Mechanisms That Underlie Expression of Estradiol-Induced Excitatory Synaptic Potentiation in the Hippocampus Differ between Males and Females.

17ß-estradiol (E2) is synthesized in the hippocampus of both sexes and acutely potentiates excitatory synapses in each sex. Previously, we found that the mechanisms for initiation of E2-induced synaptic potentiation differ between males and females, including in the molecular signaling involved. Here, we used electrical stimulation and two-photon glutamate uncaging in hippocampal slices from adult male and female rats to investigate whether the downstream consequences of distinct molecular signaling remain different between the sexes or converge to the same mechanism(s) of expression of potentiation. This showed that synaptic activity is necessary for expression of E2-induced potentiation in females but not males, which paralleled a sex-specific requirement in females for calcium-permeable AMPARs (cpAMPARs) to stabilize potentiation. Nonstationary fluctuation analysis of two-photon evoked unitary synaptic currents showed that the postsynaptic component of E2-induced potentiation occurs either through an increase in AMPAR conductance or in nonconductive properties of AMPARs (number of channels × open probability) and never both at the same synapse. In females, most synapses (76%) were potentiated via increased AMPAR conductance, whereas in males, more synapses (60%) were potentiated via an increase in nonconductive AMPAR properties. Inhibition of cpAMPARs eliminated E2-induced synaptic potentiation in females, whereas some synapses in males were unaffected by cpAMPAR inhibition; these synapses in males potentiated exclusively via increased AMPAR nonconductive properties. This sex bias in expression mechanisms of E2-induced synaptic potentiation underscores the concept of latent sex differences in mechanisms of synaptic plasticity in which the same outcome in each sex is achieved through distinct underlying mechanisms.SIGNIFICANCE STATEMENT Estrogens are synthesized in the brains of both sexes and potentiate excitatory synapses to the same degree in each sex. Despite this apparent similarity, the molecular signaling that initiates estrogen-induced synaptic potentiation differs between the sexes. Here we show that these differences extend to the mechanisms of expression of synaptic potentiation and result in distinct patterns of postsynaptic neurotransmitter receptor modulation in each sex. Such latent sex differences, in which the same outcome is achieved through distinct underlying mechanisms in males versus females, indicate that molecular mechanisms targeted for drug development may differ between the sexes even in the absence of an overt sex difference in behavior or disease.

His and Hers: Sex Differences in the Brain.

While the 1990s bestseller Men Are from Mars, Women Are from Venus addressed behavior, the neurobiological sex differences in the male and female brain remain largely a mystery. Our author-an acclaimed neuroendocrinologist at Northwestern University-tells us what we know and why we don't know more.

Latent Sex Differences in Molecular Signaling That Underlies Excitatory Synaptic Potentiation in the Hippocampus.

Excitatory synapses can be potentiated by chemical neuromodulators, including 17ß-estradiol (E2), or patterns of synaptic activation, as in long-term potentiation (LTP). Here, we investigated kinases and calcium sources required for acute E2-induced synaptic potentiation in the hippocampus of each sex and tested whether sex differences in kinase signaling extend to LTP. We recorded EPSCs from CA1 pyramidal cells in hippocampal slices from adult rats and used specific inhibitors of kinases and calcium sources. This revealed that, although E2 potentiates synapses to the same degree in each sex, cAMP-activated protein kinase (PKA) is required to initiate potentiation only in females. In contrast, mitogen-activated protein kinase, Src tyrosine kinase, and rho-associated kinase are required for initiation in both sexes; similarly, Ca2+/calmodulin-activated kinase II is required for expression/maintenance of E2-induced potentiation in both sexes. Calcium source experiments showed that L-type calcium channels and calcium release from internal stores are both required for E2-induced potentiation in females, whereas in males, either L-type calcium channel activation or calcium release from stores is sufficient to permit potentiation. To investigate the generalizability of a sex difference in the requirement for PKA in synaptic potentiation, we tested how PKA inhibition affects LTP. This showed that, although the magnitude of both high-frequency stimulation-induced and pairing-induced LTP is the same between sexes, PKA is required for LTP in females but not males. These results demonstrate latent sex differences in mechanisms of synaptic potentiation in which distinct molecular signaling converges to common functional endpoints in males and females.SIGNIFICANCE STATEMENT Chemical- and activity-dependent neuromodulation alters synaptic strength in both male and female brains, yet few studies have compared mechanisms of neuromodulation between the sexes. Here, we studied molecular signaling that underlies estrogen-induced and activity-dependent potentiation of excitatory synapses in the hippocampus. We found that, despite similar magnitude increases in synaptic strength in males and females, the roles of cAMP-regulated protein kinase, internal calcium stores, and L-type calcium channels differ between the sexes. Therefore, latent sex differences in which the same outcome is achieved through distinct underlying mechanisms in males and females include kinase and calcium signaling involved in synaptic potentiation, demonstrating that sex is an important factor in identification of molecular targets for therapeutic development based on mechanisms of neuromodulation.

