Rapid effects of valproic acid on the fetal brain transcriptome: Implications for brain development and autism.

There is an increased incidence of autism among the children of women who take the anti-epileptic, mood-stabilizing drug, valproic acid (VPA) during pregnancy; moreover, exposure to VPA in utero causes autistic-like symptoms in rodents and non-human primates. Analysis of RNA-seq data obtained from E12.5 fetal mouse brains 3 hours after VPA administration to the pregnant dam revealed that VPA rapidly and significantly increased or decreased the expression of approximately 7,300 genes. No significant sex differences in VPA-induced gene expression were observed. Expression of 399 autism risk genes was significantly altered by VPA as was expression of 255 genes that have been reported to play fundamental roles in fetal brain development but are not otherwise linked to autism. Expression of genes associated with intracellular signaling pathways, neurogenesis, and excitation-inhibition balance as well as synaptogenesis, neuronal fate determination, axon and dendritic development, neuroinflammation, circadian rhythms, and epigenetic modulation of gene expression was dysregulated by VPA. The goal of this study was to identify mouse genes that are: (a) significantly up- or down-regulated by VPA in the fetal brain and (b) known to be associated with autism and/or to play a role in embryonic neurodevelopmental processes, perturbation of which has the potential to alter brain connectivity and, consequently behavior, in the adult. The set of genes meeting these criteria provides potential targets for future hypothesis-driven studies to elucidate the proximal causes of errors in brain connectivity underlying neurodevelopmental disorders such as autism.

Rapid effects of valproic acid on the fetal brain transcriptome: Implications for brain development and autism.

There is an increased incidence of autism among the children of women who take the anti-epileptic, mood stabilizing drug, valproic acid (VPA) during pregnancy; moreover, exposure to VPA in utero causes autistic-like symptoms in rodents and non-human primates. Analysis of RNAseq data ob-tained from E12.5 fetal mouse brains 3 hours after VPA administration revealed that VPA significant-ly increased or decreased the expression of approximately 7,300 genes. No significant sex differ-ences in VPA-induced gene expression were observed. Expression of genes associated with neu-rodevelopmental disorders (NDDs) such as autism as well as neurogenesis, axon growth and syn-aptogenesis, GABAergic, glutaminergic and dopaminergic synaptic transmission, perineuronal nets, and circadian rhythms was dysregulated by VPA. Moreover, expression of 399 autism risk genes was significantly altered by VPA as was expression of 252 genes that have been reported to play fundamental roles in the development of the nervous system but are not otherwise linked to autism. The goal of this study was to identify mouse genes that are: (a) significantly up- or down-regulated by VPA in the fetal brain and (b) known to be associated with autism and/or to play a role in embryonic neurodevelopmental processes, perturbation of which has the potential to alter brain connectivity in the postnatal and adult brain. The set of genes meeting these criteria pro-vides potential targets for future hypothesis-driven approaches to elucidating the proximal underly-ing causes of defective brain connectivity in NDDs such as autism.

Association of complement component 4 with neuroimmune abnormalities in the subventricular zone in schizophrenia and autism spectrum disorders.

An early inflammatory insult is the most recognized risk factor associated with neurodevelopmental psychiatric disorders, even more so than genetic variants. Notably, complement component 4 (C4), a molecule involved in inflammatory responses, has been strongly associated with schizophrenia (SZ) and its role in other neurodevelopmental disorders, such as autism (ASD), is an area of active investigation. However, while C4 in SZ has been implicated in the context of synaptic pruning, little is known about its neuroinflammatory role. The subventricular zone (SVZ) is a region heavily involved in neurodevelopment and neuroimmune interactions through the lifespan; thus, it is a region wherein C4 may play a vital role in disease pathology. Using in situ hybridization with radioactive riboprobes and RNAscope, we identified robust astrocytic expression of C4 in the SVZ and in the septum pellucidum. C4 was also expressed in ependyma, neurons, and Ki67+ progenitor cells. Examination of mRNA levels showed elevated C4 in both ASD and SZ, with higher expression in SZ compared to controls. Targeted transcriptomic analysis of inflammatory pathways revealed a strong association of complement system genes with SZ, and to a lesser extent, ASD, as well as generalized immune dysregulation without a strong association with known infectious pathways. Analysis of differentially expressed genes (DEGs) showed that ASD DEGs were enriched in adaptive immune system functions such as Th cell differentiation, while SZ DEGs were enriched in innate immune system functions, including NF-κB and toll like receptor signaling. Moreover, the number of Ki67+ cells was significantly higher in ASD compared to SZ and controls. Taken together, these results support a role for C4 into inflammatory-neuroimmune dysregulation observed in SZ and ASD pathology.

