Microglial over-pruning of synapses during development in autism-associated SCN2A-deficient mice and human cerebral organoids.

Autism spectrum disorder (ASD) is a major neurodevelopmental disorder affecting 1 in 36 children in the United States. While neurons have been the focus of understanding ASD, an altered neuro-immune response in the brain may be closely associated with ASD, and a neuro-immune interaction could play a role in the disease progression. As the resident immune cells of the brain, microglia regulate brain development and homeostasis via core functions including phagocytosis of synapses. While ASD has been traditionally considered a polygenic disorder, recent large-scale human genetic studies have identified SCN2A deficiency as a leading monogenic cause of ASD and intellectual disability. We generated a Scn2a-deficient mouse model, which displays major behavioral and neuronal phenotypes. However, the role of microglia in this disease model is unknown. Here, we reported that Scn2a-deficient mice have impaired learning and memory, accompanied by reduced synaptic transmission and lower spine density in neurons of the hippocampus. Microglia in Scn2a-deficient mice are partially activated, exerting excessive phagocytic pruning of post-synapses related to the complement C3 cascades during selective developmental stages. The ablation of microglia using PLX3397 partially restores synaptic transmission and spine density. To extend our findings from rodents to human cells, we established a microglia-incorporated human cerebral organoid model carrying an SCN2A protein-truncating mutation identified in children with ASD. We found that human microglia display increased elimination of post-synapse in cerebral organoids carrying the SCN2A mutation. Our study establishes a key role of microglia in multi-species autism-associated models of SCN2A deficiency from mouse to human cells.

Positive Allosteric Modulator of SERCA Pump NDC-1173 Exerts Beneficial Effects in Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is an irreversible neurodegenerative disease that affects millions of people worldwide. AD does not have a cure and most drug development efforts in the AD field have been focused on targeting the amyloid pathway based on the "amyloid cascade hypothesis". However, in addition to the amyloid pathway, substantial evidence also points to dysregulated neuronal calcium (Ca2+) signaling as one of the key pathogenic events in AD, and it has been proposed that pharmacological agents that stabilize neuronal Ca2+ signaling may act as disease-modifying agents in AD. In previous studies, we demonstrated that positive allosteric regulators (PAMs) of the Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pump might act as such Ca2+ stabilizing agents. In the present study, we report the development of a novel SERCA PAM agent, compound NDC-1173. To test the effectiveness of this compound, we performed behavioral studies with the APP/PS1 transgenic AD mouse model. We also evaluated effects of this compound on expression of endoplasmic reticulum (ER) stress genes in the hippocampus of APP/PS1 mice. The results of this study support the hypothesis that the SERCA pump is a potential novel therapeutic drug target and that NDC-1173 is a promising lead molecule for developing disease-modifying agents in AD.

Generation and basic characterization of a gene-trap knockout mouse model of Scn2a with a substantial reduction of voltage-gated sodium channel Nav 1.2 expression.

Large-scale genetic studies revealed SCN2A as one of the most frequently mutated genes in patients with neurodevelopmental disorders. SCN2A encodes for the voltage-gated sodium channel isoform 1.2 (Nav 1.2) expressed in the neurons of the central nervous system. Homozygous knockout (null) of Scn2a in mice is perinatal lethal, whereas heterozygous knockout of Scn2a (Scn2a+/- ) results in mild behavior abnormalities. The Nav 1.2 expression level in Scn2a+/- mice is reported to be around 50-60% of the wild-type (WT) level, which indicates that a close to 50% reduction of Nav 1.2 expression may not be sufficient to lead to major behavioral phenotypes in mice. To overcome this barrier, we characterized a novel mouse model of severe Scn2a deficiency using a targeted gene-trap knockout (gtKO) strategy. This approach produces viable homozygous mice (Scn2agtKO/gtKO ) that can survive to adulthood, with about a quarter of Nav 1.2 expression compared to WT mice. Innate behaviors like nesting and mating were profoundly disrupted in Scn2agtKO/gtKO mice. Notably, Scn2agtKO/gtKO mice have a significantly decreased center duration compared to WT in the open field test, suggesting anxiety-like behaviors in a novel, open space. These mice also have decreased thermal and cold tolerance. Additionally, Scn2agtKO/gtKO mice have increased fix-pattern exploration in the novel object exploration test and a slight increase in grooming, indicating a detectable level of repetitive behaviors. They bury little to no marbles and have decreased interaction with novel objects. These Scn2a gene-trap knockout mice thus provide a unique model to study pathophysiology associated with severe Scn2a deficiency.

