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Glycine receptor in rat hippocampal and spinal cord neurons as a molecular target for rapid actions of 17-beta-estradiol.

Jiang P, Kong Y, Zhang XB, Wang W, Liu CF, Xu TL - Mol Pain (2009)

Bottom Line: Here we show that, in cultured rat hippocampal (HIP) and spinal dorsal horn (SDH) neurons, 17-beta-estradiol (E2) rapidly and reversibly reduced the peak amplitude of whole-cell glycine-activated currents (IGly).Moreover, the E2 effect on IGly persisted in the presence of the calcium chelator BAPTA, the protein kinase inhibitor staurosporine, the classical ER (i.e. ERalpha and ERbeta) antagonist tamoxifen, or the G-protein modulators, favoring a direct action of E2 on GlyRs.In HEK293 cells expressing various combinations of GlyR subunits, E2 only affected the IGly in cells expressing alpha2, alpha2beta or alpha3beta subunits, suggesting that either alpha2-containing or alpha3beta-GlyRs mediate the E2 effect observed in neurons.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Neuroscience and State Key Laboratory of Neuroscience, Chinese Academy of Sciences, Shanghai 200031, PR China.

ABSTRACT
Glycine receptors (GlyRs) play important roles in regulating hippocampal neural network activity and spinal nociception. Here we show that, in cultured rat hippocampal (HIP) and spinal dorsal horn (SDH) neurons, 17-beta-estradiol (E2) rapidly and reversibly reduced the peak amplitude of whole-cell glycine-activated currents (IGly). In outside-out membrane patches from HIP neurons devoid of nuclei, E2 similarly inhibited IGly, suggesting a non-genomic characteristic. Moreover, the E2 effect on IGly persisted in the presence of the calcium chelator BAPTA, the protein kinase inhibitor staurosporine, the classical ER (i.e. ERalpha and ERbeta) antagonist tamoxifen, or the G-protein modulators, favoring a direct action of E2 on GlyRs. In HEK293 cells expressing various combinations of GlyR subunits, E2 only affected the IGly in cells expressing alpha2, alpha2beta or alpha3beta subunits, suggesting that either alpha2-containing or alpha3beta-GlyRs mediate the E2 effect observed in neurons. Furthermore, E2 inhibited the GlyR-mediated tonic current in pyramidal neurons of HIP CA1 region, where abundant GlyR alpha2 subunit is expressed. We suggest that the neuronal GlyR is a novel molecular target of E2 which directly inhibits the function of GlyRs in the HIP and SDH regions. This finding may shed new light on premenstrual dysphoric disorder and the gender differences in pain sensation at the CNS level.

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Inhibitory effect of E2 on GlyR-mediated tonic current in HIP slices. (A) Left, Whole-cell voltage-clamp recording showing the current in the presence of 0.3 μM TTX, 10 μM bicuculline, 3 μM CNQX, 10 μM APV, 20 μM glycine and 0.5 mM sarcosine. Application of strychnine (2 μM) decreased the membrane current noise and revealed the tonically activated GlyR current. In the presence of E2 (10 μM), the amplitude of GlyR-mediated tonic current was significantly reduced. Right (a1–a3), Gaussian fit to all-point histograms of 30 s traces at the time point a1, a2 and a3 (A, inset). The differences among the Gaussian means are marked by the dotted lines. (B) The normalized GlyR-mediated tonic current in the absence or presence of E2 (n = 8). *P < 0.05, Paired Student's t-test, compared with the control without E2 treatment.
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Figure 6: Inhibitory effect of E2 on GlyR-mediated tonic current in HIP slices. (A) Left, Whole-cell voltage-clamp recording showing the current in the presence of 0.3 μM TTX, 10 μM bicuculline, 3 μM CNQX, 10 μM APV, 20 μM glycine and 0.5 mM sarcosine. Application of strychnine (2 μM) decreased the membrane current noise and revealed the tonically activated GlyR current. In the presence of E2 (10 μM), the amplitude of GlyR-mediated tonic current was significantly reduced. Right (a1–a3), Gaussian fit to all-point histograms of 30 s traces at the time point a1, a2 and a3 (A, inset). The differences among the Gaussian means are marked by the dotted lines. (B) The normalized GlyR-mediated tonic current in the absence or presence of E2 (n = 8). *P < 0.05, Paired Student's t-test, compared with the control without E2 treatment.

Mentions: In the developing and mature hippocampus, α2 subunit represents the primary component of the functional GlyRs [21]. This unique property enables us to examine the action of E2 on GlyRs from pyramidal neurons in CA1 region of HIP slices. Glycine concentration in cerebrospinal fluid has been estimated to be in the micromolar range [40]. In order to magnify the current, we recorded the GlyR-mediated tonic current by adding 20 μM glycine to ACSF in addition to the presence of GlyT1 inhibitor sarcosine (0.5 mM) [25]. At the same time, TTX (0.3 μM) was added in the bath to reduce random baseline current fluctuations, and 10 μM bicuculline, 10 μM DL-2-amino-5-phosphovaleric acid (APV) and 3 μM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) were added to block GABAA and ionotropic glutamate receptors, respectively. After recording a control period in the cocktail solution, E2 at 10 μM was superfused (Figure 6A). As shown in Figure 6B, the GlyR-mediated tonic current was significantly reduced to 69.3 ± 5.8% of control by E2 perfusion (P = 0.043, n = 8, Wilcoxon matched-pairs signed-ranks test), a level compatible with that obtained in the cultured neurons (Figure 1B) or HEK 293 cells expressing recombinant α2- and α2β-GlyRs (Figure 4B).


