Neurosteroid Dehydroepiandrosterone Sulphate Enhances Pain

British Journal of Anaesthesia, 123 (2): e215ee225 (2019)

doi: 10.1016/j.bja.2019.03.026 Advance Access Publication Date: 26 April 2019 Pain Mechanisms and Novel Analgesics

Neurosteroid dehydroepiandrosterone sulphate enhances pain transmission in rat spinal cord dorsal horn Goh Yamamoto1, Yoshinori Kamiya1,*, Mika Sasaki1, Miho Ikoma2, Hiroshi Baba1 and Tatsuro Kohno1,3

1Division of Anesthesiology, Niigata University Graduate School of Medical and Dental Sciences, Niigata City, Japan, 2Division of Palliative Medicine, Department of Medical Oncology, Niigata University Medical and Dental Hospital, Niigata City, Japan and 3Department of Anesthesiology, Tohoku Medical and Pharmaceutical University, Sendai City, Japan

*Corresponding author. E-mail: [email protected]

Abstract Background: The neurosteroid dehydroepiandrosterone sulphate (DHEAS) activates the sigma-1 receptor, inhibits gamma-aminobutyric acid A (GABAA) and glycine receptors, and induces hyperalgesic effects. Although its effects have been studied in various tissues of the nervous system, its synaptic mechanisms in nociceptive pathways remain to be elucidated. Methods: The threshold of mechanical hypersensitivity and spontaneous pain behaviour was assessed using the von Frey test in adult male Wistar rats after intrathecal administration of DHEAS. We also investigated the effects of DHEAS on synaptic transmission in the spinal dorsal horn using slice patch-clamp electrophysiology. Results: Intrathecally administered DHEAS elicited dose-dependent mechanical hyperalgesia and spontaneous pain behaviours (withdrawal threshold: saline; 51.0 [20.1] g, 3 mg DHEAS; 14.0 [7.8] g, P<0.01, 10 mg DHEAS; 6.9 [5.2] g, 15 min after administration, P<0.001). DHEAS at 100 mM increased the frequency of miniature postsynaptic currents in the rat 2þ dorsal spinal horn; this increase was extracellular Ca -dependent but not sigma-1 and N-methyl-D-aspartate receptor- dependent. DHEAS suppressed the frequency of miniature inhibitory postsynaptic currents in a GABAA receptor- and sigma-1 receptor-dependent manner. Conclusions: These results suggest that DHEAS participates in the pathophysiology of nociceptive synaptic transmission in the spinal cord by potentiation of glutamate release and inhibition of the GABAA receptor.

Keywords: analgesia; gamma-aminobutyric acid; glutamate; neuropathic pain; neurosteroids; nociception; spinal cord dorsal horn; synaptic transmission

e215 e216 - Yamamoto et al.

effects of intrathecally administered DHEAS on the threshold Editor’s key points of mechanical hypersensitivity and spontaneous pain behaviour and investigated the effects of DHEAS in synaptic trans- The neurosteroid dehydroepiandrosterone sulphate mission in the spinal dorsal horn in male rats. (DHEAS) induces hyperalgesic effects, but the receptor mechanisms for these effects are unknown. In a rat model of neuropathic pain, intrathecally Methods administered DHEAS elicited dose-dependent Animals mechanical hyperalgesia and spontaneous pain behaviours in male rats. All animal experiments were conducted in accordance with The effects of DHEAS on synaptic transmission in the international guidelines on the ethical use of animals, spinal dorsal horn slice showed increases in excitatory including the ARRIVE (Animal Research: Reporting of In Vivo and decreases in inhibitory synaptic transmission. Experiments) guidelines, and all efforts were made to mini- These results suggest that DHEAS participates in the mise pain or discomfort experienced by the animals. Animal pathophysiology of nociceptive synaptic transmission in housing and surgical procedures were approved by the Insti- the spinal cord by potentiation of glutamate release and tutional Animal Care and Use Committee of Niigata University