Dorsal Hippocampal Actin Polymerization Is Necessary for Activation of G-Protein-Coupled Estrogen Receptor (GPER) to Increase CA1 Dendritic Spine Density and Enhance Memory Consolidation.

Activation of the membrane estrogen receptor G-protein-coupled estrogen receptor (GPER) in ovariectomized mice via the GPER agonist G-1 mimics the beneficial effects of 17ß-estradiol (E2) on hippocampal CA1 spine density and memory consolidation, yet the cell-signaling mechanisms mediating these effects remain unclear. The present study examined the role of actin polymerization and c-Jun N-terminal kinase (JNK) phosphorylation in mediating effects of dorsal hippocampally infused G-1 on CA1 dendritic spine density and consolidation of object recognition and spatial memories in ovariectomized mice. We first showed that object learning increased apical CA1 spine density in the dorsal hippocampus (DH) within 40 min. We then found that DH infusion of G-1 increased both CA1 spine density and phosphorylation of the actin polymerization regulator cofilin, suggesting that activation of GPER may increase spine morphogenesis through actin polymerization. As with memory consolidation in our previous work (Kim et al., 2016), effects of G-1 on CA1 spine density and cofilin phosphorylation depended on JNK phosphorylation in the DH. Also consistent with our previous findings, E2-induced cofilin phosphorylation was not dependent on GPER activation. Finally, we found that infusion of the actin polymerization inhibitor, latrunculin A, into the DH prevented G-1 from increasing apical CA1 spine density and enhancing both object recognition and spatial memory consolidation. Collectively, these data demonstrate that GPER-mediated hippocampal spinogenesis and memory consolidation depend on JNK and cofilin signaling, supporting a critical role for actin polymerization in the GPER-induced regulation of hippocampal function in female mice.SIGNIFICANCE STATEMENT Emerging evidence suggests that G-protein-coupled estrogen receptor (GPER) activation mimics effects of 17ß-estradiol on hippocampal memory consolidation. Unlike canonical estrogen receptors, GPER activation is associated with reduced cancer cell proliferation; thus, understanding the molecular mechanisms through which GPER regulates hippocampal function may provide new avenues for the development of drugs that provide the cognitive benefits of estrogens without harmful side effects. Here, we demonstrate that GPER increases CA1 dendritic spine density and hippocampal memory consolidation in a manner dependent on actin polymerization and c-Jun N-terminal kinase phosphorylation. These findings provide novel insights into the role of GPER in mediating hippocampal morphology and memory consolidation, and may suggest first steps toward new therapeutics that more safely and effectively reduce memory decline in menopausal women.

Activation of androgen receptors protects intact male mice from memory impairments caused by aromatase inhibition.

