- Short report
- Open Access
Potentiation of excitatory transmission in substantia gelatinosa neurons of rat spinal cord by inhibition of estrogen receptor alpha
Molecular Painvolume 6, Article number: 92 (2010)
It has been shown that estrogen is synthesized in the spinal dorsal horn and plays a role in modulating pain transmission. One of the estrogen receptor (ER) subtypes, estrogen receptor alpha (ERα), is expressed in the spinal laminae I-V, including substantia gelatinosa (SG, lamina II). However, it is unclear how ERs are involved in the modulation of nociceptive transmission.
In the present study, a selective ERα antagonist, methyl-piperidino-pyrazole (MPP), was used to test the potential functional roles of spinal ERα in the nociceptive transmission. Using the whole-cell patch-clamp technique, we examined the effects of MPP on SG neurons in the dorsal root-attached spinal cord slice prepared from adult rats. We found that MPP increased glutamatergic excitatory postsynaptic currents (EPSCs) evoked by the stimulation of either Aδ- or C-afferent fibers. Further studies showed that MPP treatment dose-dependently increased spontaneous EPSCs frequency in SG neurons, while not affecting the amplitude. In addition, the PKC was involved in the MPP-induced enhancement of synaptic transmission.
These results suggest that the selective ERα antagonist MPP pre-synaptically facilitates the excitatory synaptic transmission to SG neurons. The nociceptive transmission evoked by Aδ- and C-fiber stimulation could be potentiated by blocking ERα in the spinal neurons. Thus, the spinal estrogen may negatively regulate the nociceptive transmission through the activation of ERα.
Several studies suggest that estrogen plays an important role in the spectrum of neural functions, such as nociception [1–4]. Estrogen is synthesized in many neurons in laminae I-III of the spinal cord [5–8], and potentiates the pain behavior . Estrogen may modulate nociceptive responses through the increase of glutamate-induced currents, the inhibition of γ-aminobutyric acid (GABA) and glycine (Gly) receptors, or the modulation of the opioid receptors in the spinal dorsal horn [9–11]. It is well known that the classical estrogen action in neurons is to activate nuclear estrogen receptor α and β (ERα/β), which cause long-term genomic effects [12, 13], or to activate cytoplasmic signaling events at or near the plasma membrane [14, 15] through either membrane-localized classical ERs [16, 17] or novel ERs . Recent studies showed that ERα is expressed in spinal laminae I-V, especially in laminae I-II, and is most abundant in the lower lumbar (L) and sacral segments [19, 20]. However, whether the ERα is involved in estrogen-mediating pain behavior remains unclear. Considering that the superficial dorsal horn of the spinal cord, especially substantia gelatinosa (SG, lamina II), plays an important role in the modulation of synaptic transmission of fine myelinated A (Aδ)- and unmyelinated C-afferent fibers [21, 22], we used a selective ERα antagonist, methyl-piperidino-pyrazole (MPP) , to examine the function of spinal ERα in nociceptive transmission in SG neurons. The dorsal root-attached spinal cord slices were prepared from adult rats and recorded with whole-cell patch-clamp technique.
Whole-cell recordings were carried out in SG neurons. Stable recordings could be maintained in vitro for more than 8 hrs; and recordings could be made from a single SG neuron up to 2 hrs. The monosynaptic, Aδ-afferent evoked excitatory postsynaptic currents (eEPSCs) with a mean amplitude of 156 ± 25 pA (50~360 pA; VH = -70 mV) were found in ~70% of recorded neurons (18/25). In 8 out of these 18 neurons (~ 45%), superfusion of MPP (10 µM) increased the peak amplitude of the Aδ-eEPSC in a reversible manner (Figure 1A). The enhancement was averaged at 130 ± 5% (n = 8) in magnitude.