inhibition of gamma-aminobutyric acid A (GABAA) Graduate School of Medical and Dental Science (Niigata City, receptors. Japan; Approval No. H25-342-4). We used adult male Wistar rats (6e8 weeks old, 200e280 g) housed under a 12-h light/dark cycle with ad libitum access to food and water. We used only Neuroactive steroids (neurosteroids) are biosynthesised and male rats to exclude the possibility of sexual cycle-modified 1 metabolised in various tissues of the nervous system, These behaviour because DHEAS is a precursor of sex steroids. steroids are known to have non-genomic effects and do not affect transcription and gene expression via steroid receptors Behavioural testing on the nucleus membrane.2 The non-genomic effects of neurosteroids involve various types of voltage- and ligand-gated A catheter was placed in the spinal cord subarachnoid space for e 30 ion channels.1,3 9 Moreover, neurotransmitter release and drug administration as described. Briefly, 0.5% lidocaine was e synaptic transmission can be modulated by neurosteroids.8 14 infiltrated at the surgical site after animals were anaesthetised Dehydroepiandrosterone (DHEA) and its sulphate derivative, with 2 vol% isoflurane. Next, the lumbar vertebrae were dehydroepiandrosterone sulphate (DHEAS), are considered exposed, the spinal process of the fifth lumbar vertebrae was the most important neurosteroids and show multiple impor- removed to access the dura, and ~2 cm of the polyethylene tant memory-enhancing, antidepressant, and anxiolytic ac- catheter (PE-10; BD Biosciences, Franklin Lakes, NJ, USA) was tivities.71516A previous study showed that DHEAS increased inserted from the gap between the fourth and fifth lumbar the frequency of excitatory postsynaptic currents (EPSCs) in vertebrae so that its tip was close to the lumbar enlargement of the prelimbic cortex17 and induced long-term potentiation- the spinal cord; the other end of the tube was closed by thermal m like effects in the hippocampus18; therefore, DHEAS may play melting. After 3 days, DHEAS (3, 10, 30 g) or 20% dimethyl important roles in synaptic plasticity. sulphoxide in saline (vehicle) was administered to the spinal DHEAS has also been studied extensively in relation to the cord subarachnoid space. The pain reaction threshold against pathophysiology of pain transduction and chronic pain modu- mechanical stimuli was measured using the von Frey test (1, 1.4, lation because of the abundance of enzymes that synthesise 2, 4, 6, 8, 10, 15, 26, 60 g) before drug administration, and at 5, 15, DHEAS (cytochrome P450 17a hydroxylase and sulphotransfer- 30, 60, and 120 min after administration. Eight stimuli were e ase) in the spinal cord.19 21 DHEAS has pivotal effects on the pain applied per filament on the left hindlimb, and the test was e state, including acute-phase pain-evoking effects19,22 24 and deemed positive if a reaction was observed more than twice. late-phase pain-suppressive effects.23,25 Yoon and col- leagues24,26 reported that intrathecally administered DHEAS Preparation of spinal cord slices decreased the threshold for mechanical and heat stimuli to the Cross-sectional slices of the spinal cord were prepared as pre- hindpaw of mice. These DHEAS-induced threshold reductions viously described.31 Briefly, the lumbar enlargement of the spi- are inhibited by sigma-1 receptor antagonists and gamma- nal cord was extracted under urethane anaesthesia (1e1.5 g kg 1 aminobutyric acid A (GABAA) receptor agonists. This suggests i.p.) and immersed in a cooled Krebs solution saturated with 95% that DHEAS can enhance pain, possibly via sigma-1 or GABAA O2 and 5% CO2. Except for the L4 dorsal root on the left side, all receptors in the dorsal horn of the spinal cord. nerve roots were removed. Cross-sectional spinal cord slices, Sigma-1 receptors are putative receptors for several neuro- approximately 650 mm thick, were prepared with the left L4 27 steroids and are densely present in the spinal dorsal horn. dorsal root using a microslicer (Dosaka EM, Kyoto, Japan). Slices Intrathecally administered selective sigma-1 ligands can induce were placed on a recording chamber and perfused (15 ml min 1) pain-like behaviours and can phosphorylate the GluN1 subunitof with a Krebs solution heated to 34e36 C. The composition of the the N-methyl-D-aspartate (NMDA) receptor in the spinal dorsal Krebs solution (mM) was as follows: NaCl, 117; KCl, 3.6; CaCl2,2.5; horn.22 DHEAS-induced GluN1 phosphorylation can be sup- MgCl2,1.2;NaH2PO4,1.2;NaHCO3, 25, and D-glucose, 11.5, 26,28 þ pressed by a sigma-1 receptor antagonist. The NMDA recep- pH¼7.35. for Ca2 measurements; and NaCl, 113.1; KCl, 3.6; tor is well known as a key molecule that regulates pain sensation, MgCl2,5;NaH2PO4,1.2;NaHCO3, 25; and D-glucose, 11, pH¼7.35. þ and phosphorylation of GluN1 increases surface expression of for Ca2 -free measurements. NMDA receptors.29 This evidence indicates that acutely administered DHEAS induces hyperalgesia because of the activation of Patch-clamp recording from dorsal horn neurones the NMDA receptor through sigma-1 receptor activation. However, the synaptic mechanisms of DHEAS in the noci- Blind whole-cell patch-clamp recording was conducted under ceptive pathway remain to be elucidated. We re-analysed the the stereomicroscope using glass microelectrodes with a tip DHEAS enhances pain transmission in spinal cord - e217