Although 17ß-estradiol (E2) is known to regulate hippocampal function, the specific contributions of hippocampally-synthesized E2 remain unclear. Infusion of the aromatase inhibitor letrozole into the dorsal hippocampus (DH) of ovariectomized mice disrupts object recognition and object placement memory consolidation, suggesting that DH-synthesized E2 is essential for memory. However, the role of DH-synthesized E2 in memory among male rodents is unknown. Here, we examined effects of aromatase inhibition on memory consolidation in male mice. Intact and gonadectomized mice were infused with vehicle or letrozole into the DH immediately post-training in object placement and object recognition tasks. Letrozole blocked memory in both tasks among gonadectomized males only, suggesting that circulating androgens, or a rise in hippocampal androgens due to aromatase inhibition, may support memory consolidation in intact males. To test this hypothesis, intact males were infused with the androgen receptor antagonist flutamide into the DH after object training. A dose-dependent impairment was observed in both tasks, indicating that blocking androgen signaling can impair memory consolidation. To test if hippocampal androgen receptor activation protected intact males from the impairing effects of letrozole, a non-impairing dose of flutamide was co-infused with letrozole. Co-administration of both drugs blocked object placement and object recognition memory consolidation, demonstrating that letrozole impairs memory in intact males only if androgen receptors are blocked. Together, these data suggest that DH-synthesized E2 and androgen receptor activation may work in concert to mediate memory consolidation in intact males, such that androgen receptor activation protects against memory impairments caused by aromatase inhibition.

Sex Differences in the Rapid Cell Signaling Mechanisms Underlying the Memory-Enhancing Effects of 17ß-Estradiol.

Little is known about how 17ß-estradiol (E2) mediates memory formation in males. In ovariectomized (OVX) mice, bilateral dorsal hippocampal (DH) infusion of E2 enhances memory consolidation in object recognition (OR) and object placement (OP) tasks in a manner dependent on activation of extracellular signal-regulated kinase (ERK) and Akt signaling. Here, bilateral DH E2 infusion enhanced memory consolidation in both tasks among OVX female, gonadally-intact male, and castrated male mice, suggesting comparable facilitation of memory consolidation in both sexes, independent of testicular hormones in males. Contrary to previous reports in OVX mice, E2 did not increase DH ERK or Akt phosphorylation in males, nor did the ERK inhibitor U0126 [1,4-diamino-2,3-dicyano-1,4-bis (o-aminophenylmercapto) butadiene] prevent E2 from enhancing memory consolidation among intact and castrated males. These data suggest that ERK activation is not necessary for E2 to enhance memory consolidation in males, and compared with previous reports in females, reveal novel sex differences in the cell-signaling pathways through which E2 facilitates memory consolidation. To explore the mechanisms underlying E2-induced memory enhancements in males, phosphorylation of the transcription factor cAMP response element binding protein (CREB) in the DH was assessed. E2 increased phospho-CREB levels in both sexes, yet U0126 did not block these increases in castrated or intact males, indicating that E2 regulates CREB phosphorylation in males via an ERK-independent mechanism. Collectively, these findings suggest that the beneficial effects of hippocampal E2 on memory consolidation in males and females are mediated by different molecular mechanisms, which has important implications for the development of treatments to reduce memory dysfunction in men and women.

Estradiol and hippocampal memory in female and male rodents.

Estrogens influence nearly every aspect of hippocampal function, including memory formation. Although this research has traditionally focused on ovariectomized females, more recent work is providing insights into the ways in which estrogens regulate hippocampal function in both sexes. This review provides an overview of estrogenic regulation of hippocampal function in female and male rodents, with a particular emphasis on memory formation. Where applicable, we discuss the involvement of specific estrogen receptors and molecular mechanisms that mediate these effects. The review concludes by suggesting gaps in the literature that need to be filled to provide greater insights into potential sex differences in the effects of estrogens on hippocampal function.

Estrogenic regulation of memory consolidation: A look beyond the hippocampus, ovaries, and females.

The potent estrogen 17ß-estradiol (E2) has long been known to regulate the hippocampus and hippocampal-dependent memories in females, and research from the past decade has begun to shed light on the molecular mechanisms through which E2 mediates memory formation in females. Although E2 can also regulate hippocampal function in males, relatively little is known about how E2 influences memory formation in males, or whether sex differences in underlying mechanisms exist. This review, based on a talk given in April 2017 at the American University symposium entitled, "Sex Differences: From Neuroscience to the Clinic and Beyond", first provides an overview of the molecular mechanisms in the dorsal hippocampus through which E2 enhances memory consolidation in ovariectomized female mice. Next, newer research is described demonstrating key roles for the prefrontal cortex and de novo hippocampal E2 synthesis to the memory-enhancing effects of E2 in females. The review then discusses the effects of de novo and exogenous E2 on hippocampal memory consolidation in both sexes, and putative sex differences in the underlying molecular mechanisms through which E2 enhances memory formation. The review concludes by discussing the importance and implications of sex differences in the molecular mechanisms underlying E2-induced memory consolidation for human health.