The monosynaptic C-afferent eEPSCs with a mean amplitude of 135 ± 31 pA (40~310 pA; VH = -70 mV) were found in ~60% of neurons (10/16). In 5 out of these 10 neurons, MPP (10 μM) treatment increased the peak amplitude of the C-eEPSC and normal Kreb's solution washed off the MPP-induced effect (Figure 1B). The averaged magnitude of the enhancement was 150 ± 6% (n = 5). In other three neurons exhibiting both Aδ- and C-eEPSCs, MPP increased the amplitude of both types of eEPSCs (Figure 1C).
Further comparison of MPP-induced enhancement between Aδ- and C-eEPSCs showed that the increase in C-eEPSC amplitude during MPP application was more pronounced than that of Aδ-EPSC (Figure 1D). In spite of the differences of their sensitivity to MPP, Aδ- and C-eEPSCs were responded with a similar time course following MPP superfusion. The current amplitudes had been changed maximally and measured at 3 min after MPP was applied.
To examine whether MPP modulated the afferent synaptic transmission through pre- or post-synaptic action, the spontaneous EPSC (sEPSC) in SG neurons during MPP treatment were analyzed. We found that superfusion of MPP (10 μM) resulted in a reversible enhancement in sEPSC frequency (Figure 2A and 2B; 234 ± 9% of control at 3 min following its application, n = 8; P < 0.001). Furthermore, the MPP-induced responses were dose-dependent. At a concentration of 1 and 5 μM, the increased sEPSC frequency was 124 ± 8% (n = 5) and 180 ± 6% (n = 5), respectively. However, the amplitude of sEPSC was not altered by the treatment with MPP (Figure 2B and 2C). The modulation on sEPSC frequency, but not on amplitude, suggests that MPP regulates nociceptive transmission through a pre-synaptic action.
To investigate whether exogenous estrogen could modulate glutamatergic excitatory synaptic transmission in SG neurons, we tested whether a treatment with 17β-estradiol could regulate the sEPSC. We found that the frequency of sEPSC was reduced by bath-applied 17β-estradiol (1 μM), and this effect could be reversed by MPP (Figure 3A, n = 6). Finally, we also found that a PKC inhibitor bisindolylmaleimide I hydrochloride (BIM) (1 μM), but not a PKA inhibitor H89 (5 μM), could reduce the MPP-induced enhancement of sEPSC frequency (Figure 3B, n = 5).
The above results suggest that nociceptive transmission could be facilitated by blocking ERα, such as a selective antagonist MPP used in the current study. In addition, the endogenous estrogen may activate ERα in spinal dorsal horn to reduce glutamatergic excitatory transmission and inhibit the nociceptive responses. Previous studies showed that ERα is expressed in the small-diameter neurons in the dorsal root ganglion (DRG), a subset of nociceptive sensory neurons [24–26]. The ERα-mediated inhibition of ATP-induced Ca2+ signaling in mouse DRG neurons  suggests that peripheral ERα negatively regulates nociceptive transmission. Moreover, ERα immunoreactivity has been found in the spinal cord. A larger numbers of ERα-immunoreactive neurons were found in the lower lumbar spinal cord segments. These ERα-containing neurons were mainly found in the spinal lamina II, and some were in laminae I, III, IV, V, and X. In the superficial layers of the medullary dorsal horn, ERα-immunoreactivity was mainly located in lamina II, which was also expressed noxious-induced Fos [19, 20, 28]. These findings provide an anatomical and neurochemical basis for the hypothesis that estrogen activates ERα directly to regulate pain transmission at the central level . Consistent with early studies, our present study shows that in the spinal dorsal horn, ERα is involved in the modulation of nociceptive Aδ- and C-afferent transmission.
