Sex differences in motor learning flexibility are accompanied by sex differences in mushroom spine pruning of the mouse primary motor cortex during adolescence.

Background: Although males excel at motor tasks requiring strength, females exhibit greater motor learning flexibility. Cognitive flexibility is associated with low baseline mushroom spine densities achieved by pruning which can be triggered by α4ßδ GABAA receptors (GABARs); defective synaptic pruning impairs this process. Methods: We investigated sex differences in adolescent pruning of mushroom spine pruning of layer 5 pyramidal cells of primary motor cortex (L5M1), a site essential for motor learning, using microscopic evaluation of Golgi stained sections. We assessed α4GABAR expression using immunohistochemical and electrophysiological techniques (whole cell patch clamp responses to 100 nM gaboxadol, selective for α4ßδ GABARs). We then compared performance of groups with different post-pubertal mushroom spine densities on motor learning (constant speed) and learning flexibility (accelerating speed following constant speed) rotarod tasks. Results: Mushroom spines in proximal L5M1 of female mice decreased >60% from PND35 (puberty onset) to PND56 (Pubertal: 2.23 ± 0.21 spines/10 µm; post-pubertal: 0.81 ± 0.14 spines/10 µm, P < 0.001); male mushroom spine density was unchanged. This was due to greater α4ßδ GABAR expression in the female (P < 0.0001) because α4 -/- mice did not exhibit mushroom spine pruning. Although motor learning was similar for all groups, only female wild-type mice (low mushroom spine density) learned the accelerating rotarod task after the constant speed task (P = 0.006), a measure of motor learning flexibility. Conclusions: These results suggest that optimal motor learning flexibility of female mice is associated with low baseline levels of post-pubertal mushroom spine density in L5M1 compared to male and female α4 -/- mice.

Manipulation of α4ßδ GABAA receptors alters synaptic pruning in layer 3 prelimbic prefrontal cortex and impairs temporal order recognition: Implications for schizophrenia and autism.

Temporal order memory is impaired in autism spectrum disorder (ASD) and schizophrenia (SCZ). These disorders, more prevalent in males, result in abnormal dendritic spine pruning during adolescence in layer 3 (L3) medial prefrontal cortex (mPFC), yielding either too many (ASD) or too few (SCZ) spines. Here we tested whether altering spine density in neural circuits including the mPFC could be associated with impaired temporal order memory in male mice. We have shown that α4ßδ GABAA receptors (GABARs) emerge at puberty on spines of L5 prelimbic mPFC (PL) where they trigger pruning. We show here that α4ßδ receptors also increase at puberty in L3 PL (P < 0.0001) and used these receptors as a target to manipulate spine density here. Pubertal injection (14 d) of the GABA agonist gaboxadol, at a dose (3 mg/kg) selective for α4ßδ, reduced L3 spine density by half (P < 0.0001), while α4 knock-out increased spine density âˆ¼ 40 % (P < 0.0001), mimicking spine densities in SCZ and ASD, respectively. In both cases, performance on the mPFC-dependent temporal order recognition task was impaired, resulting in decreases in the discrimination ratio which assesses preference for the novel object: -0.39 ± 0.15, gaboxadol versus 0.52 ± 0.09, vehicle; P = 0.0002; -0.048 ± 0.10, α4 KO versus 0.49 ± 0.04, wild-type; P < 0.0001. In contrast, the number of approaches was unaltered, reflecting unchanged locomotion. These data suggest that altering α4ßδ GABAR expression/activity alters spine density in L3 mPFC and impairs temporal order memory to mimic changes in ASD and SCZ. These findings may provide insight into these disorders.

Atg4b Overexpression Extends Lifespan and Healthspan in Drosophila melanogaster.