Sex differences in hippocampal function.

Sex differences in the function of the hippocampus have been observed in numerous mammalian species. However, the magnitude, extent, and specificity of these differences are unclear because they can depend on factors including age, methodology, and environment. This Review will discuss seminal studies examining sex differences in hippocampal memory, neuronal morphology, synaptic plasticity, and cell signaling in humans and rodents. We also describe possible organizational and activational effects of sex steroid hormones during early development, puberty, and adulthood that may lead to sex differences observed in the hippocampus. We conclude by discussing the implications of sex differences in hippocampal function for mental health. © 2016 Wiley Periodicals, Inc.

Beta-hydroxy-beta-methylbutyrate ameliorates aging effects in the dendritic tree of pyramidal neurons in the medial prefrontal cortex of both male and female rats.

Beta-hydroxy-beta-methylbutyrate (HMB), a supplement commonly used to maintain muscle in elderly and clinical populations, has been unexplored in the aging brain. In both healthy aging humans and rat models, there are cognitive deficits associated with age-related dendritic shrinkage within the prefrontal cortex. The present study explores the effects of relatively short- and long-term (7 and 31 weeks) oral HMB supplementation starting at 12 months of age in male and female rats on the dendritic tree of layer 5 pyramidal neurons in the medial prefrontal cortex. Since female rats continue to secrete ovarian hormones after reaching reproductive senescence, middle-aged female rats were ovariectomized to model humans. As expected, there were fewer spines and a retraction of dendritic material in the apical and basilar trees in old age controls of both sexes compared with their middle-aged counterparts. However, these losses did not occur in the HMB-treated rats in either dendrites or the total number of dendritic spines. Thus, HMB forestalled the effects of aging on the dendritic tree of this population of neurons.

Gonadectomy before puberty increases the number of neurons and glia in the medial prefrontal cortex of female, but not male, rats.

The human prefrontal cortex, important for executive functions, loses gray matter throughout the adolescent period. In rats, our laboratory demonstrated that a loss of neurons between adolescence and adulthood partially underlies the loss of volume, and this loss is greater in females than males. Here, we examine whether being deprived of gonadal hormones before puberty through adulthood influences the number of neurons in the medial prefrontal cortex (mPFC). Prior to puberty, the testes or ovaries were removed in male and female rats. In adulthood, the number of neurons and glia in the mPFC were quantified using unbiased stereology, and the volume of the frontal white matter was measured. Prepubertal ovariectomy resulted in a higher number of neurons and glia and a larger volume of white matter compared to sham control littermates. Castrated males were not different from sham males on any measure. Thus ovarian hormones secreted after puberty influence the cellular composition of the medial prefrontal cortex.

Dendritic remodeling in the adolescent medial prefrontal cortex and the basolateral amygdala of male and female rats.

There is recent evidence of continuing development throughout adolescence in two neural areas involved in emotion and cognition, the basolateral amygdala (BLN) and the medial prefrontal cortex (mPFC). Previous research from our laboratory has demonstrated a cellular loss in both of these brain regions in rats between postnatal day (P) 35 and 90. This study investigates dendritic changes in pyramidal neurons of the BLN and Layer 5 of the mPFC at P20 (juvenile), 35 (puberty), and 90 (adulthood) in hooded rats of both sexes. Dendritic branching and dendritic spines were quantified in Golgi-Cox impregnated tissue. Between P20 and 35, dendritic length and complexity, as well as the density of dendritic spines, increased in both structures. Between P35 and 90, dendritic spines in the mPFC neurons significantly decreased in both sexes, while a loss of basilar dendrites was only detected in females. In the BLN, there was an increase in the number of branches between P35 and 90 without an increase in the total length of the dendritic tree. BLN spine density also remained stable during this period. These results show that the dendritic tree grows prior to puberty while dendritic remodeling and pruning occurs after puberty in both of these neural areas. This late development may lead to susceptibilities to psychopathologies and addictions that often develop at this time.

