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

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.

Figure 1

E₂-induced inhibition of I_Gly in cultured SDH and HIP neurons. (A) Representative traces of current induced by 100 μM glycine in the presence or absence of E₂ at various concentrations in cultured HIP (upper) and SDH (bottom) neurons. The neurons were pre-treated with E₂ for 30 s before E₂ and glycine were co-applied. (B) Summarized data illustrating the concentration dependence of E₂ inhibition (n = 4–8) as shown in A. (C) E₂ significantly inhibited I_Gly recorded from the outside-out patches (n = 5). The upper traces show the representative I_Gly recorded from outside-out patches in the presence and absence of E₂. ***P < 0.001, Paired Student's t-test, compared with control without adding E₂. (D) The concentration-response curves of I_Gly in the presence and absence of 10 μM E₂. For each neuron recorded, the current was normalized to the peak amplitude of I_Gly induced by 100 μM glycine alone (✰) from the same neuron and each point represents the average value of 5–9 neurons.

Figure 2

E₂-induced inhibition of I_Gly is independent of intracellular signaling pathways and classical estrogen receptors (ERs). (A) Sample traces illustrating the inhibitory effects of E₂ on the peak I_Gly under the conditions of intracellular application 15 mM BAPTA (A1), 5 μM staurosporine (A2), 0.5 mM GTP-γ-S (A3) and 0.5 mM GTP-β-S (A4), respectively. A5, Effect of E₂ on I_Gly after incubation of neurons with tamoxifen for 2 h. A6, Effect of 17-α-E₂ on I_Gly. (B) Pooled data summarizing the effect of E₂ on I_Gly under various conditions shown in A. Each column represents the average values from 4–6 neurons, ***P < 0.001, Paired Student's t-test, compared with control without adding E₂ or 17-α-E₂ (dashed line). NS indicates no significant difference in this and the following figures.

Figure 3

Interactions of E₂ and PGN on I_Gly. (A) Sample traces illustrating the additive effect of E₂ and PGN on I_Gly. (B) Summary of results from all experiments similar to those shown in A (n = 4–5). Sum (1) is the expected linear summation of the inhibition induced by 10 μM E₂ and 1 μM PGN; Sum (2) is the expected linear summation of the inhibition induced by 10 μM E₂ and 10 μM PGN. P > 0.05, Unpaired Student's t-test.

Figure 4

Inhibitory effect of E₂ on recombinant GlyRs. (A) Sample traces demonstrating the effects of 10 μM E₂ on various homomeric and heteromeric GlyRs. (B) Summary of results from all experiments similar to those shown in A. E₂ selectively inhibited the peak amplitude of I_Gly mediated by α2-containing GlyRs and α3β heteromeric GlyR. Each column represents the average value of 6–11 neurons. ***P < 0.001, Paired Student's t-test, compared with the control without E₂ treatment (dashed line).

Figure 5

Developmental regulation of E₂ inhibition on I_Gly. Hippocampal and spinal cord neurons in culture were used for electrophysiological recording following 6–8, 13–15 and 20–23 days of in vitro (DIV) differentiation to examine the developmental dependence of E₂ inhibition on I_Gly. Each column represents the average value of 5–8 neurons. * P < 0.05 and *** P < 0.001 (Student's paired t test, n = 8), compared to control without adding E₂; # P < 0.05 (one-way ANOVA, n = 8), comparing DIV 20–23 with either DIV 6–8 or DIV 13–15.

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.