Functional G-protein-coupled receptor 35 is expressed by neurons in the CA1 field of the hippocampus.

The G-protein-coupled receptor 35 (GPR35) was de-orphanized after the discovery that kynurenic acid (KYNA), an endogenous tryptophan metabolite, acts as an agonist of this receptor. Abundant evidence supports that GPR35 exists primarily in peripheral tissues. Here, we tested the hypothesis that GPR35 exists in the hippocampus and influences the neuronal activity. Fluorescence immunohistochemical staining using an antibody anti-NeuN (a neuronal marker), an antibody anti-GFAP (a glial marker), and an antibody anti-GPR35 revealed that neurons in the stratum oriens, stratum pyramidale, and stratum radiatum of the CA1 field of the hippocampus express GPR35. To determine the presence of functional GPR35 in the neurocircuitry, we tested the effects of various GPR35 agonists on the frequency of spontaneous action potentials recorded as fast current transients (CTs) from stratum radiatum interneurons (SRIs) under cell-attached configuration in rat hippocampal slices. Bath application of the GPR35 agonists zaprinast (1-10 µM), dicumarol (50-100 µM), pamoic acid (500-1000 µM), and amlexanox (3 µM) produced a concentration- and time-dependent reduction in the frequency of CTs. Superfusion of the hippocampal slices with the GPR35 antagonist ML145 (1 µM) increased the frequency of CTs and reduced the inhibitory effect of zaprinast. Bath application of phosphodiesterase 5 inhibitor sildenafil (1 or 5 µM) was ineffective, whereas a subsequent application of zaprinast was effective in reducing the CT frequency. The present results demonstrate for the first time that functional GPR35s are expressed by CA1 neurons and suggest that these receptors can be molecular targets for controlling neuronal activity in the hippocampus.

Estrogen promotes germ cell and seminiferous tubule development in the baboon fetal testis.

The foundation for development of the male reproduction system occurs in utero, but relatively little is known about the regulation of primate fetal testis maturation. Our laboratories have shown that estrogen regulates key aspects of the physiology of pregnancy and fetal development. Therefore, in the present study, we characterized and quantified germ cells and Sertoli cells in the fetal baboon testis in late normal gestation (i.e., Day 165; term is 184 days) and in baboons administered the aromatase inhibitor letrozole throughout the second half of gestation to assess the impact of endogenous estrogen on fetal testis development. In untreated baboons, the seminiferous cords were comprised of undifferentiated (i.e., type A) spermatogonia classified by their morphology as dark (Ad) or pale (Ap), gonocytes (precursors of type A spermatogonia), unidentified cells (UI), and Sertoli cells. In letrozole-treated baboons, serum estradiol levels were decreased by 95%. The number per milligram of fetal testis (x10(4)) of Ad spermatogonia (0.42 +/- 0.11) was 45% lower (P = 0.03), and that of gonocytes (0.58 +/- 0.06) and UI (0.45 +/- 0.12) was twofold greater (P < 0.01 and P = 0.06, respectively), than in untreated baboons. Moreover, in the seminiferous cords of estrogen-deprived baboons, the basement membrane appeared fragmented, the germ cells and Sertoli cells appeared disorganized, and vacuoles were present. We conclude that endogenous estrogen promotes fetal testis development and that the changes in the germ cell population in the estrogen-deprived baboon fetus may impair spermatogenesis and fertility in adulthood.

Estrogen and estrogen receptor-{beta} (ER{beta})-selective ligands induce galanin expression within gonadotropin hormone-releasing hormone-immunoreactive neurons in the female rat brain.