Considering Sex as a Biological Variable Will Be Valuable for Neuroscience Research.

The recently implemented National Institutes of Health policy requiring that grant applicants consider sex as a biological variable in the design of basic and preclinical animal research studies has prompted considerable discussion within the neuroscience community. Here, we present reasons to be optimistic that this new policy will be valuable for neuroscience, and we suggest some ways for neuroscientists to think about incorporating sex as a variable in their research.

Sex differences in cerebellar synaptic transmission and sex-specific responses to autism-linked Gabrb3 mutations in mice.

Neurons of the cerebellar nuclei (CbN) transmit cerebellar signals to premotor areas. The cerebellum expresses several autism-linked genes, including GABRB3, which encodes GABAA receptor ß3 subunits and is among the maternal alleles deleted in Angelman syndrome. We tested how this Gabrb3 m-/p+ mutation affects CbN physiology in mice, separating responses of males and females. Wild-type mice showed sex differences in synaptic excitation, inhibition, and intrinsic properties. Relative to females, CbN cells of males had smaller synaptically evoked mGluR1/5-dependent currents, slower Purkinje-mediated IPSCs, and lower spontaneous firing rates, but rotarod performances were indistinguishable. In mutant CbN cells, IPSC kinetics were unchanged, but mutant males, unlike females, showed enlarged mGluR1/5 responses and accelerated spontaneous firing. These changes appear compensatory, since mutant males but not females performed indistinguishably from wild-type siblings on the rotarod task. Thus, sex differences in cerebellar physiology produce similar behavioral output, but provide distinct baselines for responses to mutations.

Acute inhibition of neurosteroid estrogen synthesis suppresses status epilepticus in an animal model.

Status epilepticus (SE) is a common neurological emergency for which new treatments are needed. In vitro studies suggest a novel approach to controlling seizures in SE: acute inhibition of estrogen synthesis in the brain. Here, we show in rats that systemic administration of an aromatase (estrogen synthase) inhibitor after seizure onset strongly suppresses both electrographic and behavioral seizures induced by kainic acid (KA). We found that KA-induced SE stimulates synthesis of estradiol (E2) in the hippocampus, a brain region commonly involved in seizures and where E2 is known to acutely promote neural activity. Hippocampal E2 levels were higher in rats experiencing more severe seizures. Consistent with a seizure-promoting effect of hippocampal estrogen synthesis, intra-hippocampal aromatase inhibition also suppressed seizures. These results reveal neurosteroid estrogen synthesis as a previously unknown factor in the escalation of seizures and suggest that acute administration of aromatase inhibitors may be an effective treatment for SE.

17ß-Estradiol Acutely Potentiates Glutamatergic Synaptic Transmission in the Hippocampus through Distinct Mechanisms in Males and Females.