Autophagy plays important but complex roles in aging, affecting health and longevity. We found that, in the general population, the levels of ATG4B and ATG4D decreased during aging, yet they are upregulated in centenarians, suggesting that overexpression of ATG4 members could be positive for healthspan and lifespan. We therefore analyzed the effect of overexpressing Atg4b (a homolog of human ATG4D) in Drosophila, and found that, indeed, Atg4b overexpression increased resistance to oxidative stress, desiccation stress and fitness as measured by climbing ability. The overexpression induced since mid-life increased lifespan. Transcriptome analysis of Drosophila subjected to desiccation stress revealed that Atg4b overexpression increased stress response pathways. In addition, overexpression of ATG4B delayed cellular senescence, and improved cell proliferation. These results suggest that ATG4B have contributed to a slowdown in cellular senescence, and in Drosophila, Atg4b overexpression may have led to improved healthspan and lifespan by promoting a stronger stress response. Overall, our study suggests that ATG4D and ATG4B have the potential to become targets for health and lifespan interventions.

dNAGLU Extends Life Span and Promotes Fitness and Stress Resistance in Drosophila.

To identify new factors that promote longevity and healthy aging, we studied Drosophila CG13397, an ortholog of the human NAGLU gene, a lysosomal enzyme overexpressed in centenarians. We found that the overexpression of CG13397 (dNAGLU) ubiquitously, or tissue specifically, in the nervous system or fat body could extend fly life span. It also extended the life span of flies overexpressing human Aß42, in a Drosophila Alzheimer's disease (AD) model. To investigate whether dNAGLU could influence health span, we analyzed the effect of its overexpression on AD flies and found that it improved the climbing ability and stress resistance, including desiccation and hunger, suggesting that dNAGLU improved fly health span. We found that the deposition of Aß42 in the mushroom body, which is the fly central nervous system, was reduced, and the lysosomal activity in the intestine was increased in dNAGLU over-expressing flies. When NAGLU was overexpressed in human U251-APP cells, which expresses a mutant form of the Aß-precursor protein (APP), APP-p.M671L, these cells exhibited stronger lysosomal activity and and enhanced expression of lysosomal pathway genes. The concentration of Aß42 in the cell supernatant was reduced, and the growth arrest caused by APP expression was reversed, suggesting that NAGLU could play a wider role beyond its catalytic activity to enhance lysosomal activity. These results also suggest that NAGLU overexpression could be explored to promote healthy aging and to prevent the onset of neurodegenerative diseases, including AD.

Preventing adolescent synaptic pruning in mouse prelimbic cortex via local knockdown of α4ßδ GABAA receptors increases anxiety response in adulthood.

Anxiety is increasingly reported, especially in adolescent females. The etiology is largely unknown, which limits effective treatment. Layer 5 prelimbic cortex (L5PL) increases anxiety responses but undergoes adolescent synaptic pruning, raising the question of the impact of pruning on anxiety. Here we show that preventing L5PL pruning increases anxiety in response to an aversive event in adolescent and adult female mice. Spine density of Golgi-stained neurons decreased ~ 63% from puberty (~ PND35, vaginal opening) to post-puberty (PND56, P < 0.0001). Expression of α4ßδ GABAA receptors (GABARs) transiently increased tenfold in L5PL at puberty (P < 0.00001), but decreased post-pubertally. Both global and local knockdown of these receptors during puberty prevented pruning, increasing spine density post-pubertally (P < 0.0001), an effect reversed by blocking NMDA receptors (NMDARs). Pubertal expression of the NMDAR-dependent spine protein kalirin7 decreased (50%, P < 0.0001), an effect prevented by α4 knock-out, suggesting that α4ßδ-induced reductions in kalirin7 underlie pruning. Increased spine density due to local α4 knockdown at puberty decreased open arm time on the elevated plus maze post-pubertally (62%, P < 0.0001) in response to an aversive stimulus, suggesting that increases in L5PL synapses increase anxiety responses. These findings suggest that prelimbic synaptic pruning is necessary to limit anxiety in adulthood and may suggest novel therapies.

Increased Dendritic Branching of and Reduced δ-GABAA Receptor Expression on Parvalbumin-Positive Interneurons Increase Inhibitory Currents and Reduce Synaptic Plasticity at Puberty in Female Mouse CA1 Hippocampus.