Previous studies showed that the enzyme aromatase catalyzed the formation of estrogen from testosterone in the gonads and other tissues, such as many nociceptive neurons in the spinal laminae I-III. Moreover, the specific nonsteroidal aromatase inhibitor, vorozole, was found to inhibit the phosphorylation of aromatase in the spinal cord and induce an acute inhibition of the endogenous spinal estrogen synthesis, which could consequently lead to the inhibition of nociceptive responses [6–8]. Some studies showed that estrogen rapidly potentiated glutamate (kainate)-induced currents through a second-messenger cascade . G-protein coupled to inwardly rectifying K channels could be inhibited by the estrogen-induced reduction of the potency of GABA and opioid receptor agonists [18, 29]. Moreover, estrogen inhibited the GABA and Gly receptors or modulated the opioid receptor in the spinal dorsal horn [10, 11]. The estrogen-induced potentiation of kainate currents and inhibition of GABA and Gly receptors may play a role in the activation of the central pain pathways [30–32]. Our present results show that activation of ERα by estrogen may inhibit nociceptive transmission through a PKC signaling pathway. Therefore, the ERα-mediated negative regulation of nociceptive transmission may be balanced by the effect of estrogen on other receptors in some certain extent.
In conclusion, we propose that estrogen may inhibit the nociceptive transmission via the ERα in the spinal dorsal horn. Our results may help to understand the functions and mechanisms of estrogen in pain modulation, and suggest that ERα may be a potential target in relieving pain syndrome.
Materials and methods
Spinal slice preparation and whole-cell recording
Transverse spinal cord slices (~600 μm, L4 or L5 segment) with an attached dorsal root from adult rats (male, 6-8 weeks old) were prepared with a vibrating microslicer and perfused in the oxygen-bubbled Krebs' solution (in mM: 117 NaCl, 3.6 KCl, 2.5 CaCl2, 1.2 MgCl2, 1.2 NaH2PO4, 25 NaHCO3, and 11 D-glucose) for a blind ruptured patch-clamp recording as our previous study . Resistance of the patch electrodes was typically 4~10 MΩ. The internal eletrode solution contained (in mM: 135 K-gluconate, 0.5 CaCl2, 2 MgCl2, 5 KCl, 5 EGTA, 5 HEPES and 5 D-glucose). Currents were filtered at 2 kHz and digitized at 5 kHz (Axopatch 200B amplifier, Molecular Devices) and were analyzed by using pCLAMP8.5 program. The membrane potential was hold at -70 mV. To evoke Aδ- and C-fiber activation, the dorsal root stimulation was delivered with a suction electrode which was linked to a constant-current stimulator (Digitimer). Monosynaptic eEPSC was studied in the presence of 20 μM bicuculline and 2 μM strychnine. Frequency and amplitude of sEPSC were analyzed with Axograph (Molecular Devices). Afferent Aδ- or C-fibers were identified by the basis of the conduction velocity (CV) of afferent fibres (Aδ: 2~12 m/s; C: <1.2 m/s) calculated from the latency of EPSC from a stimulus artifact, the length of dorsal root, and the stimulus threshold (Aδ: 10~60 μA; C: 180~620 μA). The Aδ and C responses were considered as monosynaptic in origin when the latency remained constant and there was no failure during stimulation at 20 Hz for the Aδ-fiber evoked EPSCs, and at 2 Hz for the C-fiber evoked EPSCs. Drugs were applied through a superfusion exchange of the solutions in the recording chamber. The drugs used in the present studies included strychnine, bicuculline, MPP, 17β-estradiol (Sigma, USA), BIM and H89 (Calbiochem, USA). All drugs except for MPP, 17β-estradiol and H89 (where dimethyl sulphoxide was used as a solvent) were dissolved in distilled water at 1000 times the concentration in stock and kept at -20°C. On the experimental days, they were diluted to the desired concentration within Kreb's solution.
Data analysis and statistics
All numerical data were presented as the mean ± S.E.M. Statistical significance was determined as P < 0.05 using the student's paired t-test, n refers to the number of neurons studied.
Amandusson A, Hallbeck M, Hallbeck AL, Hermanson O, Blomqvist A: Estrogen-induced alterations of spinal cord enkephalin gene expression. Pain 1999,83(2):243–8. 10.1016/S0304-3959(99)00109-8
Bradshaw HB, Berkley KJ: Estrous changes in responses of rat gracile nucleus neurons to stimulation of skin and pelvic viscera. J Neurosci 2000,20(20):7722–7.
