Sex-specific Difference of Hippocampal Synaptic Plasticity in Response to Sex Neurosteroids.

Numerous studies provide increasing evidence, which supports the ideas that every cell in the brain of males may differ from those in females due to differences in sex chromosome complement as well as in response to hormonal effects. In this study, we address the question as to whether actions of neurosteroids, thus steroids, which are synthesized and function within the brain, contribute to sex-specific hippocampal synaptic plasticity. We have previously shown that predominantly in the female hippocampus, does inhibition of the conversion of testosterone to estradiol affect synaptic transmission. In this study, we show that testosterone and its metabolite dihydrotestosterone are essential for hippocampal synaptic transmission specifically in males. This also holds true for the density of mushroom spines and of spine synapses. We obtained similar sex-dependent results using primary hippocampal cultures of male and female animals. Since these cultures originated from perinatal animals, our findings argue for sex-dependent differentiation of hippocampal neurons regarding their responsiveness to sex neurosteroids up to birth, which persist during adulthood. Hence, our in vitro findings may point to a developmental effect either directly induced by sex chromosomes or indirectly by fetal testosterone secretion during the perinatal critical period, when developmental sexual priming takes place.

Sex-Dependent Regulation of Aromatase-Mediated Synaptic Plasticity in the Basolateral Amygdala.

The basolateral amygdala (BLA) integrates sensory input from cortical and subcortical regions, a function that requires marked synaptic plasticity. Here we provide evidence that cytochrome P450 aromatase (AROM), the enzyme converting testosterone to 17ß-estradiol (E2), contributes to the regulation of this plasticity in a sex-specific manner. We show that AROM is expressed in the BLA, particularly in the basolateral nucleus (BL), in male and female rodents. Systemic administration of the AROM inhibitor letrozole reduced spine synapse density in the BL of adult female mice but not in the BL of male mice. Similarly, in organotypic corticoamygdalar slice cultures from immature rats, treatment with letrozole significantly reduced spine synapses in the BL only in cultures derived from females. In addition, letrozole sex-specifically altered synaptic properties in the BL: in acute slices from juvenile (prepubertal) female rats, wash-in of letrozole virtually abolished long-term potentiation (LTP), whereas it did not prevent the generation of LTP in the slices from males. Together, these data indicate that neuron-derived E2 modulates synaptic plasticity in rodent BLA sex-dependently. As protein expression levels of AROM, estrogen and androgen receptors did not differ between males and females and were not sex-specifically altered by letrozole, the findings suggest sex-specific mechanisms of E2 signaling.SIGNIFICANCE STATEMENT The basolateral amygdala (BLA) is a key structure of the fear circuit. This research reveals a sexually dimorphic regulation of synaptic plasticity in the BLA involving neuronal aromatase, which produces the neurosteroid 17ß-estradiol (E2). As male and female neurons in rodent BLA responded differently to aromatase inhibition both in vivo and in vitro, our findings suggest that E2 signaling in BLA neurons is regulated sex-dependently, presumably via mechanisms that have been established during sexual determination. These findings could be relevant for the understanding of sex differences in mood disorders and of the side effects of cytochrome P450 aromatase inhibitors, which are frequently used for breast cancer therapy.

The serine protease inhibitor neuroserpin is required for normal synaptic plasticity and regulates learning and social behavior.

The serine protease inhibitor neuroserpin regulates the activity of tissue-type plasminogen activator (tPA) in the nervous system. Neuroserpin expression is particularly prominent at late stages of neuronal development in most regions of the central nervous system (CNS), whereas it is restricted to regions related to learning and memory in the adult brain. The physiological expression pattern of neuroserpin, its high degree of colocalization with tPA within the CNS, together with its dysregulation in neuropsychiatric disorders, suggest a role in formation and refinement of synapses. In fact, studies in cell culture and mice point to a role for neuroserpin in dendritic branching, spine morphology, and modulation of behavior. In this study, we investigated the physiological role of neuroserpin in the regulation of synaptic density, synaptic plasticity, and behavior in neuroserpin-deficient mice. In the absence of neuroserpin, mice show a significant decrease in spine-synapse density in the CA1 region of the hippocampus, while expression of the key postsynaptic scaffold protein PSD-95 is increased in this region. Neuroserpin-deficient mice show decreased synaptic potentiation, as indicated by reduced long-term potentiation (LTP), whereas presynaptic paired-pulse facilitation (PPF) is unaffected. Consistent with altered synaptic plasticity, neuroserpin-deficient mice exhibit cognitive and sociability deficits in behavioral assays. However, although synaptic dysfunction is implicated in neuropsychiatric disorders, we do not detect alterations in expression of neuroserpin in fusiform gyrus of autism patients or in dorsolateral prefrontal cortex of schizophrenia patients. Our results identify neuroserpin as a modulator of synaptic plasticity, and point to a role for neuroserpin in learning and memory.

Hippocampal synaptic connectivity in phenylketonuria.