Parvalbumin positive (PV+) interneurons play a pivotal role in cognition and are known to be regulated developmentally and by ovarian hormones. The onset of puberty represents the end of a period of optimal learning when impairments in synaptic plasticity are observed in the CA1 hippocampus of female mice. Therefore, we tested whether the synaptic inhibitory current generated by PV+ interneurons is increased at puberty and contributes to these deficits in synaptic plasticity. To this end, the spontaneous inhibitory postsynaptic current (sIPSC) was recorded using whole-cell patch-clamp techniques from CA1 pyramidal cells in the hippocampal slice before (PND 28-32) and after the onset of puberty in female mice (~PND 35-44, assessed by vaginal opening). sIPSC frequency and amplitude were significantly increased at puberty, but these measures were reduced by 1 µM DAMGO [1 µM, (D-Ala2, N-MePhe4, Gly-ol)-enkephalin], which silences PV+ activity via µ-opioid receptor targets. At puberty, dendritic branching of PV+ interneurons in GAD67-GFP mice was increased, while expression of the δ subunit of the GABAA receptor (GABAR) on these interneurons decreased. Both frequency and amplitude of sIPSCs were significantly increased in pre-pubertal mice with reduced δ expression, suggesting a possible mechanism. Theta burst induction of long-term potentiation (LTP), an in vitro model of learning, is impaired at puberty but was restored to optimal levels by DAMGO administration, implicating inhibition via PV+ interneurons as one cause. Administration of the neurosteroid/stress steroid THP (30 nM, 3α-OH, 5α-pregnan-20-one) had no effect on sIPSCs. These findings suggest that phasic inhibition generated by PV+ interneurons is increased at puberty when it contributes to impairments in synaptic plasticity. These results may have relevance for the changes in cognitive function reported during early adolescence.

Pubertal hormones increase hippocampal expression of α4ßδ GABAA receptors.

CA1 hippocampal expression of α4ßδ GABAA receptors (GABARs) increases at the onset of puberty in female mice, an effect dependent upon the decline in hippocampal levels of the neurosteroid THP (3α-OH-5α-pregnan-20-one) which occurs at this time. The present study further characterized the mechanisms underlying α4ßδ expression, assessed in vivo. Blockade of pubertal levels of 17ß-estradiol (E2) (formestane, 0.5 mg/kg, i.p. 3 d) reduced α4 and δ expression by 75-80% (P < 0.05) in CA1 hippocampus of female mice, assessed using Western blot techniques. Conversely, E2 administration increased α4 and δ expression by 50-100% in adults, an effect enhanced by more than 2-fold by concomitant administration of the 5α-reductase blocker finasteride (50 mg/kg, i.p., 3d, P < 0.05), suggesting that both declining THP levels and increasing E2 levels before puberty trigger α4ßδ expression. This effect was blocked by ICI 182,780 (20 mg/kg, s.c., 3 d), a selective blocker of E2 receptor-α (ER-α). These results suggest that both the rise in circulating levels of E2 and the decline in hippocampal THP levels at the onset of puberty trigger maximal levels of α4ßδ expression in the CA1 hippocampus.

α4ßδ GABAA Receptors Trigger Synaptic Pruning and Reduce Dendritic Length of Female Mouse CA3 Hippocampal Pyramidal Cells at Puberty.

Synaptic pruning during adolescence is critical for optimal cognition. The CA3 hippocampus contains unique spine types and plays a pivotal role in pattern separation and seizure generation, where sex differences exist, but adolescent pruning has only been studied in the male. Thus, for the present study we assessed pruning of specific spine types in the CA3 hippocampus during adolescence and investigated a possible mechanism in the female mouse. To this end, we used Golgi-impregnated brains from pubertal (∼PND 35, assessed by vaginal opening) and post-pubertal (PND 56) mice. Spine density was assessed from z-stack (0.1-µm steps) images taken using a Nikon DS-U3 camera through a Nikon Eclipse Ci-L microscope and analyzed with NIS Elements. Spine density decreased significantly (P < 0.05) during adolescence, with 50-60% decreases in mushroom and stubby spine-types (P < 0.05, ∼PND35 vs. PND56) in non-proestrous mice. This was associated with decreases in kalirin-7, a spine protein which stabilizes the cytoskeleton and is required for spine maintenance. Because our previous findings suggest that pubertal increases in α4ßδ GABAA receptors (GABARs) trigger pruning in CA1, we investigated their role in CA3. α4 expression in CA3 hippocampus increased 4-fold at puberty (P < 0.05), assessed by immunostaining and verified electrophysiologically by an increased response to gaboxadol (100 nM), which is selective for α4ßδ. Knock-out of α4 prevented the pubertal decrease in kalirin-7 and synaptic pruning and also increased the dendritic length, demonstrating a functional link. These data suggest that pubertal α4ßδ GABARs alter dendritic morphology and trigger pruning in female CA3 hippocampus.

