Ovarian steroids increase glutamatergic related gene expression in serotonin neurons of macaques

Abstract

Dendritic spines are the elementary structural units of neuronal plasticity and their proliferation and stabilization involve components of glutamate neurotransmission. In a model of hormone replacement therapy (HT), we sought the effect of estradiol (E) and progesterone (P) on gene expression related to glutamate neurotransmission in a laser captured preparation enriched for serotonin neurons from rhesus macaques. Microarray analysis was conducted (n=2 animals/treatment) and then confirmed for pivotal genes with qRT-PCR on additional laser captured material (n=3 animals/treatment). Ovariectomized rhesus macaques were treated with either placebo, E or E+P via Silastic implants for 1month prior to euthanasia. The midbrain was obtained, sectioned and immunostained for TPH. TPH-positive neurons were laser captured using an Arcturus Laser Dissection Microscope (Pixel II). RNA from laser captured serotonin neurons (n=2 animals/treatment) was hybridized to Rhesus Affymetrix GeneChips for screening purposes. There was a 2-fold or greater change in the expression of 28 probe sets related to glutamate processes in E and E+P treated animals. Quantitative (q) RT-PCR was conducted for 11 genes with a custom Taqman PCR array containing monkey specific primers and analyzed with ANOVA followed by Bonferroni's test. The log of the relative expression values indicated that in general, the responses to E and E+P were similar. Comparison of the relative expression or log relative expression in Ovx-controls to combined E and E+P treated groups with t-tests showed a significant increase in AMPA1 (GRIA1), AMPA2 (GRIA2), AMPA4 (GRIA4), NMDA2a (GRIN2A), metabotrophic glutamate receptor (GRM1), glutamine synthetase (GLUL), glutamate dehydrogenase (GLUD), glutamate cysteine ligase modifier subunit (GCLM), the glutamate transporter 2 (SLC1A2) and the glutamate transporter 3 (SLC1A3) with steroid treatment. There was no effect of steroid treatment on gene expression of the glutamate cysteine ligase catalytic subunit (GCLC). These data suggest that ovarian steroids target gene expression of ionotrophic and metabotrophic glutamate receptors in serotonin neurons. These receptors are present on dendritic spines and are necessary for spine maturation. The mRNAs coding for glutamate-related enzymes and transporters are likely derived from astrocytes or glutamate-containing terminals. Their induction by ovarian steroids indicates a complex upregulation of multiple components in the glutamate cycle and antioxidation, in addition to spine proliferation.