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.

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.

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.

Estradiol facilitates the release of neuropeptide Y to suppress hippocampus-dependent seizures.

About one-third of women with epilepsy have a catamenial seizure pattern, in which seizures fluctuate with the menstrual cycle. Catamenial seizures occur more frequently when the ratio of circulating estradiol to progesterone is high, suggesting that estradiol is proconvulsant. We used adult female rats to test how estradiol-induced suppression of GABAergic inhibition in the hippocampus affects behavioral seizures induced by kainic acid. As expected, estradiol decreased the latency to initiate seizures, indicating increased seizure susceptibility. At the same time, however, estradiol also shortened the duration of late-stage seizures, indicating decreased seizure severity. Additional analyses showed that the decrease in seizure severity was attributable to greater release of the anticonvulsant neuropeptide, neuropeptide Y (NPY). First, blocking hippocampal NPY during seizures eliminated the estradiol-induced decrease in seizure duration. Second, light and electron microscopic studies indicated that estradiol increases the potentially releasable pool of NPY in inhibitory presynaptic boutons and facilitates the release of NPY from inhibitory boutons during seizures. Finally, the presence of estrogen receptor-alpha on large dense-core vesicles (LDCVs) in the hippocampus suggests that estradiol could facilitate neuropeptide release by acting directly on LDCVs themselves. Understanding how estradiol regulates NPY-containing LDCVs could point to molecular targets for novel anticonvulsant therapies.