electrode resistance of 5e10 MU from the substantia gelatinosa (SG) neurones. The composition of the glass microelectrode solution (mM) was as follows: K-gluconate, 135; KCl, 5; CaCl2, 0.5; MgCl2, 2; ethylene glycol-bis(b-aminoethyl ether)- 0 0 N,N,N ,N -tetraacetic acid (EGTA), 5; 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid (HEPES), 5; tetraethylammonium (TEA), 5; and ATP-Mg, 5, pH¼XX for the current clamp method; and Cs-sulphate, 110; CaCl2, 0.5; MgCl2, 2; EGTA, 5; HEPES, 5; TEA, 5; and ATP-Mg, 5, pH¼XX for the voltage clamp method.

Drug application and experimental protocol The volume of cerebrospinal fluid was assumed to be 300 ml; therefore, the predicted concentration was assumed to be 100 mM when 10 mg of DHEAS (Sigma-Aldrich, St. Louis, MO, USA) was administered to the spinal cord subarachnoid space. DHEAS was dissolved in Krebs solution before administration. A 60 pA current was applied for 1000 ms to measure the action potential using the current clamp method. The holding Fig. 1. Paw withdrawal threshold in response to von Frey potential during measurements was e70 mV when observing stimulation is reduced in a concentration-dependent manner by EPSCs and 0 mV when observing inhibitory postsynaptic intrathecal injection of DHEAS (vehicle, DHEAS 3 mg, DHEAS 10 currents (IPSCs). Miniature EPSCs (mEPSCs), and miniature mg). The data are presented as mean (SEM). n¼5 in each group; IPSCs (mIPSCs) were measured in the presence of 1 mM *P<0.05, **P<0.01, ***P<0.001 by two-way ANOVA. ANOVA, analysis of tetrodotoxin (TTX; Wako Pure Chemical Industries, Osaka, variance; DHEAS, dehydroepiandrosterone sulphate; SEM, stan- Japan). Evoked EPSCs, which were obtained by stimulating the dard error of the mean. dorsal root by a suction electrode, were measured after stimulation of C nerve fibres at 1 mA for 500 ms and stimulation of Ad nerve fibres at 100 mA for 50 ms. The resulting DHEAS potentiated excitability in spinal dorsal horn current was amplified by Axopatch 200B (Molecular Devices, neurones Sunnyvale, CA, USA), A/D converted using Digidata 1440A (Molecular Devices), recorded on a computer, and analysed We investigated the mechanism of DHEAS action on SG neu- with pCLAMP 10.4 (Molecular Devices) and Minianalysis 6.0 rones using the whole-cell patch-clamp technique. In the (Synaptosoft, Leonia, NJ, USA). All other drugs were form current clamp mode, several series of action potentials were Sigma-Aldrich. observed with 60 pA of current injection for 1000 ms. The number of action potentials significantly increased after a 5- min superfusion of 100 mM DHEAS (control, 2.0 [1e8]; DHEAS, Statistical analysis 6.5 [1e17], P¼0.013; Fig. 2). Data are expressed as mean (standard error of mean) for behavioural experiments and median (range) for electrophysiological experiments. For behavioural experiments, DHEAS increased both amplitude and frequency of miniature EPSCs two-way analysis of variance (ANOVA) followed by Bonferroni corrections were used, and the corresponding data obtained DHEAS (100 mM) increased cell excitability