Liu NJ, Gintzler AR: Prolonged ovarian sex steroid treatment of male rats produces antinociception: identification of sex-based divergent analgesic mechanisms. Pain 2000,85(1–2):273–81. 10.1016/S0304-3959(99)00278-X
Craft RM: Modulation of pain by estrogens. Pain 2007,132(Suppl 1):S3–12. 10.1016/j.pain.2007.09.028
Blomqvist A: Sex hormones and pain: a new role for brain aromatase? J Comp Neurol 2000,423(4):549–51. 10.1002/1096-9861(20000807)423:4<549::AID-CNE1>3.0.CO;2-B
Evrard H, Baillien M, Foidart A, Absil P, Harada N, Balthazart J: Localization and controls of aromatase in the quail spinal cord. J Comp Neurol 2000,423(4):552–64. 10.1002/1096-9861(20000807)423:4<552::AID-CNE2>3.0.CO;2-S
Evrard HC, Balthazart J: Aromatase (estrogen synthase) activity in the dorsal horn of the spinal cord: functional implications. Ann N Y Acad Sci 2003, 1007: 263–71. 10.1196/annals.1286.025
Evrard HC, Balthazart J: Rapid regulation of pain by estrogens synthesized in spinal dorsal horn neurons. J Neurosci 2004,24(33):7225–9. 10.1523/JNEUROSCI.1638-04.2004
Gu Q, Korach KS, Moss RL: Rapid action of 17β-estradiol on kainate-induced currents in hippocampal neurons lacking intracellular estrogen receptors. Endocrinology 1999,140(2):660–6. 10.1210/en.140.2.660
Jiang P, Kong Y, Zhang XB, Wang W, Liu CF, Xu TL: Glycine receptor in rat hippocampal and spinal cord neurons as a molecular target for rapid actions of 17-β-estradiol. Mol Pain 2009, 5: 2. 10.1186/1744-8069-5-2
Li W, Jin X, Covey DF, Steinbach JH: Neuroactive steroids and human recombinant rho1 GABA C receptors. J Pharmacol Exp Ther 2007,323(1):236–47. 10.1124/jpet.107.127365
Maggi A, Ciana P, Belcredito S, Vegeto E: Estrogens in the nervous system: mechanisms and nonreproductive functions. Annu Rev Physiol 2004, 66: 291–313. 10.1146/annurev.physiol.66.032802.154945
Nadal A, Diaz M, Valverde MA: The estrogen trinity: membrane, cytosolic, and nuclear effects. News Physiol Sci 2001, 16: 251–5.
Collins P, Webb C: Estrogen hits the surface. Nat Med 1999,5(10):1130–1. 10.1038/13453
Edwards DP: Regulation of signal transduction pathways by estrogen and progesterone. Annu Rev Physiol 2005, 67: 335–76. 10.1146/annurev.physiol.67.040403.120151
Levin ER: Cellular functions of plasma membrane estrogen receptors. Steroids 2002,67(6):471–5. 10.1016/S0039-128X(01)00179-9
Abraham IM, Todman MG, Korach KS, Herbison AE: Critical in vivo roles for classical estrogen receptors in rapid estrogen actions on intracellular signaling in mouse brain. Endocrinology 2004,145(7):3055–61. 10.1210/en.2003-1676
Qiu J, Bosch MA, Tobias SC, Grandy DK, Scanlan TS, Ronnekleiv OK, Kelly MJ: Rapid signaling of estrogen in hypothalamic neurons involves a novel G-protein-coupled estrogen receptor that activates protein kinase C. J Neurosci 2003,23(29):9529–40.
