced in the corpus callosum and external capsule. Transmission electron microscopy showed that these changes were associated with thinning of the myelin sheath in the corpus callosum. Myelin thinning was independent of changes in axon number or diameter but was associated with effects on mature oligodendrocytes, including aberrant intracellular distribution of myelin basic protein. Finally, we demonstrated effects on cognitive phenotypes sensitive to both disruptions in myelin and callosal circuitry.

13-cis-Retinoic acid alters intracellular serotonin, increases 5-HT1A receptor, and serotonin reuptake transporter levels in vitro.

In addition to their established role in nervous system development, vitamin A and related retinoids are emerging as regulators of adult brain function. Accutane (13-cis-retinoic acid, isotretinoin) treatment has been reported to increase depression in humans. Recently, we showed that chronic administration of 13-cis-retinoic acid (13-cis-RA) to adolescent male mice increased depression-related behaviors. Here, we have examined whether 13-cis-RA regulates components involved in serotonergic neurotransmission in vitro. We used the RN46A-B14 cell line, derived from rat embryonic raphe nuclei. This cell line synthesizes serotonin (5-hydroxytryptamine, 5-HT) and expresses the 5-HT(1A) receptor and the serotonin reuptake transporter (SERT). Cells were treated with 0, 2.5, or 10 microM 13-cis-RA for 48 or 96 hrs, and the levels of 5-HT; its metabolite, 5-hydroxyindoleacetic acid (5HIAA); 5-HT(1A) receptor; and SERT were determined. Treatment with 13-cis-RA for 96 hrs increased the intracellular levels of 5-HT and tended to increase intra-cellular 5HIAA levels. Furthermore, 48 hrs of treatment with 2.5 and 10 microM 13-cis-RA significantly increased 5-HT(1A) protein to 168.5 +/- 20.0% and 148.7 +/- 2.2% of control respectively. SERT protein levels were significantly increased to 142.5 +/- 11.1% and 119.2 +/- 3.6% of control by 48 hrs of treatment with 2.5 and 10 microM of 13-cis-RA respectively. Increases in both 5-HT(1A) receptor and SERT proteins may lead to decreased serotonin availability at synapses. Such an effect of 13-cis-RA could contribute to the increased depression-related behaviors we have shown in mice.

Rescue of long-term memory after reconsolidation blockade.

Memory reconsolidation is considered to be the process whereby stored memories become labile on recall, allowing updating. Blocking the restabilization of a memory during reconsolidation is held to result in a permanent amnesia. The targeted knockdown of either Zif268 or Arc levels in the brain, and inhibition of protein synthesis, after a brief recall results in a non-recoverable retrograde amnesia, known as reconsolidation blockade. These experimental manipulations are seen as key proof for the existence of reconsolidation. However, here we demonstrate that despite disrupting the molecular correlates of reconsolidation in the hippocampus, rodents are still able to recover contextual memories. Our results challenge the view that reconsolidation is a separate memory process and instead suggest that the molecular events activated initially at recall act to constrain premature extinction.

Genetic risk for schizophrenia: convergence on synaptic pathways involved in plasticity.

Recent large-scale genomic studies have revealed two broad classes of risk alleles for schizophrenia: a polygenic component of risk mediated through multiple common risk variants and rarer more highly penetrant submicroscopic chromosomal deletions and duplications, known as copy number variants. The focus of this review is on the emerging findings from the latter and subsequent exome sequencing data of smaller, deleterious single nucleotide variants and indels. In these studies, schizophrenia patients were found to have enriched de novo mutations in genes belonging to the postsynaptic density at glutamatergic synapses, particularly components of the N-methyl-D-aspartate receptor signaling complex, including the PSD-95 complex, activity-regulated cytoskeleton-associated protein interactors, the fragile X mental retardation protein complex, voltage-gated calcium channels, and genes implicated in actin cytoskeletal dynamics. The convergence of these implicated genes onto a coherent biological pathway at the synapse, with a specific role in plasticity, provides a significant advance in understanding pathogenesis and points to new targets for biological investigation. We consider the implications of these studies in the context of existing genetic data and the potential need to reassess diagnostic boundaries of neuropsychiatric disorders before discussing ways forward for more directed mechanistic studies to develop stratified, novel therapeutic approaches in the future.