The Ubx Polycomb response element bypasses an unpaired Fab-8 insulator via cis transvection in Drosophila.

Chromatin insulators or boundary elements protect genes from regulatory activities from neighboring genes or chromatin domains. In the Drosophila Abdominal-B (Abd-B) locus, the deletion of such elements, such as Frontabdominal-7 (Fab-7) or Fab-8 led to dominant gain of function phenotypes, presumably due to the loss of chromatin barriers. Homologous chromosomes are paired in Drosophila, creating a number of pairing dependent phenomena including transvection, and whether transvection may affect the function of Polycomb response elements (PREs) and thus contribute to the phenotypes are not known. Here, we studied the chromatin barrier activity of Fab-8 and how it is affected by the zygosity of the transgene, and found that Fab-8 is able to block the silencing effect of the Ubx PRE on the DsRed reporter gene in a CTCF binding sites dependent manner. However, the blocking also depends on the zygosity of the transgene in that the barrier activity is present when the transgene is homozygous, but absent when the transgene is heterozygous. To analyze this effect, we performed chromatin immunoprecipitation and quantitative PCR (ChIP-qPCR) experiments on homozygous transgenic embryos, and found that H3K27me3 and H3K9me3 marks are restricted by Fab-8, but they spread beyond Fab-8 into the DsRed gene when the two CTCF binding sites within Fab-8 were mutated. Consistent with this, the mutation reduced H3K4me3 and RNA Pol II binding to the DsRed gene, and consequently, DsRed expression. Importantly, in heterozygous embryos, Fab-8 is unable to prevent the spread of H3K27me3 and H3K9me3 marks from crossing Fab-8 into DsRed, suggesting an insulator bypass. These results suggest that in the Abd-B locus, deletion of the insulator in one copy of the chromosome could lead to the loss of insulator activity on the homologous chromosome, and in other loci where chromosomal deletion created hemizygous regions of the genome, the chromatin barrier could be compromised. This study highlights a role of homologous chromosome pairing in the regulation of gene expression in the Drosophila genome.

Retrotransposons Are the Major Contributors to the Expansion of the Drosophila ananassae Muller F Element.

The discordance between genome size and the complexity of eukaryotes can partly be attributed to differences in repeat density. The Muller F element (∼5.2 Mb) is the smallest chromosome in Drosophila melanogaster, but it is substantially larger (>18.7 Mb) in D. ananassae To identify the major contributors to the expansion of the F element and to assess their impact, we improved the genome sequence and annotated the genes in a 1.4-Mb region of the D. ananassae F element, and a 1.7-Mb region from the D element for comparison. We find that transposons (particularly LTR and LINE retrotransposons) are major contributors to this expansion (78.6%), while Wolbachia sequences integrated into the D. ananassae genome are minor contributors (0.02%). Both D. melanogaster and D. ananassae F-element genes exhibit distinct characteristics compared to D-element genes (e.g., larger coding spans, larger introns, more coding exons, and lower codon bias), but these differences are exaggerated in D. ananassae Compared to D. melanogaster, the codon bias observed in D. ananassae F-element genes can primarily be attributed to mutational biases instead of selection. The 5' ends of F-element genes in both species are enriched in dimethylation of lysine 4 on histone 3 (H3K4me2), while the coding spans are enriched in H3K9me2. Despite differences in repeat density and gene characteristics, D. ananassae F-element genes show a similar range of expression levels compared to genes in euchromatic domains. This study improves our understanding of how transposons can affect genome size and how genes can function within highly repetitive domains.