Glycine receptor in rat hippocampal and spinal cord neurons as a molecular target for rapid actions of 17-beta-estradiol.

Jiang P, Kong Y, Zhang XB, Wang W, Liu CF, Xu TL - Mol Pain (2009)

Inhibitory effect of E2 on GlyR-mediated tonic current in HIP slices. (A) Left, Whole-cell voltage-clamp recording showing the current in the presence of 0.3 μM TTX, 10 μM bicuculline, 3 μM CNQX, 10 μM APV, 20 μM glycine and 0.5 mM sarcosine. Application of strychnine (2 μM) decreased the membrane current noise and revealed the tonically activated GlyR current. In the presence of E2 (10 μM), the amplitude of GlyR-mediated tonic current was significantly reduced. Right (a1–a3), Gaussian fit to all-point histograms of 30 s traces at the time point a1, a2 and a3 (A, inset). The differences among the Gaussian means are marked by the dotted lines. (B) The normalized GlyR-mediated tonic current in the absence or presence of E2 (n = 8). *P < 0.05, Paired Student's t-test, compared with the control without E2 treatment.
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Related In: Results  -  Collection

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Figure 6: Inhibitory effect of E2 on GlyR-mediated tonic current in HIP slices. (A) Left, Whole-cell voltage-clamp recording showing the current in the presence of 0.3 μM TTX, 10 μM bicuculline, 3 μM CNQX, 10 μM APV, 20 μM glycine and 0.5 mM sarcosine. Application of strychnine (2 μM) decreased the membrane current noise and revealed the tonically activated GlyR current. In the presence of E2 (10 μM), the amplitude of GlyR-mediated tonic current was significantly reduced. Right (a1–a3), Gaussian fit to all-point histograms of 30 s traces at the time point a1, a2 and a3 (A, inset). The differences among the Gaussian means are marked by the dotted lines. (B) The normalized GlyR-mediated tonic current in the absence or presence of E2 (n = 8). *P < 0.05, Paired Student's t-test, compared with the control without E2 treatment.
Mentions: In the developing and mature hippocampus, α2 subunit represents the primary component of the functional GlyRs [21]. This unique property enables us to examine the action of E2 on GlyRs from pyramidal neurons in CA1 region of HIP slices. Glycine concentration in cerebrospinal fluid has been estimated to be in the micromolar range [40]. In order to magnify the current, we recorded the GlyR-mediated tonic current by adding 20 μM glycine to ACSF in addition to the presence of GlyT1 inhibitor sarcosine (0.5 mM) [25]. At the same time, TTX (0.3 μM) was added in the bath to reduce random baseline current fluctuations, and 10 μM bicuculline, 10 μM DL-2-amino-5-phosphovaleric acid (APV) and 3 μM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) were added to block GABAA and ionotropic glutamate receptors, respectively. After recording a control period in the cocktail solution, E2 at 10 μM was superfused (Figure 6A). As shown in Figure 6B, the GlyR-mediated tonic current was significantly reduced to 69.3 ± 5.8% of control by E2 perfusion (P = 0.043, n = 8, Wilcoxon matched-pairs signed-ranks test), a level compatible with that obtained in the cultured neurons (Figure 1B) or HEK 293 cells expressing recombinant α2- and α2β-GlyRs (Figure 4B).

Bottom Line: Here we show that, in cultured rat hippocampal (HIP) and spinal dorsal horn (SDH) neurons, 17-beta-estradiol (E2) rapidly and reversibly reduced the peak amplitude of whole-cell glycine-activated currents (IGly).Moreover, the E2 effect on IGly persisted in the presence of the calcium chelator BAPTA, the protein kinase inhibitor staurosporine, the classical ER (i.e. ERalpha and ERbeta) antagonist tamoxifen, or the G-protein modulators, favoring a direct action of E2 on GlyRs.In HEK293 cells expressing various combinations of GlyR subunits, E2 only affected the IGly in cells expressing alpha2, alpha2beta or alpha3beta subunits, suggesting that either alpha2-containing or alpha3beta-GlyRs mediate the E2 effect observed in neurons.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Neuroscience and State Key Laboratory of Neuroscience, Chinese Academy of Sciences, Shanghai 200031, PR China.

ABSTRACT
Glycine receptors (GlyRs) play important roles in regulating hippocampal neural network activity and spinal nociception. Here we show that, in cultured rat hippocampal (HIP) and spinal dorsal horn (SDH) neurons, 17-beta-estradiol (E2) rapidly and reversibly reduced the peak amplitude of whole-cell glycine-activated currents (IGly). In outside-out membrane patches from HIP neurons devoid of nuclei, E2 similarly inhibited IGly, suggesting a non-genomic characteristic. Moreover, the E2 effect on IGly persisted in the presence of the calcium chelator BAPTA, the protein kinase inhibitor staurosporine, the classical ER (i.e. ERalpha and ERbeta) antagonist tamoxifen, or the G-protein modulators, favoring a direct action of E2 on GlyRs. In HEK293 cells expressing various combinations of GlyR subunits, E2 only affected the IGly in cells expressing alpha2, alpha2beta or alpha3beta subunits, suggesting that either alpha2-containing or alpha3beta-GlyRs mediate the E2 effect observed in neurons. Furthermore, E2 inhibited the GlyR-mediated tonic current in pyramidal neurons of HIP CA1 region, where abundant GlyR alpha2 subunit is expressed. We suggest that the neuronal GlyR is a novel molecular target of E2 which directly inhibits the function of GlyRs in the HIP and SDH regions. This finding may shed new light on premenstrual dysphoric disorder and the gender differences in pain sensation at the CNS level.

Show MeSH
Related in: MedlinePlus