Altered brain gene expression but not steroid biochemistry in a genetic mouse model of neurodevelopmental disorder.

BACKGROUND: The 39,XY*O mouse, which lacks the orthologues of the ADHD and autism candidate genes STS (steroid sulphatase) and ASMT (acetylserotonin O-methyltransferase), exhibits behavioural phenotypes relevant to developmental disorders. The neurobiology underlying these phenotypes is unclear, although there is evidence for serotonergic abnormalities in the striatum and hippocampus. METHODS: Using microarray and quantitative gene expression analyses, and gas chromatography-mass spectrometry, we compared brain gene expression and steroid biochemistry in wildtype (40,XY) and 39,XY*O adult mice to identify non-obvious genetic and endocrine candidates for between-group differences in behaviour and neurochemistry. We also tested whether acute STS inhibition by COUMATE in wildtype (40,XY) adult male mice recapitulated any significant gene expression or biochemical findings from the genetic comparison. Data were analysed by unpaired t-test or Mann Whitney U-test depending on normality, with a single factor of KARYOTYPE. RESULTS: Microarray analysis indicated seven robust gene expression differences between the two groups (Vmn2r86, Sfi1, Pisd-ps1, Tagap1, C1qc, Metap1d, Erdr1); Erdr1 and C1qc expression was significantly reduced in the 39,XY*O striatum and hippocampus, whilst the expression of Dhcr7 (encoding 7-dehydrocholesterol reductase, a modulator of serotonin system development), was only reduced in the 39,XY*O hippocampus. None of the confirmed gene expression changes could be recapitulated by COUMATE administration. We detected ten free, and two sulphated steroids in 40,XY and 39,XY*O brain; surprisingly, the concentrations of all of these were equivalent between groups. CONCLUSIONS: Our data demonstrate that the mutation in 39,XY*O mice: i) directly disrupts expression of the adjacent Erdr1 gene, ii) induces a remarkably limited suite of downstream gene expression changes developmentally, with several of relevance to associated neurobehavioural phenotypes and iii) does not elicit large changes in brain steroid biochemistry. It is possible that individuals with STS/ASMT deficiency exhibit a similarly specific pattern of gene expression changes to the 39,XY*O mouse, and that these contribute towards their abnormal neurobiology. Future work may focus on whether complement pathway function, mitochondrial metabolism and cholesterol biosynthesis pathways are perturbed in such subjects.

Genetic and pharmacological modulation of the steroid sulfatase axis improves response control; comparison with drugs used in ADHD.

Maladaptive response control is a feature of many neuropsychiatric conditions, including attention deficit hyperactivity disorder (ADHD). As ADHD is more commonly diagnosed in males than females, a pathogenic role for sex-linked genes has been suggested. Deletion or point mutation of the X-linked STS gene, encoding the enzyme steroid sulfatase (STS) influences risk for ADHD. We examined whether deletion of the Sts gene in the 39,X(Y*)O mouse model, or pharmacological manipulation of the STS axis, via administration of the enzyme substrate dehydroepiandrosterone sulfate or the enzyme inhibitor COUMATE, influenced behavior in a novel murine analog of the stop-signal reaction time task used to detect inhibitory deficits in individuals with ADHD. Unexpectedly, both the genetic and pharmacological treatments resulted in enhanced response control, manifest as highly specific effects in the ability to cancel a prepotent action. For all three manipulations, the effect size was comparable to that seen with the commonly used ADHD therapeutics methylphenidate and atomoxetine. Hence, converging genetic and pharmacological evidence indicates that the STS axis is involved in inhibitory processes and can be manipulated to give rise to improvements in response control. While the precise neurobiological mechanism(s) underlying the effects remain to be established, there is the potential for exploiting this pathway in the treatment of disorders where failures in behavioral inhibition are prominent.