α4ßδ GABAA receptors reduce dendritic spine density in CA1 hippocampus and impair relearning ability of adolescent female mice: Effects of a GABA agonist and a stress steroid.

Synaptic pruning underlies the transition from an immature to an adult CNS through refinements of neuronal circuits. Our recent study indicates that pubertal synaptic pruning is triggered by the inhibition generated by extrasynaptic α4ßδ GABAA receptors (GABARs) which are increased for 10 d on dendritic spines of CA1 pyramidal cells at the onset of puberty (PND 35-44) in the female mouse, suggesting α4ßδ GABARs as a novel target for the regulation of adolescent synaptic pruning. In the present study we used a pharmacological approach to further examine the role of these receptors in altering spine density during puberty of female mice and the impact of these changes on spatial learning, assessed in adulthood. Two drugs were chronically administered during the pubertal period (PND 35-44): the GABA agonist gaboxadol (GBX, 0.1mg/kg, i.p.), to enhance current gated by α4ßδ GABARs and the neurosteroid/stress steroid THP (3α-OH-5ß-pregnan-20-one, 10mg/kg, i.p.) to decrease expression of α4ßδ. Spine density was determined on PND 56 with Golgi staining. Spatial learning and relearning were assessed using the multiple object relocation task and an active place avoidance task on PND 56. Pubertal GBX decreased spine density post-pubertally by 70% (P<0.05), while decreasing α4ßδ expression with THP increased spine density by twofold (P<0.05), in both cases, with greatest effects on the mushroom spines. Adult relearning ability was compromised in both hippocampus-dependent tasks after pubertal administration of either drug. These findings suggest that an optimal spine density produced by α4ßδ GABARs is necessary for optimal cognition in adults.

CTCF interacts with the lytic HSV-1 genome to promote viral transcription.

CTCF is an essential chromatin regulator implicated in important nuclear processes including in nuclear organization and transcription. Herpes Simplex Virus-1 (HSV-1) is a ubiquitous human pathogen, which enters productive infection in human epithelial and many other cell types. CTCF is known to bind several sites in the HSV-1 genome during latency and reactivation, but its function has not been defined. Here, we report that CTCF interacts extensively with the HSV-1 DNA during lytic infection by ChIP-seq, and its knockdown results in the reduction of viral transcription, viral genome copy number and virus yield. CTCF knockdown led to increased H3K9me3 and H3K27me3, and a reduction of RNA pol II occupancy on viral genes. Importantly, ChIP-seq analysis revealed that there is a higher level of CTD Ser2P modified RNA Pol II near CTCF peaks relative to the Ser5P form in the viral genome. Consistent with this, CTCF knockdown reduced the Ser2P but increased Ser5P modified forms of RNA Pol II on viral genes. These results suggest that CTCF promotes HSV-1 lytic transcription by facilitating the elongation of RNA Pol II and preventing silenced chromatin on the viral genome.

Role of α4-containing GABAA receptors in limiting synaptic plasticity and spatial learning of female mice during the pubertal period.

Expression of α4ßδ GABAA receptors (GABARs) increases at the onset of puberty on dendritic spines of CA1 hippocampal pyramidal cells. These receptors reduce activation of NMDA receptors (NMDARs), impair induction of long-term potentiation (LTP) and reduce hippocampal-dependent spatial learning. These effects are not seen in the δ-/- mouse, implicating α4ßδ GABARs. Here we show that knock-out of α4 also restores synaptic plasticity and spatial learning in female mice at the onset of puberty (verified by vaginal opening). To this end, field excitatory post-synaptic potentials (fEPSPs) were recorded from the stratum radiatum of CA1 hippocampus in the slice from +/+ and α4-/- pubertal mice (PND 35-44). Induction of LTP, in response to stimulation of the Schaffer collaterals with theta burst stimulation (TBS), was unsuccessful in the +/+ hippocampus, but reinstated by α4 knock-out (~65% potentiation) but not by blockade of α5-GABARs with L-655,708 (50nM). In order to compare spatial learning in the two groups of mice, animals were trained in an active place avoidance task where the latency to first enter a shock zone is a measure of learning. α4-/- mice had significantly longer latencies by the third learning trial, suggesting better spatial learning, compared to +/+ animals, who did not reach the criterion for learning (120s latency). These findings suggest that knock-out of the GABAR α4 subunit restores synaptic plasticity and spatial learning at puberty and is consistent with the concept that the dendritic α4ßδ GABARs which emerge at puberty selectively impair CNS plasticity. This article is part of a Special Issue entitled SI: Adolescent plasticity.