Estradiol (E2) acutely potentiates glutamatergic synaptic transmission in the hippocampus of both male and female rats. Here, we investigated whether E2-induced synaptic potentiation occurs via presynaptic and/or postsynaptic mechanisms and which estrogen receptors (ERs) mediate E2's effects in each sex. Whole-cell voltage-clamp recordings of mEPSCs in CA1 pyramidal neurons showed that E2 increases both mEPSC frequency and amplitude within minutes, but often in different cells. This indicated that both presynaptic and postsynaptic mechanisms are involved, but that they occur largely at different synapses. Two-photon (2p) glutamate uncaging at individual dendritic spines showed that E2 increases the amplitude of uncaging-evoked EPSCs (2pEPSCs) and calcium transients (2pCaTs) at a subset of spines on a dendrite, demonstrating synapse specificity of E2's postsynaptic effects. All of these results were essentially the same in males and females. However, additional experiments using ER-selective agonists indicated sex differences in the mechanisms underlying E2-induced potentiation. In males, an ERß agonist mimicked the postsynaptic effects of E2 to increase mEPSC, 2pEPSC, and 2pCaT amplitude, whereas in females, these effects were mimicked by an agonist of G protein-coupled ER-1. The presynaptic effect of E2, increased mEPSC frequency, was mimicked by an ERα agonist in males, whereas in females, an ERß agonist increased mEPSC frequency. Thus, E2 acutely potentiates glutamatergic synapses similarly in both sexes, but distinct ER subtypes mediate the presynaptic and postsynaptic aspects of potentiation in each sex. This indicates a latent sex difference in which different molecular mechanisms converge to the same functional endpoint in males versus females. SIGNIFICANCE STATEMENT: Some sex differences in the brain may be latent differences, in which the same functional endpoint is achieved through distinct underlying mechanisms in males versus females. Here we report a latent sex difference in molecular regulation of excitatory synapses in the hippocampus. The steroid 17ß-estradiol is known to acutely potentiate glutamatergic synaptic transmission in both sexes. We find that this occurs through a combination of increased presynaptic glutamate release probability and increased postsynaptic sensitivity to glutamate in both sexes, but that distinct estrogen receptor subtypes underlie each aspect of potentiation in each sex. These results indicate that therapeutics targeting a specific estrogen receptor subtype or its downstream signaling would likely affect synaptic transmission differently in the hippocampus of each sex.

Sex Differences in Molecular Signaling at Inhibitory Synapses in the Hippocampus.

The possibility that mechanisms of synaptic modulation differ between males and females has far-reaching implications for understanding brain disorders that vary between the sexes. We found recently that 17ß-estradiol (E2) acutely suppresses GABAergic inhibition in the hippocampus of female rats through a sex-specific estrogen receptor α (ERα), mGluR, and endocannabinoid-dependent mechanism. Here, we define the intracellular signaling that links ERα, mGluRs, and endocannabinoids in females and identify where in this pathway males and females differ. Using a combination of whole-cell patch-clamp recording and biochemical analyses in hippocampal slices from young adult rats, we show that E2 acutely suppresses inhibition in females through mGluR1 stimulation of phospholipase C, leading to inositol triphosphate (IP3) generation, activation of the IP3 receptor (IP3R), and postsynaptic endocannabinoid release, likely of anandamide. Analysis of sex differences in this pathway showed that E2 stimulates a much greater increase in IP3 levels in females than males, whereas the group I mGluR agonist DHPG increases IP3 levels equivalently in each sex. Coimmunoprecipitation showed that ERα-mGluR1 and mGluR1-IP3R complexes exist in both sexes but are regulated by E2 only in females. Independently of E2, a fatty acid amide hydrolase inhibitor, which blocks breakdown of anandamide, suppressed >50% of inhibitory synapses in females with no effect in males, indicating tonic endocannabinoid release in females that is absent in males. Together, these studies demonstrate sex differences in both E2-dependent and E2-independent regulation of the endocannabinoid system and suggest that manipulation of endocannabinoids in vivo could affect physiological and behavioral responses differently in each sex. SIGNIFICANCE STATEMENT: Many brain disorders vary between the sexes, yet the degree to which this variation arises from differential experience versus intrinsic biological sex differences is unclear. In this study, we demonstrate intrinsic sex differences in molecular regulation of a key neuromodulatory system, the endocannabinoid system, in the hippocampus. Endocannabinoids are involved in diverse aspects of physiology and behavior that involve the hippocampus, including cognitive and motivational state, responses to stress, and neurological disorders such as epilepsy. Our finding that molecular regulation of the endocannabinoid system differs between the sexes suggests mechanisms through which experiences or therapeutics that engage endocannabinoids could affect males and females differently.

Quantitative analysis of long-form aromatase mRNA in the male and female rat brain.