Biological mechanisms associated with increased perseveration and hyperactivity in a genetic mouse model of neurodevelopmental disorder.

Chromosomal deletions at Xp22.3 appear to influence vulnerability to the neurodevelopmental disorders attention deficit hyperactivity disorder (ADHD) and autism. 39,X(Y*)O mice, which lack the murine orthologue of the Xp22.3 ADHD candidate gene STS (encoding steroid sulfatase), exhibit behavioural phenotypes relevant to such disorders (e.g. hyperactivity), elevated hippocampal serotonin (5-HT) levels, and reduced serum levels of dehydroepiandrosterone (DHEA). Here we initially show that 39,X(Y*)O mice are also deficient for the recently-characterised murine orthologue of the Xp22.3 autism candidate gene ASMT (encoding acetylserotonin-O-methyltransferase). Subsequently, to specify potential behavioural correlates of elevated hippocampal 5-HT arising due to the genetic lesion, we compared 39,X(Y*)O MF1 mice to 40,XY MF1 mice on behavioural tasks taxing hippocampal and/or 5-HT function (a 'foraging' task, an object-location task, and the 1-choice serial reaction time task of impulsivity). Although Sts/Asmt deficiency did not influence foraging behaviour, reactivity to familiar objects in novel locations, or 'ability to wait', it did result in markedly increased response rates; these rates correlated with hippocampal 5-HT levels and are likely to index behavioural perseveration, a frequent feature of neurodevelopmental disorders. Additionally, we show that whilst there was no systematic relationship between serum DHEA levels and hippocampal 5-HT levels across 39,X(Y*)O and 40,XY mice, there was a significant inverse linear correlation between serum DHEA levels and activity. Our data suggest that deficiency for genes within Xp22.3 could influence core behavioural features of neurodevelopmental disorders via dissociable effects on hippocampal neurochemistry and steroid hormone levels, and that the mediating neurobiological mechanisms may be investigated in the 39,X(Y*)O model.

The influence of sex-linked genetic mechanisms on attention and impulsivity.

It is now generally agreed that there are inherent sex differences in healthy individuals across a number of neurobiological domains (including brain structure, neurochemistry, and cognition). Moreover, there is a burgeoning body of evidence highlighting sex differences within neuropsychiatric populations (in terms of the rates of incidence, clinical features/progression, neurobiology and pathology). Here, we consider the extent to which attention and impulsivity are sexually dimorphic in healthy populations and the extent to which sex might modulate the expression of disorders characterised by abnormalities in attention and/or impulsivity such as attention deficit hyperactivity disorder (ADHD), autism and addiction. We then discuss general genetic mechanisms that might underlie sex differences in attention and impulsivity before focussing on specific positional and functional candidate sex-linked genes that are likely to influence these cognitive processes. Identifying novel sex-modulated molecular targets should ultimately enable us to develop more effective therapies in disorders associated with attentional/impulsive dysfunction.

Altered serotonergic function may partially account for behavioral endophenotypes in steroid sulfatase-deficient mice.