Pubertal Expression of α4ßδ GABAA Receptors Reduces Seizure-Like Discharges in CA1 Hippocampus.

More than half of children with epilepsy outgrow their seizures, yet the underlying mechanism is unknown. GABAergic inhibition increases at puberty in female mice due to expression of extrasynaptic α4ßδ GABAA receptors (GABARs). Therefore, we tested the role of these receptors in regulating seizure-like discharges in CA1 hippocampus using a high K(+) (8.5 mM) seizure model. Spontaneous field potentials were recorded from hippocampus of pre-pubertal (~28-32 PND) and pubertal (~35-44 PND) female wild-type or α4-/- mice. The coastline length, a measure of burst intensity, was assessed. 8.5 mM K(+) induced seizure-like discharges in over 60% of pre-pubertal slices, but only in 7% of pubertal slices, where the coastline length was reduced by 70% (P = 0.04). However, the pubertal decrease in seizure-like discharges was not seen in the α4-/-, implicating α4ßδ GABARs as the cause of the decreased seizure-like activity during puberty. Administration of THIP or DS2, to selectively increase α4ßδ current, reduced activity in 8.5 mM K(+) at puberty, while blockade of α5-GABARs had no effect. GABAergic current was depolarizing but inhibitory in 8.5 mM K(+), suggesting a mechanism for the effects of α4ßδ and α5-GABARs, which exhibit different polarity-dependent desensitization. These data suggest that α4ßδ GABARs are anti-convulsant during adolescence.

Synaptic pruning in the female hippocampus is triggered at puberty by extrasynaptic GABAA receptors on dendritic spines.

Adolescent synaptic pruning is thought to enable optimal cognition because it is disrupted in certain neuropathologies, yet the initiator of this process is unknown. One factor not yet considered is the α4ßδ GABAA receptor (GABAR), an extrasynaptic inhibitory receptor which first emerges on dendritic spines at puberty in female mice. Here we show that α4ßδ GABARs trigger adolescent pruning. Spine density of CA1 hippocampal pyramidal cells decreased by half post-pubertally in female wild-type but not α4 KO mice. This effect was associated with decreased expression of kalirin-7 (Kal7), a spine protein which controls actin cytoskeleton remodeling. Kal7 decreased at puberty as a result of reduced NMDAR activation due to α4ßδ-mediated inhibition. In the absence of this inhibition, Kal7 expression was unchanged at puberty. In the unpruned condition, spatial re-learning was impaired. These data suggest that pubertal pruning requires α4ßδ GABARs. In their absence, pruning is prevented and cognition is not optimal.

Flumazenil decreases surface expression of α4ß2δ GABAA receptors by increasing the rate of receptor internalization.

Increases in expression of α4ßδ GABAA receptors (GABARs), triggered by fluctuations in the neurosteroid THP (3α-OH-5α[ß]-pregnan-20-one), are associated with changes in mood and cognition. We tested whether α4ßδ trafficking and surface expression would be altered by in vitro exposure to flumazenil, a benzodiazepine ligand which reduces α4ßδ expression in vivo. We first determined that flumazenil (100 nM-100 µM, IC50=∼1 µM) acted as a negative modulator, reducing GABA (10 µM)-gated current in the presence of 100 nM THP (to increase receptor efficacy), assessed with whole cell patch clamp recordings of recombinant α4ß2δ expressed in HEK-293 cells. Surface expression of recombinant α4ß2δ receptors was detected using a 3XFLAG reporter at the C-terminus of α4 (α4F) using confocal immunocytochemical techniques following 48 h exposure of cells to GABA (10 µM)+THP (100 nM). Flumazenil (10 µM) decreased surface expression of α4F by ∼60%, while increasing its intracellular accumulation, after 48 h. Reduced surface expression of α4ß2δ after flumazenil treatment was confirmed by decreases in the current responses to 100 nM of the GABA agonist gaboxadol. Flumazenil-induced decreases in surface expression of α4ß2δ were prevented by the dynamin blocker, dynasore, and by leupeptin, which blocks lysosomal enzymes, suggesting that flumazenil is acting to increase endocytosis and lysosomal degradation of the receptor. Flumazenil increased the rate of receptor removal from the cell surface by 2-fold, assessed using botulinum toxin B to block insertion of new receptors. These findings may suggest new therapeutic strategies for regulation of α4ß2δ expression using flumazenil.