In humans, lack of phenylalanine hydroxylase (Pah) activity results in phenylketonuria (PKU), which is associated with the development of severe mental retardation after birth. The underlying mechanisms, however, are poorly understood. Mutations of the Pah gene in Pah(enu2)/c57bl6 mice result in elevated levels of phenylalanine in serum similar to those in humans suffering from PKU. In our study, long-term potentiation (LTP) and paired-pulse facilitation, measured at CA3-CA1 Schaffer collateral synapses, were impaired in acute hippocampal slices of Pah(enu2)/c57bl6 mice. In addition, we found reduced expression of presynaptic proteins, such as synaptophysin and the synaptosomal-associated protein 25 (SNAP-25), and enhanced expression of postsynaptic marker proteins, such as synaptopodin and spinophilin. Stereological counting of spine synapses at the ultrastructural level revealed higher synaptic density in the hippocampus, commencing at 3 weeks and persisting up to 12 weeks after birth. Consistent effects were seen in response to phenylalanine treatment in cultures of dissociated hippocampal neurones. Most importantly, in the hippocampus of Pah(enu2)/c57bl6 mice, we found a significant reduction in microglia activity. Reorganization of hippocampal circuitry after birth, namely synaptic pruning, relies on elimination of weak synapses by activated microglia in response to neuronal activity. Hence, our data strongly suggest that reduced microglial activity in response to impaired synaptic transmission affects physiological postnatal remodelling of synapses in the hippocampus and may trigger the development of mental retardation in PKU patients after birth.

Aromatase inhibition abolishes LTP generation in female but not in male mice.

Inhibitors of aromatase, the final enzyme of estradiol synthesis, are suspected of inducing memory deficits in women. In previous experiments, we found hippocampal spine synapse loss in female mice that had been treated with letrozole, a potent aromatase inhibitor. In this study, we therefore focused on the effects of letrozole on long-term potentiation (LTP), which is an electrophysiological parameter of memory and is known to induce spines, and on phosphorylation of cofilin, which stabilizes the spine cytoskeleton and is required for LTP in mice. In acute slices of letrozole-treated female mice with reduced estradiol serum concentrations, impairment of LTP started as early as after 6 h of treatment and progressed further, together with dephosphorylation of cofilin in the same slices. Theta-burst stimulation failed to induce LTP after 1 week of treatment. Impairment of LTP was followed by spine and spine synapse loss. The effects were confirmed in vitro by using hippocampal slice cultures of female mice. The sequence of effects in response to letrozole were similar in ovariectomized female and male mice, with, however, differences as to the degree of downregulation. Our data strongly suggest that impairment of LTP, followed by loss of mushroom spines and spine synapses in females, may have implications for memory deficits in women treated with letrozole.

Postembryonic development of centrally generated flight motor patterns in the hawkmoth, Manduca sexta.

This study analyses the maturation of centrally generated flight motor patterns during metamorphosis of Manduca sexta. Bath application of the octopamine agonist chlordimeform to the isolated central nervous system of adult moths reliably induces fictive flight patterns in wing depressor and elevator motoneurons. Pattern maturation is investigated by chlordimeform application at different developmental stages. Chlordimeform also induces motor patterns in larval ganglia, which differ from fictive flight, indicating that in larvae and adults, octopamine affects different networks. First changes in motoneuron activity occur at the pupal stage P10. Rhythmic motor output is induced in depressor, but not in elevator motoneurons at P12. Adult-like fictive flight activity in motoneurons is observed at P16 and increases in speed and precision until emergence 2 days later. Pharmacological block of chloride channels with picrotoxin also induces fictive flight in adults, suggesting that the pattern-generating network can be activated by the removal of inhibition, and that proper network function does not rely on GABA(A) receptors. Our results suggest that the flight pattern-generating network becomes gradually established between P12 and P16, and is further refined until adulthood. These findings are discussed in the context of known physiological and structural CNS development during Manduca metamorphosis.

Sexual dimorphism in estrogen-induced synaptogenesis in the adult hippocampus.

It has long been known that estradiol influences synaptic plasticity in the female hippocampus. The density of dendritic spines varies during the estrous cycle and correlates positively with varying levels of estradiol in serum. In accordance, ovariectomy results in a loss of spines that can be rescued by estradiol treatment in animals, suggesting that estradiol originating from the ovaries induces spine formation in the hippocampus. More recent studies point to a role of hippocampus-derived estradiol in synaptogenesis in the female hippocampus, rather than of estradiol of ovarian origin. In our studies, we have shown that inhibition of hippocampal estrogen synthesis results in spine synapse loss in female animals and, more importantly, also in ovariectomized animals. Surprisingly, inhibition of local estradiol synthesis had no effect on synapse formation in males, in spite of a similar capacity to synthesize estradiol in male and female hippocampal neurons. In females, neuro-sexual steroid production is promoted by hypothalamic, cyclic GnRH release and likely underlies the estrus cyclicity of spine synapse density in the hippocampus. As a result, peripheral serum concentrations of estradiol determine the amount of estradiol synthesis in the hippocampus. This paradigm may also be true in males. In support of this hypothesis, we found that the content of estradiol in hippocampal tissue is higher in female compared to that in male animals, with low levels of estradiol in serum and tonic and acyclical GnRH release. In summary, our data point to important sex-specific differences in sexual steroid-induced synaptogenesis.

Endocrine regulation of estrogen synthesis in the hippocampus?

Estradiol synthesis in the ovaries is regulated via feedback mechanisms mediated by gonadotrophin-releasing hormone (GnRH) and gonadotrophins, secreted by the hypothalamus and the pituitary, respectively. Estradiol synthesis also takes place in the hippocampus. In hippocampal slice cultures of female animals, GnRH regulates estradiol synthesis dose-dependently. Hence, both hippocampal and ovarian estradiol synthesis are synchronized by GnRH. Hippocampus-derived estradiol is essential to synapse stability and maintenance because it stabilizes the spine cytoskeleton of hippocampal neurons. Inhibition of hippocampal estradiol synthesis in mice, however, results in loss of spines and spine synapses in females, but not in males. Stereotaxic application of GnRH to the hippocampus of female rats confirms the regulatory role of GnRH on estradiol synthesis and synapse density in the female hippocampus in vivo. This regulatory role of GnRH necessarily results in estrus cyclicity of spine density in the hippocampus of females.