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. 2013 Mar 10;367(1-2):85-97.
doi: 10.1016/j.mce.2012.12.021. Epub 2013 Jan 7.

mRNA expression of ion channels in GnRH neurons: subtype-specific regulation by 17β-estradiol

Martha A Bosch  1 , Karen J Tonsfeldt, Oline K Rønnekleiv
Affiliations

mRNA expression of ion channels in GnRH neurons: subtype-specific regulation by 17β-estradiol

Martha A Bosch et al. Mol Cell Endocrinol. .

Abstract

Burst firing of neurons optimizes neurotransmitter release. GnRH neurons exhibit burst firing activity and T-type calcium channels, which are vital for burst firing activity, are regulated by 17β-estradiol (E2) in GnRH neurons. To further elucidate ion channel expression and E2 regulation during positive and negative feedback on GnRH neurosecretion, we used single cell RT-PCR and real-time qPCR to quantify channel mRNA expression in GnRH neurons. GFP-GnRH neurons expressed numerous ion channels important for burst firing activity. E2-treatment sufficient to induce an LH surge increased mRNA expression of HCN1 channels, which underlie the pacemaker current, the calcium-permeable Ca(V)1.3, Ca(V)2.2, Ca(V)2.3 channels, and TRPC4 channels, which mediate the kisspeptin excitatory response. E2 also decreased mRNA expression of SK3 channels underlying the medium AHP current. Therefore, E2 exerts fundamental changes in ion channel expression in GnRH neurons, to prime them to respond to incoming stimuli with increased excitability at the time of the surge.