Drosophila muller f elements maintain a distinct set of genomic properties over 40 million years of evolution.

The Muller F element (4.2 Mb, ~80 protein-coding genes) is an unusual autosome of Drosophila melanogaster; it is mostly heterochromatic with a low recombination rate. To investigate how these properties impact the evolution of repeats and genes, we manually improved the sequence and annotated the genes on the D. erecta, D. mojavensis, and D. grimshawi F elements and euchromatic domains from the Muller D element. We find that F elements have greater transposon density (25-50%) than euchromatic reference regions (3-11%). Among the F elements, D. grimshawi has the lowest transposon density (particularly DINE-1: 2% vs. 11-27%). F element genes have larger coding spans, more coding exons, larger introns, and lower codon bias. Comparison of the Effective Number of Codons with the Codon Adaptation Index shows that, in contrast to the other species, codon bias in D. grimshawi F element genes can be attributed primarily to selection instead of mutational biases, suggesting that density and types of transposons affect the degree of local heterochromatin formation. F element genes have lower estimated DNA melting temperatures than D element genes, potentially facilitating transcription through heterochromatin. Most F element genes (~90%) have remained on that element, but the F element has smaller syntenic blocks than genome averages (3.4-3.6 vs. 8.4-8.8 genes per block), indicating greater rates of inversion despite lower rates of recombination. Overall, the F element has maintained characteristics that are distinct from other autosomes in the Drosophila lineage, illuminating the constraints imposed by a heterochromatic milieu.

Neurosteroid effects at α4ßδ GABAA receptors alter spatial learning and synaptic plasticity in CA1 hippocampus across the estrous cycle of the mouse.

Fluctuations in circulating levels of ovarian hormones have been shown to regulate cognition (Sherwin and Grigorova, 2011. Fertil. Steril. 96, 399-403; Shumaker et al., 2004. JAMA. 291, 2947-2958), but increases in estradiol on the day of proestrus yield diverse outcomes: In vivo induction of long-term potentiation (LTP), a model of learning, is reduced in the morning, but optimal in the afternoon (Warren et al., 1995. Brain Res. 703, 26-30). The mechanism underlying this discrepancy is not known. Here, we show that impairments in both CA1 hippocampal LTP and spatial learning observed on the morning of proestrus are due to increased dendritic expression of α4ßδ GABAA receptors (GABARs) on CA1 pyramidal cells, as assessed by electron microscopic (EM) techniques, compared with estrus and diestrus. LTP induction and spatial learning were robust, however, when assessed on the morning of proestrus in α4-/- mice, implicating these receptors in mediating impaired plasticity. Although α4ßδ expression remained elevated on the afternoon of proestrus, increases in 3α-OH-THP (3α-OH-5α-pregnan-20-one) decreased inhibition by reducing outward current through α4ßδ GABARs (Shen et al., 2007. Nat. Neurosci. 10, 469-477), in contrast to the usual effect of this steroid to enhance inhibition. Proestrous levels of 3α-OH-THP reversed the deficits in LTP and spatial learning, an effect prevented by the inactive metabolite 3ß-OH-THP (10 mg/kg, i.p.), which antagonizes actions of 3α-OH-THP. In contrast, administration of 3α-OH-THP (10 mg/kg, i.p.) on the morning of proestrus improved spatial learning scores 150-300%. These findings suggest that cyclic fluctuations in ovarian steroids can induce changes in cognition via α4ßδ GABARs that are dependent upon 3α-OH-THP. This article is part of a Special Issue entitled SI: Brain and Memory.