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. 2016 May;231(5):1057-64.
doi: 10.1002/jcp.25198. Epub 2015 Sep 30.

Genes in the GABA Pathway Increase in the Lateral Thalamus of Sprague-Dawley Rats During the Proestrus/Estrus Phase

Mikhail Umorin  1 , Crystal Stinson  1 , Larry L Bellinger  1 , Phillip R Kramer  1
Affiliations

Genes in the GABA Pathway Increase in the Lateral Thalamus of Sprague-Dawley Rats During the Proestrus/Estrus Phase

Mikhail Umorin et al. J Cell Physiol. 2016 May.

Abstract

Pain can vary over the estrous cycle as a result of changes in estradiol concentration but the mechanism causing this variation is unclear. Because the thalamus is important in pain control, gene expression in the lateral thalamus (ventral posteromedial, ventral posterolateral, reticular thalamic nuclei) was screened at different phases of the estrous cycle. Gene expression changes in Sprague-Dawley rats were further analyzed by real-time PCR and ELISA and plasma estradiol levels were measured by RIAs at different phases of the estrous cycle. Our results indicated that both the RNA and protein expression of glutamate decarboxylase 1 and 2 (GAD1, GAD2), GABA(A) receptor-associated protein like 1 (GABARAPL1), and vesicular GABA transporter (VGAT) significantly increased in the lateral thalamus when plasma estradiol levels were elevated. Estradiol levels were elevated during the proestrus and estrus phases of the estrous cycle. Estrogen receptor α (ERα) was observed to be co-localized in thalamic cells and thalamic infusion of an ERα antagonist significantly reduced GAD1 and VGAT transcript. GAD1, GAD2, GABARAPL1, and VGAT have been shown to effect neuronal responses suggesting that attenuation of pain during the estrous cycle can be dependent, in part, through estradiol induced changes in thalamic gene expression.

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Figures

Figure 1
Figure 1. Plasma 17 β-estradiol concentration in rats during the estrous cycle
Vaginal smears were performed and plasma was collected within one hour. Plasma estradiol was measured by RIA. Panel A is from Experiment #1 where transcript levels were measured in the thalamic tissue by RT-PCR. Panel B is from Experiment #3 where thalamic protein content was quantitated by ELISA. Significant differences of p<0.05 are indicated by an asterisk. Seven animals were in each of the four estrous cycle phases.
Figure 2
Figure 2. Transcript levels of GAD1, GAD2, GABARAPL1 and VGAT in the thalamus of cycling female rats
Estrous cycle phases were determined by vaginal smears and the thalamic tissue was isolated within one hour. RT-PCR analysis of the thalamic tissue was then performed. Panel A shows GAD1 transcript levels, Panel B shows GAD2 transcript expression, Panel C shows GABARAPL1 transcript expression and Panel D shows VGAT transcript expression. Significant differences of p<0.05 are indicated by an asterisk. There were 7 animals in each of the three estrous phases except estrus where there were 8 animals.
Figure 3
Figure 3. Thalamic gene expression plotted against the plasma estradiol concentration
Panel A shows GAD1 transcript levels, Panel B shows GAD2 transcript expression, Panel C shows GABARAPL1 transcript expression and Panel D shows VGAT transcript expression plotted on the x-axis. The estradiol concentration was plotted on the y-axis. There were 7 animals in each of the three estrous phases except estrus where there were 8 animals.
Figure 4
Figure 4. Protein content of GAD1, GAD2, GABARAPL1 and VGAT in the thalamus of cycling female rats
Estrous cycle phases were determined by vaginal smears and the thalamic tissue was isolated within one hour. Protein content was measured by ELISA. Panel A shows GAD1 protein levels, Panel B shows GAD2 protein expression, Panel C shows GABARAPL1 protein expression and Panel D shows VGAT protein expression. Significant differences of p<0.05 are indicated by an asterisk. There were 5 animals in each of the 4 estrous phases.
Figure 5
Figure 5. Lateral thalamic immuno-fluorescent staining for ERα and GAD2 (GAD65)
A frozen section from a diestrus rat was stained for ERα (red) and GAD2 (green). Panel A shows a cell with ERα and GAD2 co-localized (open arrow) and panel E is a different region on the same section showing a cell expressing ERα (arrowhead) and a cell expressing GAD2 (arrow). The ERα signal is shown in panels B and F, the GAD2 signal is shown in panels C and G and in panels D and H the nuclei are stained with Hoechst 33342. Image is taken from along the border of the reticular thalamic nucleus and the ventral posterolateral nucleus. Bar equals 50 μm.
Figure 6
Figure 6. Thalamic injection of an ERα antagonist reduced expression of VGAT and GAD1 during estrus
The lateral region of the thalamus was cannulated unilaterally and two weeks after surgery vehicle or the ERα antagonist MPP dihydrochloride was infused on the day of proestrus. Rats were sacrificed the next day (i.e., estrus) and the injected thalamus (ipsilateral) and non-injected thalamus (contralateral) were isolated. The estrus phase was confirmed by measuring blood plasma estradiol levels and vaginal smears. Real time PCR was performed using the thalamic tissues and the amount of GAD1, GAD2, GABARAPL1 and VGAT was measured and reported as the fold change in transcript, contralateral thalamus versus ipsilateral thalamus. A value of less than 0 indicates that gene expression on the ipsilateral side was reduced versus the contralateral side. A value of 1 (dotted line) indicates the transcript levels were the same on the ipsilateral and contralateral sides. There were 4 vehicle injected rats and 4 MPP dihydrochloride injected rats.
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