The effects of long-term treatment with estradiol and medroxyprogesterone acetate on tyrosine hydroxylase fibers and neuron number in the medial prefrontal cortex of aged female rats.

Menopausal women often initiate hormone treatment to alleviate the symptoms of menopause. Research suggests that these treatments also affect cognition, and studies in young animals indicate that hormone treatment can alter several neuroanatomical measures. However, very little is known about the effects of long-term hormone treatment on the aging female brain. This study investigated the effects of hormone treatment on neuron number and tyrosine hydroxylase (TH) in the rat medial prefrontal cortex (mPFC). Female Long Evans rats were ovariectomized at middle age (12-13 months) and placed in one of four groups: no replacement (NR) (n = 12), 17ß-estradiol (E(2)) (n = 12), E(2) and progesterone (n = 7), or E(2) and medroxyprogesterone acetate (MPA) (n = 10). Animals were euthanized at 20 months, and the brains were Nissl stained; a subset was immunostained for TH [NR (n = 5); E(2) (n = 6); E(2) + MPA (n = 4); E(2) + progesterone (n = 6)]. E(2) was administered through the drinking water, and progestagens were administered via pellets inserted at the nape of the neck. Neuron number and TH fiber density were quantified in the mPFC. Hormone treatment did not alter neuron number. Treatment with E(2) and MPA resulted in greater TH densities than NR in layer 1 (P < 0.05). In layers 2/3, animals receiving E(2) had greater TH densities than NR animals (P < 0.01). These results indicate that long-term hormone treatments alter dopaminergic fibers and potentially the functioning of the aging mPFC.

Estrogen effects on the forced swim test differ in two outbred rat strains.

Changes in reproductive hormones, such as estrogen, play a role in mood regulation. The present study examined strain differences (Long-Evans vs. Wistar-Hannover) in the behavioral and biochemical effects of estrogen manipulation. Adult ovariectomized female rats were treated with estradiol, vehicle, or withdrawn from estradiol. The two strains demonstrated differential behavioral responses to short-term estradiol administration in the forced swim test; estradiol induced an antidepressant-like effect in Long-Evans rats but not in Wistar rats. Conversely, withdrawal from estradiol resulted in a depressive-like state in the Wistar rats but not in the Long-Evans rats. Western blot analyses found no differences in estrogen receptors α and ß within the hippocampus or the frontal cortex, two brain areas strongly implicated in affective disorders. These data demonstrate the importance of strain as a variable when interpreting behavioral effects of estrogen.

Sex differences in the effects of ethanol pre-exposure during adolescence on ethanol-induced conditioned taste aversion in adult rats.

Alcohol use, which typically begins during adolescence and differs between males and females, is influenced by both the rewarding and aversive properties of the drug. One way adolescent alcohol use may modulate later consumption is by reducing alcohol's aversive properties. Here, we used a conditioned taste aversion (CTA) paradigm to determine if pre-exposure to alcohol (ethanol) during adolescence would attenuate ethanol-induced CTA assessed in adulthood in a sex-dependent manner. Male and female Long-Evans rats were given intraperitoneal (i.p.) injections of saline or 3.0g/kg ethanol in a binge-like pattern during postnatal days (PD) 35-45. In adulthood (>PD 100), rats were given access to 0.1% saccharin, followed by saline or ethanol (1.0 or 1.5g/kg, i.p.), over four conditioning sessions. We found sex differences in ethanol-induced CTA, with males developing a more robust aversion earlier in conditioning. Sex differences in the effects of pre-exposure were also evident: males, but not females, showed an attenuated CTA in adulthood following ethanol pre-exposure, which occurred approximately nine weeks earlier. Taken together, these findings indicate that males are more sensitive to the aversive properties of ethanol than females. In addition, the ability of pre-exposure to the ethanol US to attenuate CTA is enhanced in males compared to females.