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Figures

Figure 1
Figure 1
Mean serum levels of LH in OVX, E2-treated mice housed under 12 h light, 12 h dark lighting condition and killed at one of four time points, ZT4, ZT11, ZT12, ZT13. Lights were turned on at ZT0 and turned off at ZT12. Serum LH levels at ZT11, 12 and 13 were elevated compared to LH levels at ZT4; *, p<0.05, **, p<0.01, Student’s t-test. The number of animals at each time point is indicated.
Figure 2
Figure 2
qPCR amplification for GnRH, GPR54 and SK3 in GnRH neurons. Amplification of cDNA synthesized from differing amounts of RNA in individual GnRH neurons and pools of 2, 4, and 8 cells (GnRH and GPR54) as well as pools of 5 and 10 cells for SK3. cDNA from GnRH single cells and pools was diluted 1:10 for GnRH qPCR measurements. GPR54 and SK3 were quantified in full strength cDNA. A. Cycle number was plotted against normalized fluorescence intensity (delta Rn) to visualize the PCR amplification. Cycle threshold (CT; dashed line) is the point in the amplification at which the sample values were calculated. B. Linear regression analysis plotting crossing threshold (CT) versus the log scale of number of cells collected in each pool (mean ± SEM; n = number of individual cells or pools of cells, which were 5–6 for GnRH and GPR54, and 10–13 for SK3. GnRH, GPR54 and SK3 produced slope values of −3.02, −3.17, and −3.23 respectively, which corresponds to a doubling between each cycle with high linearity (Pearson’s coefficient r2= 0.99). β-actin showed a similar linear relationship between individual cells and cell pools (slope −3.00, r2 = 0.98; data not shown). The amplification efficiency for each primer pair is listed in Table 1. These efficiencies allowed us to use the ΔΔCT method for quantification. C. Bar graphs illustrating the quantitative analysis of relative mRNA expression of GnRH in individual GnRH neurons and in pools of 2, 4 and 8 cells (one-way ANOVA; a-a, b-b, p<0.001; n=6 individual cells and 5–6 pools). D. Bar graphs illustrating the quantitative analysis of relative mRNA expression of GPR54 in individual GnRH neurons and in pools of 2, 4 and 8 cells (one-way ANOVA; a-a, b-b, c-c, p<0.001; n = 6 individual cells and 6 pools). E. Bar graphs illustrating the quantitative analysis of relative mRNA expression of SK3 in GnRH neuronal pools containing 5 and 10 cells (Student’s t-test; ***, p<0.001, n = 10–13 pools).
Figure 3
Figure 3
Expression of HCN channels in GnRH neurons from intact proestrus/estrus EGFP-GnRH mice. A, Representative gels illustrating the mRNA expression of HCN channel subtypes 1–4. The expected sizes for the PCR products are as follows: for HCN1, 136 bp; for HCN2, 97 bp; for HCN3, 118 bp; for HCN4, 123 bp; and for GnRH, 239 bp. As a negative control, a cell reacted without reverse transcriptase (−RT) did not express any of the transcripts. POA tissue RNA was also included as positive control (+, with RT) and negative control (−, without RT). MM, molecular markers. B, Summary bar graphs of the percentage expression of HCN1, HCN2, HCN3, and HCN4. 12 cells/animal from 4–6 animals were analyzed by sc-PCR, and the mean number of neurons expressing HCN channel subtypes from each animal was determined. Bar graphs represent the mean ± SEM of the percentage of GnRH neurons expressing each HCN subtype per animal. C, Quantitative real-time PCR assay with amplification curves for HCN1, HCN2 and HCN3 subunits. Cycle number was plotted against the normalized fluorescence intensity (delta Rn) to visualize the PCR amplification. The cycle threshold (CT, arrow) is the point in the amplification at which the sample values were calculated. The amplification efficiencies for each primer pair is listed in Table 1. These efficiencies allowed us to use the ΔΔCT method for quantification. D, The melting curves depict single-product melting at 82, 83 and 84°C for HCN1, HCN2 and HCN3, respectively, illustrating that only one product was formed for each transcript in GnRH 5-cell pools. E, Bar-graphs illustrating the relative mRNA expression of HCN1, HCN2, HCN3 and HCN4. a-a, p<0.01, b-b, p<0.05; Student’s t-test. HCN4 was measured in 10-cell pools (n=2) and was often below the level of detectability and was, therefore, not included in the statistical analysis. The number of animals is indicated.
Figure 4
Figure 4
Expression of HVA channels in GnRH neurons from intact proestrus/estrus EGFP-GnRH mice. A, Representative gels illustrating the mRNA expression of HVA channel subtypes. The expected sizes for the PCR products are as follows: for CaV1.2 (L), 126 bp; CaV1.3 (L), 116 bp; CaV2.1 (P/Q), 75 bp; CaV2.2 (N), 169 bp; CaV2.3 (R), 107 bp. As a negative control, a cell reacted without reverse transcriptase (−RT) did not express any of the transcripts. POA tissue RNA was also included as positive control (+, with RT) and negative control (−, without RT). MM, molecular markers. B, Summary bar graphs of the percentage expression of HVA channel subtypes in GnRH neurons. 12 cells/animal from 4 animals were analyzed by sc-PCR, and the mean number of neurons expressing HVA channel subtypes from each animal was determined. Bar graphs represent the mean ± SEM of the percentage of GnRH neurons expressing each HVA subtype per animal. C, Quantitative real-time PCR assay with amplification curves for CaV2.3, CaV1.3, CaV2.1, CaV1.2, and CaV2.2. Cycle number was plotted against the normalized fluorescence intensity (delta Rn) to visualize the PCR amplification. The cycle threshold (CT, arrow) is the point in the amplification at which the sample values were calculated. The amplification efficiencies for each primer pair is listed in Table 1. D, The melting curves depict single-product melting at 82, 80, 81, 80 and 83°C for CaV1.2, 1.3, 2.1, 2.2 and 2.3, respectively, illustrating that only one product was formed for each transcript in GnRH 5- or 10-cell pools. E, Bar-graphs illustrating the relative mRNA expression of the various CaV channel subtypes. R-type (CaV2.3) mRNA expression was significantly higher than that of the other channel subtypes (***, p<0.001; one-way ANOVA). L-type (CaV1.3) mRNA expression was significantly higher than CaV1.2, 2.1 and 2.2 mRNA levels (**, p<0.01; one way ANOVA). The number of animals is indicated.