In vitro studies show that estrogens acutely modulate synaptic function in both sexes. These acute effects may be mediated in vivo by estrogens synthesized within the brain, which could fluctuate more rapidly than circulating estrogens. For this to be the case, brain regions that respond acutely to estrogens should be capable of synthesizing them. To investigate this question, we used quantitative real-time PCR to measure expression of mRNA for the estrogen-synthesizing enzyme, aromatase, in different brain regions of male and female rats. Importantly, because brain aromatase exists in two forms, a long form with aromatase activity and a short form with unknown function, we targeted a sequence found exclusively in long-form aromatase. With this approach, we found highest expression of aromatase mRNA in the amygdala followed closely by the bed nucleus of the stria terminalis (BNST) and preoptic area (POA); we found moderate levels of aromatase mRNA in the dorsal hippocampus and cingulate cortex; and aromatase mRNA was detectable in brainstem and cerebellum, but levels were very low. In the amygdala, gonadal/hormonal status regulated aromatase expression in both sexes; in the BNST and POA, castration of males down-regulated aromatase, whereas there was no effect of estradiol in ovariectomized females. In the dorsal hippocampus and cingulate cortex, there were no differences in aromatase levels between males and females or effects of gonadal/hormonal status. These findings demonstrate that long-form aromatase is expressed in brain regions that respond acutely to estrogens, such as the dorsal hippocampus, and that gonadal/hormonal regulation of aromatase differs among different brain regions.

Estradiol regulates large dense core vesicles in the hippocampus of adult female rats.

Previous work has shown that the steroid hormone estradiol facilitates the release of anticonvulsant neuropeptides from inhibitory neurons in the hippocampus to suppress seizures. Because neuropeptides are packaged in large dense core vesicles, estradiol may facilitate neuropeptide release through regulation of dense core vesicles. In the current study, we used serial section electron microscopy in the hippocampal CA1 region of adult female rats to test three hypotheses about estradiol regulation of dense core vesicles: (1) Estradiol increases the number of dense core vesicles in axonal boutons, (2) Estradiol increases the size of dense core vesicles in axonal boutons, (3) Estradiol shifts the location of dense core vesicles toward the periphery of axonal boutons, potentially lowering the threshold for neuropeptide release during seizures. We found that estradiol increases the number and size of dense core vesicles in inhibitory axonal boutons, consistent with increased neuropeptide content, but does not shift the location of dense core vesicles closer to the bouton periphery. These effects were specific to large dense core vesicles (>80 nm) in inhibitory boutons. Estradiol had no effects on small dense core vesicles or dense core vesicles in excitatory boutons. Our results indicate that estradiol suppresses seizures at least in part by increasing the potentially releasable pool of neuropeptides in the hippocampus, and that estradiol facilitation of neuropeptide release involves a mechanism other than mobilization of dense core vesicles toward sites of release.

Distribution and posttranslational modification of synaptic ERα in the adult female rat hippocampus.

Acute 17ß-estradiol (E2) signaling in the brain is mediated by extranuclear estrogen receptors. Here we used biochemical methods to investigate the distribution, posttranslational modification, and E2 regulation of estrogen receptor-α (ERα) in synaptosomal fractions isolated by differential centrifugation from the adult female rat hippocampus. We find that ERα is concentrated presynaptically and is highly enriched with synaptic vesicles. Immunoisolation of vesicles using vesicle subtype-specific markers showed that ERα is associated with both glutamate and γ-aminobutyric acid-containing neurotransmitter vesicles as well as with some large dense core vesicles. Experiments using broad spectrum and residue-specific phosphatases indicated that a portion of ERα in synaptosomal fractions is phosphorylated at serine/threonine residues leading to a mobility shift in SDS-PAGE and creating a double band on Western blots. The phosphorylated form of ERα runs in the upper of the two bands and is particularly concentrated with synaptic vesicles. Finally, we used E2 with or without the acyl protein thioesterase 1 inhibitor, Palmostatin B, to show that 20 min of E2 treatment of hippocampal slices depletes ERα from the synaptosomal membrane by depalmitoylation. We found no evidence that E2 regulates phosphorylation of synaptosomal ERα on this time scale. These studies begin to fill the gap between detailed molecular characterization of extranuclear ERα in previous in vitro studies and acute E2 modulation of hippocampal synapses in the adult brain.