in SG neurones. To in electrophysiological experiments were tested using the determine the effects of DHEAS on excitatory synapses in the Wilcoxon signed-rank test. Statistical significance was spinal dorsal horn, we investigated the effects of DHEAS on defined as P<0.05. GraphPad Prism (version 7, GraphPad mEPSCs. Superfusion of 100 mM DHEAS for 5 min increased Software, San Diego, CA, USA) was used for statistical mEPSC frequency and amplitude without the baseline inward analysis. current (frequency: control, 16.5 [1.6e36.8] Hz; DHEAS, 36.3 [13.8e48.6] Hz, P¼0.008; amplitude: control, 9.3 [4.6e13.6] pA; DHEAS, 12.76 [5.0e20.5] pA, P¼0.008; Fig. 3aec). Results Excitatory synaptic currents were elicited via two glutamate receptors, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic Intrathecally administered DHEAS evoked dose- acid (AMPA) and NMDA receptors. Then, we investigated dependent mechanical hypersensitivity changes in amplitude and frequency of mEPSCs with an NMDA Intrathecally administered DHEAS caused dose-dependent receptor inhibitor to determine if the NMDA receptor is influ- mechanical hyperalgesia 15 min after administration as enced by DHEAS. In the presence of 50 mM AP5, an NMDA re- compared with saline administration (saline: 51.0 [20.1] g; 3 mg ceptor antagonist, 100 mM DHEAS significantly increased the DHEAS: 14.0 [7.8] g, P<0.01; 10 mg DHEAS: 6.9 [5.2] g, P<0.00; frequency, but not the amplitude, of mEPSCs (frequency: con- Fig. 1). The mechanical threshold returned to the pre- trol, 11.3 [1.3e32.2] Hz; DHEAS, 18.1 [2.3e45.9] Hz, P¼0.02; administration value 120 min after the drug was adminis- amplitude: control, 7.4 [4.8e12.7] pA; DHEAS, 7.3 [5.4e13.5] pA, tered (Fig. 1). Intrathecally administered DHEAS (30 mg) elicited P¼0.13, Fig. 3d and e). spontaneous pain behaviour (flinching, licking, and biting the DHEAS is a putative ligand of the sigma-1 receptor.32 To hindlimb and tail); therefore, the mechanical threshold could determine the involvement of this receptor in the effects of not be measured in this group. DHEAS on mEPSCs, we investigated the effects of BD1047, a e218 - Yamamoto et al.

Fig. 2. DHEAS potentiates neuronal excitability in the spinal dorsal horn. (a) Current injection (60 pA, 1000 ms) elicited action potential(s) in spinal dorsal horn neurones under current clamp mode. (b) DHEAS (100 mM, 5 min) increased the number of action potentials induced by current injection. (c) Summary of the data. DHEAS superfusion to spinal cord slices signiﬁcantly increased the number of action potentials (n¼9). Boxes show inter-quartile range (IQR), middle lines show medians, and whiskers show the range of values. DHEAS, dehydroepiandrosterone sulphate.

sigma-1 receptor antagonist, on mEPSCs. In the presence of As both GABAA and glycine receptors are involved in IPSCs, 100 mM BD1047, 100 mM DHEAS increased the frequency, but we attempted to clarify which of these receptors is involved in not the amplitude, of mEPSCs (frequency: control, 10.3 DHEAS inhibition of IPSCs. In the presence of 10 mM bicucul-