Figure 5
Figure 5
TRPC channel subtype distribution by real-time PCR. A, qPCR assay with amplification curves for TRPC1, TRPC4 and TRPC5 subunits. (TRPC1 and TRPC4 were analyzed in 5-cell pools and TRPC5 in 10-cell pools). Cycle number was plotted against the normalized fluorescence intensity (delta Rn) to visualize the PCR amplification. The cycle threshold (CT, arrow) is the point in the amplification at which the sample values were calculated. The amplification efficiencies for each primer pair is listed in Table 1. B, Melting curves depict single-product melting at 77, 78, and 81°C for TRPC1, TRPC4 and TRPC5, respectively, illustrating that only one product was formed for each transcript in GnRH neuronal pools. C, Bar-graphs illustrating the relative mRNA expression of TRPC1, TRPC4 and TRPC5 (*** p<0.001 TRPC4 compared to TRPC1 and TRPC5). The number of animals is indicated.
Figure 6
Figure 6
SK channel subtype distribution in GnRH neurons. A, Representative gels illustrating the mRNA expression of SK channel subtypes 1–3. The expected sizes for the PCR products are as follows: for SK1, 181 bp; for SK2, 124 bp; for SK3, 111 bp. As a negative control, a cell reacted without reverse transcriptase (−RT) did not express any of the transcripts. POA tissue RNA was also included as positive control (+, with RT) and negative control (−, without RT). MM, molecular markers. B, Summary bar graphs of the percentage expression of SK1, SK2 and SK3 subunits. 12 cells/animal from 4 animals were analyzed by sc-PCR, and the mean number of neurons expressing SK channel subtypes from each animal was determined. Bar graphs represent the mean ± SEM of the percentage of GnRH neurons expressing each SK subtype per animal. Note SK1 was not detected in mouse GnRH neurons.
Figure 7
Figure 7
Expression of GPR54 mRNA in GnRH neurons from intact proestrus/estrus EGFP-GnRH mice. A, Representative gels illustrating the mRNA expression of GPR54 and GnRH in EGFP-GnRH mice. The expected sizes for the PCR products are as follows: for GnRH, 239 bp; for GPR54,245 bp; As a negative control, a cell reacted without reverse transcriptase (−RT) did not express any of the transcripts. POA tissue RNA was also included as positive control (+, with RT) and negative control (−, without RT). MM, molecular markers. B, Summary bar graphs of the percentage expression of GnRH and GPR54 in EGFP-GnRH neurons. 12 cells/animal from 4 animals were analyzed by sc-PCR, and the mean number of neurons expressing GnRH and GPR54 mRNAs from each animal was determined. Bar graphs represent the mean ± SEM of the percentage of GnRH neurons expressing GnRH and GPR54 per animal. Essentially, all EGFP neurons harvested expressed GnRH and GPR54. C, qPCR analysis of of GnRH and GPR54 mRNA expression in GnRH neurons. Bar-graphs illustrating the relative mRNA expression of GnRH and GPR54 (***, p<0.001; Student’s t-test; the number of animals is indicated).
Figure 8
Figure 8
E2 regulates HCN1 channel mRNA expression in GnRH neurons. A,B, Quantitative real-time PCR measurements of HCN1, HCN2 and HCN3 mRNAs in GnRH neuronal pools (3–4 pools per animal) from oil- and E2-treated mice (n=3–7 animals per group) obtained during the morning, ZT4 (A) or during the evening, ZT11 (B). The expression values were calculated via the ΔΔCT method and normalized to the mean ΔCT of the oil-treated samples. Bar graphs represent the mean ± SEM. *, p<0.05, oil versus E2.
Figure 9
Figure 9
E2 regulates HVA channel mRNAs in GnRH neurons. A,B, Quantitative real-time PCR measurements of CaV1.2, CaV1.3, CaV2.1 and CaV2.3 mRNAs during the morning, ZT4 (A) and CaV1.3, CaV2.2 and CaV2.3 mRNAs during the evening, ZT11 (B) in GnRH neuronal pools (3–4 pools of 5 or 10 (CaV1.2, CaV2.2) cells per animal) from oil- and E2-treated mice (n=3–6 animals per group). The expression values were calculated via the ΔΔCT method, normalized to β-actin and relative to the oil control values. Bar graphs represent the mean ± SEM. *, p<0.05, oil versus E2.
Figure 10
Figure 10
E2 regulates TRPC4 mRNA in GnRH neurons. A, B, Quantitative real-time PCR measurements of TRPC1 and TRPC4 mRNAs during the morning, ZT4 (A) and TRPC4 during the evening, ZT11 (B) in GnRH neuronal pools (3–4 pools of 5 cells per animal) from oil- and E2-treated mice (n=5–7 animals per group). The expression values were calculated via the ΔΔCT method, normalized to β-actin and relative to the oil control values. Bar graphs represent the mean ± SEM. *, p<0.05, oil versus E2.
Figure 11
Figure 11
E2 decreases SK3 mRNA in GnRH neurons. Quantitative real-time PCR measurements of SK3 mRNA during the morning, ZT4 (A) and SK3 mRNA during the evening, ZT11 in GnRH neuronal pools (3–4 pools of 5 cells per animal) from oil- and E2-treated mice (n=5 animals per group). The expression values were calculated via the ΔΔCT method, normalized to β-actin and each E2 value was calculated relative to the respective oil control value. Bar graphs represent the mean ± SEM. *, p<0.05, **, p<0.01, oil versus E2.
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