Estradiol alters only GAD67 mRNA levels in ischemic rat brain with no consequent effects on GABA

Abstract

The present study tested the hypothesis that estradiol reduces tissue infarction after middle cerebral artery occlusion (MCAO) in estradiol-deficient females by augmenting glutamic acid decarboxylase (GAD) expression and thus activity, leading to increases in gamma-amino-butyric acid (GABA) tissue levels. Glutamic acid decarboxylase is the principal enzyme for GABA synthesis and has two isoforms, GAD65 and GAD67, which differ in size and cellular distribution. Rats were ovariectomized 7 to 8 days before receiving no hormone, placebo, or 25 microg estradiol via subcutaneous implant 7 to 10 days before harvesting tissue in either ischemic cohorts after 2 h of MCAO (end-ischemia) or in nonischemic cohorts. Selected cortical and striatal regions were microdissected from harvested brains. GAD65/67 mRNA levels were determined by microlysate ribonuclease protection assay. End-ischemic GABA concentrations were determined by HPLC. Steroid treatment selectively decreased ischemic cortical GAD67 mRNA levels. In most brain regions evaluated, regional GABA concentrations increased with ischemia regardless of treatment. Estradiol blocked MCAO-induced increases in GABA concentration only in dorsomedial cortex. These data suggest that estradiol repletion in ischemic rat brain selectively decreases GAD67 mRNA levels but does not alter steady-state GABA concentrations. It may be that estradiol under ischemic conditions is attenuating GABA metabolism rather than enhancing synthesis or is augmenting other aspects of GABAergic transmission such as GABA transporters and receptors.

Figure 1

Rats were euthanized via decapitation under halothane anesthesia either at the end of 2 h of middle cerebral artery occlusion (MCAO) or at 9 days after placebo/hormone implantation. Brains were harvested and frozen on dry ice and stored at −80°C for later microdissection. Serial 300 μm coronal sections of the brains were cut in a cryostat at −9°C, thaw-mounted onto glass microscope slides, and immediately refrozen. Using a micropunch dissection technique, brain samples were removed with 17-gauge sharpened stainless-steel hypodermic tubing (1067 μm internal lumen diameter) from selected regions of the cerebral cortex and caudate-putamen in each hemisphere. Selection of these brain regions was based on previous regional cerebral blood flow (CBF) studies (Rusa et al, 1999) to sample representative ischemic regions. Dorsomedial cortex, 1 and 2; dorsolateral cortex, 3 and 4; dorsal striatum, 5 and 6; lateral striatum, 7 and 8. Odd numbers are from ipsilateral or ischemic regions, even numbers are from contralateral or nonischemic regions.

Figure 2

The effect of estradiol on GAD_65/67 mRNA levels in selected brain regions was determined by ribonuclease protection assay (RPA) in naïve, ovariectomized female Wistar rats treated with either placebo (OVX) or 25 μg estradiol pellets (E25) (n = 6 per group). All animals were ovariectomized at least 7 to 8 days before hormone treatments. All pellets were placed subcutaneously at 7 to 10 days before euthanasia. GAD_65/67 mRNA levels were not increased in naïve rat brain by estradiol treatment. All values are mean±s.d.

Figure 3

The effect of estradiol and ischemia on GAD_65/67 mRNA levels in selected brain regions was determined by ribonuclease protection assay (RPA) in ovariectomized female Wistar rats treated with either placebo (OVX, n = 5) or 25 μg estradiol pellets (E25, n = 2). All animals were ovariectomized at least 7 to 8 days before hormone treatments. All hormone pellets were placed subcutaneously at 7 to 10 days before euthanasia. Preischemic estradiol administration in ovariectomized rats reduced GAD₆₇, but not GAD₆₅, mRNA levels in cortical and lateral striatal ischemic brain regions. All values are mean±s.d. *P<0.05 from OVX.

Figure 4

γ-Amino-butyric acid (GABA) concentrations in microdissected brain tissue from ovariectomized female Wistar rats with no hormone (OVX, n = 10) or 25 μg estradiol pellet (E25, n = 11) after 2 h of middle cerebral artery occlusion (MCAO) were determined by HPLC. Control samples were taken from contralateral or nonischemic hemisphere. Middle cerebral artery occlusion samples were taken from ipsilateral or ischemic hemisphere. All animals were ovariectomized at least 7 to 8 days before hormone treatments. All hormone pellets were placed subcutaneously at 7 to 10 days before MCAO. All values are mean±s.d. *P<0.001 from corresponding control. **P<0.01 from corresponding control. ***P<0.05 from OVX MCAO.