Delayed alternation in adolescent and adult male and female rats.

The prefrontal cortex continues to develop throughout adolescence in several species, and our laboratory has demonstrated that during adolescence there is a decrease in neurons in the rat medial prefrontal cortex (mPFC). A PFC-dependent task, the delayed alternation task, was used in the present study to examine the function of the mPFC while it is still maturing in rats of both sexes. A deficit was found in adolescents when compared to adults during 15- and 60-s delays but not at other delays (5, 10, 30, and 90 s). Furthermore, adolescents committed more perseverative errors. No significant sex differences occurred at any delay for either age group; however, in the no delay training sessions, adolescent males reached criterion faster than females. These results indicate that performance on a mPFC-dependent task improves between adolescence and adulthood.

The effects of pre-pubertal gonadectomy and binge-like ethanol exposure during adolescence on ethanol drinking in adult male and female rats.

The pubertal surge in gonadal hormones that occurs during adolescence may impact the long-term effects of early alcohol exposure and sex differences in drinking behavior in adulthood. We investigated this hypothesis by performing sham or gonadectomy surgeries in Long-Evans rats around post-natal day (P) 20. From P35-45, males and females were given saline or 3.0 g/kg ethanol using a binge-like model of exposure (8 injections total). As adults (P100), they were trained to self-administer ethanol via a sucrose-fading procedure and then given access to different unsweetened concentrations (5-20%, w/v) for 5 days/concentration. We found that during adolescence, ethanol-induced intoxication was similar in males and females that underwent sham surgery. In gonadectomized males and females, however, the level of intoxication was greater following the last injection compared to the first. During adulthood, females drank more sucrose per body weight than males and binge-like exposure to ethanol reduced sucrose consumption in both sexes. These effects were not seen in gonadectomized rats. Ethanol consumption was higher in saline-exposed females compared to males, with gonadectomy reversing this sex difference by increasing consumption in males and decreasing it in females. Exposure to ethanol during adolescence augmented ethanol consumption in both sexes, but this effect was statistically significant only in gonadectomized females. Together, these results support a role for gonadal hormones during puberty in the short- and long-term effects of ethanol on behavior and in the development of sex differences in consummatory behavior during adulthood.

Different effects of subchronic doses of 17-beta estradiol in two ethologically based models of anxiety utilizing female rats.

Estrogen may have differing effects on 'anxiety' responses under different conditions. The current study tested the effects of estrogen on anxiety-like behavior when administered for 6-7 days in ovariectomized (OVX) female rats. Two animal paradigms were utilized; the elevated plus maze (EPM), measuring changes in innate fear of exploration of open spaces; and the social interaction test (SIT), measuring the exploration of a novel, same gender partner. In the EPM, estradiol-treated OVX females both entered and spent more time in the open arms than control OVX females, indicating an anxiolytic-like action of estradiol. In contrast, estradiol treated OVX females interacted less with the partner animal in the SIT compared with controls suggesting anxiogenic-like effects. The possible anxiogenic effect of estradiol in the SIT is supported by two findings: (1) the effect is reversed by the anxiolytic drug alprazolam and (2) estrogen did not affect locomotion and therefore, the reduced social interaction is not due to reduced activity. Acute administration of progesterone (5 mg/kg), which has anxiolytic properties, did not reverse estradiol-induced social interaction deficits, suggesting that lack of progesterone did not account for estradiol's anxiogenic effects. These results, while seemingly contradictory when interpreted within a unified concept of anxiety, may well reflect the ethological roles of reproductive hormones and their effects on different types of exploratory anxiety.