Among the many factors that integrate the activity of the GnRH neuronal system, estrogens play the most important role. In females, estrogen, in addition to the negative feedback, also exhibits a positive feedback influence upon the activity and output of GnRH neurons to generate the preovulatory LH surge and ovulation. Until recently, the belief has been that the GnRH neurons do not contain estrogen receptors (ERs) and that the action of estrogen upon GnRH neurons is indirect involving several, estrogen-sensitive neurotransmitter and neuromodulator systems that trans-synaptically regulate the activity of the GnRH neurons. Based on our recent findings that GnRH neurons of the female rat coexpress galanin, that galanin is a potent GnRH-releasing peptide, and that ERbeta is present in GnRH neurons, we have evaluated the effect of 17beta-estradiol and two ERbeta-selective agonists (WAY-200070, WAY-166818) on the expression of galanin within GnRH neurons. By combining immunocytochemistry for GnRH and in situ hybridization histochemistry for galanin, we demonstrate that 17beta-estradiol (20 mug/kg, sc) stimulates galanin expression within GnRH-immunoreactive neurons in a time-dependent manner. A significant increase was observed 2 h after its administration to ovariectomized rats. However, a more robust expression required 3-d treatment regimen. Treatment with the beta-selective ligands resulted in similar observations, although no statistical analysis is available for the 2 hr survival. These observations strongly suggest that estrogen and the ERbeta-selective ligands stimulate galanin expression within GnRH neurons via ERbeta, although an indirect mechanism via interneurons still cannot be ruled out.

Distribution of estrogen receptor alpha and beta in the mouse central nervous system: in vivo autoradiographic and immunocytochemical analyses.

Although the distribution of estrogen receptor beta (ERbeta) immunoreactivity in the rat central nervous has been reported, no such data are available in the mouse. The present study used in vivo autoradiography utilizing a (125)I-estrogen that has equal binding affinity for both receptors as well as immunohistochemistry for ERbeta and ERalpha, to investigate and compare the distribution of the two ERs in the mouse CNS. The use specific antisera against ERalpha and ERbeta allowed us to evaluate the contribution of these receptors to the binding detected with autoradiography. In addition, data were collected in ovariectomized wildtype and ERalpha KO (knockout) mice to examine developmental regulation of ERbeta expression by ERalpha. These studies revealed that in the mouse CNS, combining immunoreactivity for ERalpha with that for ERbeta accounted for all regions where binding was seen using autoradiography. Therefore, these data strongly suggest that the major contributors of estrogen binding in the mouse CNS are ERalpha and ERbeta. Together, these data provide an anatomical foundation for future studies and advance our understanding of estrogen action in the CNS. Moreover, since the immunocytochemical images were similar in wildtype and ERalpha KO mice, these studies suggest that the lack of ERalpha does not influence the expression of ERbeta in the central nervous system.

Global transcription profiling of estrogen activity: estrogen receptor alpha regulates gene expression in the kidney.

Estrogen receptors (ERs) are expressed in numerous organs, although only a few organs are considered classical targets for estrogens. We have completed a systematic survey of estrogen regulation of approximately 10,000 genes in 13 tissues from wild-type and ERbetaKO mice treated sc with vehicle or 17beta-estradiol (E2) for 6 wk. The uterus and pituitary had the greatest number of genes regulated by E2, whereas the kidney had the third largest number of regulated genes. In situ hybridizations localized E2 regulation in the kidney to the juxtamedullary region of the cortex in both the mouse and rat. The ED(50) for gene inductions in the kidney was 3 micro g/kg.d, comparable with the 2.4 micro g/kg.d ED(50) for c-fos induction in the uterus. E2 regulations in the kidney were intact in ERbetaKO mice, and the ERalpha-selective agonist propylpyrazole triol acted similarly to E2, together suggesting an ERalpha-mediated mechanism. Several genes were induced within 2 h of E2 treatment, suggesting a direct activity of ERalpha within the kidney. Finally, the combination of the activation function (AF)1-selective agonist tamoxifen plus ERalphaKO(CH) mice expressing an AF1-deleted version of ERalpha allowed delineation of genes with differing requirements for AF1 or AF2 activity in the kidney.