The X-linked gene STS encodes the steroid hormone-modulating enzyme steroid sulfatase. Loss-of-function of STS, and variation within the gene, have been associated with vulnerability to developing attention deficit hyperactivity disorder (ADHD), a neurodevelopmental condition characterized by inattention, severe impulsivity, hyperactivity, and motivational deficits. ADHD is commonly comorbid with a variety of disorders, including obsessive-compulsive disorder. The neurobiological role of steroid sulfatase, and therefore its potential role in ADHD and associated comorbidities, is currently poorly understood. The 39,X(Y)*O mouse, which lacks the Sts gene, exhibits several behavioral abnormalities relevant to ADHD including inattention and hyperactivity. Here, we show that, unexpectedly, 39,X(Y)*O mice achieve higher ratios than wild-type mice on a progressive ratio (PR) task thought to index motivation, but that there is no difference between the two groups on a behavioral task thought to index compulsivity (marble burying). High performance liquid chromatography analysis of monoamine levels in wild type and 39,X(Y)*O brain tissue regions (the frontal cortex, striatum, thalamus, hippocampus, and cerebellum) revealed significantly higher levels of 5-hydroxytryptamine (5-HT) in the striatum and hippocampus of 39,X(Y)*O mice. Significant correlations between hippocampal 5-HT levels and PR performance, and between striatal 5-HT levels and locomotor activity strongly implicate regionally-specific perturbations of the 5-HT system as a neurobiological candidate for behavioral differences between 40,XY and 39,X(Y)*O mice. These data suggest that inactivating mutations and functional variants within STS might exert their influence on ADHD vulnerability, and disorder endophenotypes through modulation of the serotonergic system.

Steroid sulfatase-deficient mice exhibit endophenotypes relevant to attention deficit hyperactivity disorder.

Attention Deficit Hyperactivity Disorder (ADHD) is a common neurodevelopmental condition characterised by inattention, impulsivity and hyperactivity; it is frequently co-morbid with anxiety and conduct disorders, sleep perturbation and abnormal consummatory behaviours. Recent studies have implicated the neurosteroid-modulating enzyme steroid sulfatase (STS) as a modulator of ADHD-related endophenotypes. The effects of steroid sulfatase deficiency on homecage activity, feeding/drinking behaviours, anxiety-related behaviours (assayed in light-dark box and open field paradigms), social dominance and serum steroid hormone levels were determined by comparing 40,XY and 39,X(Y*)O mice. Subsequently, mice administered the steroid sulfatase inhibitor COUMATE acutely were compared to vehicle-treated mice on behavioural tasks sensitive to enzyme deficiency to dissociate between its developmental and ongoing effects. 39,X(Y*)O mice exhibited heightened reactivity to a novel environment, hyperactivity in the active phase, and increased water (but not food) consumption relative to 40,XY mice during a 24h period; the former group also demonstrated evidence for heightened emotional reactivity. There was no difference in social dominance between the 40,XY and 39,X(Y*)O mice. COUMATE administration had no effect on homecage activity, water consumption or anxiety measures in the open field. 39,X(Y*)O mice exhibited significantly lower dehydroepiandrosterone (DHEA) serum levels than 40,XY mice, but equivalent corticosterone levels. Together with previous data, the present results suggest that steroid sulfatase may influence core and associated ADHD behavioural endophenotypes via both developmental and ongoing mechanisms, and that the 39,X(Y*)O model may represent a useful tool for elucidating the neurobiological basis of these endophenotypes.

Chronic treatment with 13-cis-retinoic acid changes aggressive behaviours in the resident-intruder paradigm in rats.

Retinoids, vitamin A related compounds, have an established role in the development of the nervous system and are increasingly recognized to play a role in adult brain function. The synthetic retinoid, 13-cis-retinoic acid (13-cis-RA, Roaccutane) is widely used to treat severe acne but has been linked to an increased risk of neuropsychiatric side effects, including depression. Here we report that chronic administration with 13-cis-RA (1 mg/kg i.p. daily, 7-14 days) in adult rats reduced aggression- and increased flight-related behaviours in the resident-intruder paradigm. However, in the forced swim, sucrose consumption and open field tests treatment for up to 6 weeks with 13-cis-RA did not modify behaviour in adult or juvenile animals. The behavioural change observed in the resident-intruder paradigm is directly opposite to that observed with chronic antidepressant administration. These findings indicate that when a suitably sensitive behavioural test is employed then chronic administration of 13-cis-RA in adult rats induces behavioural changes consistent with a pro-depressant action.