Estradiol acutely suppresses inhibition in the hippocampus through a sex-specific endocannabinoid and mGluR-dependent mechanism.

The steroid 17ß-estradiol (E2) is well known to influence hippocampal functions such as memory, affective behaviors, and epilepsy. There is growing awareness that in addition to responding to ovarian E2, the hippocampus of both males and females synthesizes E2 as a neurosteroid that could acutely modulate synaptic function. Previous work on acute E2 actions in the hippocampus has focused on excitatory synapses. Here, we show that E2 rapidly suppresses inhibitory synaptic transmission in hippocampal CA1. E2 acts through the α form of the estrogen receptor to stimulate postsynaptic mGluR1-dependent mobilization of the endocannabinoid anandamide, which retrogradely suppresses GABA release from CB1 receptor-containing inhibitory presynaptic boutons. Remarkably, this effect of E2 is sex specific, occurring in females but not in males. Acute E2 modulation of endocannabinoid tone and consequent suppression of inhibition provide a mechanism by which neurosteroid E2 could modulate hippocampus-dependent behaviors in a sex-specific manner.

Ultrastructural analysis of sex differences in nucleus accumbens synaptic connectivity.

Despite robust sex differences in behavioral responses to drugs of abuse, relatively little is known about structural sex differences in synaptic connectivity of reward circuits such as in the nucleus accumbens (NAc). Previously, we showed that distal dendritic spine density on medium spiny neurons in the NAc is higher in females than males, suggesting that sex differences in NAc excitatory synapses could play a role in differential behavioral responses to drugs. In the current study, we used electron microscopy and stereological counting methods to evaluate dendritic spine and shaft synapses, as well as tyrosine hydroxylase-immunoreactive (TH-IR) profiles, in the NAc core of male and female rats. We found an unanticipated rostro-caudal gradient in spine synapse density in females but not males, resulting in a sex difference favoring females in the caudal NAc core. The volume of the NAc was not different between males and females. We also found that the percentage of spines with large spine heads was greater in females in the rostral core. The density of shaft synapses was low compared to spine synapses, and sex differences were minor. The density of TH-IR profiles was not different between males and females, but females had a higher proportion of spines with large heads near TH suggesting a potential sex difference in dopaminergic modulation of large spine synapses. These findings underscore the importance of including both males and females in studies of reward circuitry, and of considering variation along the rostro-caudal axis of the NAc in future studies.

Quantitative mapping of cocaine-induced ΔFosB expression in the striatum of male and female rats.

ΔFosB plays a critical role in drug-induced long-term changes in the brain. In the current study, we evaluated locomotor activity in male and female rats treated with saline or cocaine for 2 weeks and quantitatively mapped ΔFosB expression in the dorsal striatum and nucleus accumbens of each animal by using an anti-FosB antibody that recognizes ΔFosB isoforms preferentially. Behavioral analysis showed that while there was little difference between males and females that sensitized to cocaine, nonsensitizing rats showed a large sex difference. Nonsensitizing males showed low behavioral activation in response to cocaine on the first day of treatment, and their activity remained low. In contrast, nonsensitizing females showed high activation on the first day of treatment and their activity remained high. Western blot and immunohistochemical analyses indicated that basal levels of ΔFosB were higher in the nucleus accumbens than the dorsal striatum, but that the effect of cocaine on ΔFosB was greater in the dorsal striatum. Immunostaining showed that the effect of cocaine in both the dorsal striatum and nucleus accumbens was primarily to increase the intensity of ΔFosB immunoreactivity in individual neurons, rather than to increase the number of cells that express ΔFosB. Detailed mapping of ΔFosB-labeled nuclei showed that basal ΔFosB levels were highest in the medial portion of the dorsal striatum and dorsomedial accumbens, particularly adjacent to the lateral ventricle, whereas the cocaine-induced increase in ΔFosB was most pronounced in the lateral dorsal striatum, where basal ΔFosB expression was lowest. Sex differences in ΔFosB expression were small and independent of cocaine treatment. We discuss implications of the sex difference in locomotor activation and regionally-specific ΔFosB induction by cocaine.