Vanderhorst VG, Gustafsson JA, Ulfhake B: Estrogen receptor-α and -β immunoreactive neurons in the brainstem and spinal cord of male and female mice: relationships to monoaminergic, cholinergic, and spinal projection systems. J Comp Neurol 2005,488(2):152–79. 10.1002/cne.20569
Vanderhorst VG, Terasawa E, Ralston HJ: Estrogen receptor-α immunoreactive neurons in the brainstem and spinal cord of the female rhesus monkey: species-specific characteristics. Neuroscience 2009,158(2):798–810. 10.1016/j.neuroscience.2008.10.017
Kumamoto E, Shimoji K, Yoshimura M: Baclofen inhibits more effectively C-afferent than Aδ-afferent glutamatergic transmission in substantia gelatinosa neurons of adult rat spinal cord slices. Pain 2000,86(3):273–82. 10.1016/S0304-3959(00)00255-4
Bird GC, Han JS, Fu Y, Adwanikar H, Willis WD, Neugebauer V: Pain-related synaptic plasticity in spinal dorsal horn neurons: role of CGRP. Mol Pain 2006, 2: 31. 10.1186/1744-8069-2-31
Sun J, Huang YR, Harrington WR, Sheng S, Katzenellenbogen JA, Katzenellenbogen BS: Antagonists selective for estrogen receptor α. Endocrinology 2002,143(3):941–7. 10.1210/en.143.3.941
Taleghany N, Sarajari S, DonCarlos LL, Gollapudi L, Oblinger MM: Differential expression of estrogen receptor alpha and beta in rat dorsal root ganglion neurons. J Neurosci Res 1999,57(5):603–15. 10.1002/(SICI)1097-4547(19990901)57:5<603::AID-JNR3>3.0.CO;2-R
Papka RE, Storey-Workley M: Estrogen receptor-α and -β coexist in a subpopulation of sensory neurons of female rat dorsal root ganglia. Neurosci Lett 2002,319(2):71–4. 10.1016/S0304-3940(01)02562-9
Papka RE, Mowa CN: Estrogen receptors in the spinal cord, sensory ganglia, and pelvic autonomic ganglia. Int Rev Cytol 2003, 231: 91–127. full_text
Chaban VV, Micevych PE: Estrogen receptor-α mediates estradiol attenuation of ATP-induced Ca2+ signaling in mouse dorsal root ganglion neurons. J Neurosci Res 2005,81(1):31–7. 10.1002/jnr.20524
Amandusson S, Blomqvist A: Estrogen receptor-α expression in nociceptive-responsive neurons in the medullary dorsal horn of the female rat. Eur J Pain 2009,14(3):245–8. 10.1016/j.ejpain.2009.05.008
Kelly MJ, Loose MD, Ronnekleiv OK: Estrogen suppresses μ-opioid and GABA B -mediated hyperpolarization of hypothalamic arcuate neurons. J Neurosci 1992,12(7):2745–50.
Li P, Wilding TJ, Kim SJ, Calejesan AA, Huettner JE, Zhuo M: Kainate-receptor-mediated sensory synaptic transmission in mammalian spinal cord. Nature 1999,397(6715):161–4. 10.1038/16469
Blednov YA, Stoffel M, Alva H, Harris RA: A pervasive mechanism for analgesia: activation of GIRK2 channels. Proc Natl Acad Sci USA 2003,100(1):277–82. 10.1073/pnas.012682399
Mitrovic I, Margeta-Mitrovic M, Bader S, Stoffel M, Jan LY, Basbaum AI: Contribution of GIRK2-mediated postsynaptic signaling to opiate and α2-adrenergic analgesia and analgesic sex differences. Proc Natl Acad Sci USA 2003,100(1):271–6. 10.1073/pnas.0136822100
Wang HB, Zhao B, Zhong YQ, Li KC, Li ZY, Wang Q, Lu YJ, Zhang ZN, He SQ, Zheng HC, Wu SX, Hökfelt TGM, Bao L, Zhang X: Coexpression of δ- and μ-opioid receptors in nociceptive sensory neurons. Proc Natl Acad Sci USA 2010,107(29):13117–22. 10.1073/pnas.1008382107
This work was supported by NNSFC 30621062, MOST 2006CB806600, CASKSCX2-YW-R-31. Dr. Kai-Cheng Li was supported by grant 06R214160 and 2007KIP402.
The authors declare that they have no competing interests.
XZ and KCL conceived and designed the study. YQZ performed the experiments. All authors read and approved the final manuscript.