Sex differences and effects of cocaine on excitatory synapses in the nucleus accumbens.

Human and animal studies indicate that drugs of abuse affect males and females differently, but the mechanism(s) underlying sex differences are unknown. The nucleus accumbens (NAc) is central in the neural circuitry of addiction and medium spiny neurons (MSNs) in the NAc show drug-induced changes in morphology and physiology including increased dendritic spine density. We previously showed in drug-naïve rats that MSN dendritic spine density is higher in females than males. In this study, we investigated sex differences in the effects of cocaine on locomotor activity as well as MSN dendritic spine density and excitatory synaptic physiology in rats treated for 5 weeks followed by 17-21 days of abstinence. Females showed a greater locomotor response to cocaine and more robust behavioral sensitization than males. Spine density was also higher in females and, particularly in the core of the NAc, the magnitude of the cocaine-induced increase in spine density was greater in females. Interestingly, in cocaine-treated females but not males, cocaine-induced behavioral activation during treatment was correlated with spine density measured after treatment. Miniature EPSC (mEPSC) frequency in core MSNs also was higher in females, and increased with cocaine in both the core and shell of females more than males. We found no differences in mEPSC amplitude or paired-pulse ratio of evoked EPSCs, suggesting that sex differences and cocaine effects on mEPSC frequency reflect differences in excitatory synapse number per neuron rather than presynaptic release probability. These studies are the first to demonstrate structural and electrophysiological differences between males and females that may drive sex differences in addictive behavior.

Estradiol potentiation of NR2B-dependent EPSCs is not due to changes in NR2B protein expression or phosphorylation.

The hormone, 17ß-estradiol (E2), influences the structure and function of synapses in the CA1 region of the hippocampus. E2 increases the density of dendritic spines and excitatory synapses on CA1 pyramidal cells, increases CA1 cells' sensitivity to excitatory synaptic input mediated by the NMDA receptor (NMDAR), enhances NMDAR-dependent long-term potentiation, and improves hippocampus-dependent working memory. Smith and McMahon (2006 J Neurosci 26:8517-8522) reported that the larger NMDAR-mediated excitatory postsynaptic currents (EPSCs) recorded after E2 treatment are due primarily to an increased contribution of NR2B-containing NMDARs. We used a combination of electrophysiology, Western blot, and immunofluorescence to investigate two potential mechanisms by which E2 could enhance NR2B-dependent EPSCs: An increase in NMDAR subunit protein levels and/or a change(s) in NR2B phosphorylation. Our studies confirmed the E2-induced increase in NR2B-dependent EPSC amplitude, but we found no evidence that E2 affects protein levels for the NR1, NR2A, or NR2B subunit of the NMDAR, nor that E2 affects phosphorylation of NR2B. Our findings suggest that the effects of E2 on NMDAR-dependent synaptic physiology in the hippocampus likely result from recruitment of NR2B-containing NMDARs to synapses rather than from increased expression of NMDARs or changes in their phosphorylation state.

Estradiol acutely potentiates hippocampal excitatory synaptic transmission through a presynaptic mechanism.

Although recent evidence suggests that the hippocampus is a source of 17ß-estradiol (E2), the physiological role of this neurosteroid E2, as distinct from ovarian E2, is unknown. One likely function of neurosteroid E2 is to acutely potentiate excitatory synaptic transmission, but the mechanism of this effect is not well understood. Using whole-cell voltage-clamp recording of synaptically evoked EPSCs in adult rat hippocampal slices, we show that, in contrast to the conclusions of previous studies, E2 potentiates excitatory transmission through a presynaptic mechanism. We find that E2 acutely potentiates EPSCs by increasing the probability of glutamate release specifically at inputs with low initial release probability. This effect is mediated by estrogen receptor ß (ERß) acting as a monomer, whereas ERα is not required. We further show that the E2-induced increase in glutamate release is attributable primarily to increased individual vesicle release probability and is associated with higher average cleft glutamate concentration. These two findings together argue strongly that E2 promotes multivesicular release, which has not been shown before in the adult hippocampus. The rapid time course of acute EPSC potentiation and its concentration dependence suggest that locally synthesized neurosteroid E2 may activate this effect in vivo.