[1.2e44.5]; DHEAS, 15.5 [2.4e82.7] Hz, P¼0.03; amplitude: con- line, a GABAA receptor antagonist, DHEAS did not show any trol, 8.0 [6.4e23.6]; DHEAS, 8.4 [5.0e24.0] pA, P¼0.54; Fig. 3f and influence on mIPSC frequency and amplitude (frequency: g). control, 1.5 [0.8e6.8]; DHEAS, 1.3 [0.4e6.4] Hz, P¼0.20; ampli- þ The NMDA receptor is a Ca2 -permeable ion channel,33 and tude: control, 6.6 [4.3e23.6]; DHEAS, 8.0 [3.8e19.2] pA, P¼0.9; þ Ca2 is important for cell excitability and synaptic plasticity. Fig. 5d and e). In contrast, DHEAS reduced both frequency and To confirm the effect of DHEAS on excitatory synaptic trans- amplitude of mIPSCs in the presence of 2 mM strychnine, a mission in the spinal dorsal horn, we investigated the effects glycine receptor antagonist (frequency: control, 2.6 [0.9e9.0]; þ of DHEAS on mEPSCs in a Ca2 -free environment. We found DHEAS, 1.2 [0.2e3.4] Hz, P¼0.003, amplitude: control, 8.2 that 100 mM DHEAS did not elicit frequency or amplitude [3.9e17.0]; DHEAS, 5.3 [2.6e12.8] pA, P¼0.002; Fig. 5f and g). potentiation of mEPSCs (frequency: control, 5.1 [0.9e7.9); Taken together, bicuculine, but not strychnine, occluded the DHEAS, 4.2 (1.0e9.7) Hz, P¼0.87; amplitude: control, 6.7 inhibitory effect on mIPSCs by DHEAS. [3.9e7.3]; DHEAS, 6.1 [4.0e7.2] pA, P¼0.11; Fig. 3h and i). DHEAS is a sigma-1 receptor agonist, and activation of the sigma-1 receptor elicited neuronal excitability via activation of the NMDA receptor.26 However, it is unclear if activation of the Evoked EPSCs were not influenced by DHEAS sigma-1 receptor influences inhibitory synaptic transmission. In slice preparations, dorsal root stimulation elicited evoked In the presence of 100 mM BD1047, DHEAS did not influence the EPSCs. Evoked EPSCs are thought to be induced by post- frequency of mIPSCs. However, it suppressed the amplitude of þ þ synaptic Ca2 influx by voltage-dependent Ca2 channels, and mIPSCs (frequency: control, 5.2 [1.5e13.6]; DHEAS, 5.4 [1.4e8.5] in this environment, the effects of presynaptic ligand-gated Hz, P¼0.67, amplitude: control, 10.5 [9.3e23.7]; DHEAS, 9.7 ion channels are negligible. DHEAS did not influence the [6.9e15.8] pA, P¼0.016; Fig. 5h and i). evoked EPSCs by electrical stimulation of the dorsal root using a suction electrode in Ad or C nerve fibres (Ad fibres: control, 95.8 [51.7e120.3]; DHEAS, 93.8 [39.6e153.9] pA, P¼0.63; C fibres: Discussion control, 90.3 (38.0e264.5); DHEAS, 81.8 [34.9e259.3] pA, P¼0.13; Intrathecally administered DHEAS induced dose-dependent Fig. 4). mechanical hyperalgesia and spontaneous pain behaviours in a non-neuropathic pain model in rats. Results from in vitro patch- clamp recording experiments in rat spinal dorsal horn showed DHEAS suppressed the frequency of mIPSCs that DHEAS increased the number of action potentials with

A previous study showed that DHEAS also inhibits GABAA re- depolarisation in the current clamp mode, which indicates that ceptors and suppressed spontaneous inhibitory postsynaptic DHEAS increases excitability in dorsal horn neurones. Under a currents (sIPSCs) in cultured midbrain neurones of rats.34 We voltage-clamp mode with tetrodotoxin to suppress spontaneous investigated the effects of DHEAS on mIPSCs in the spinal action potentials in recorded neurones, DHEAS increased mEPSC dorsal horn. With superfusion of 1 mM TTX, 100 mM DHEAS frequency and amplitude. This facilitation of mEPSC frequency decreased the frequency, but not the amplitude, of mIPSCs by DHEAS may be mediated by NMDA- and sigma-1 receptor- (frequency: control, 4.1 [1.4e10.3]; DHEAS, 2.8 [1.4e5.6] Hz, independent presynaptic mechanisms because the frequency of P¼0.018; amplitude: control, 9.4 [7.1e12.7]; DHEAS, 8.7 mEPSCs induced by DHEAS superfusion was not suppressed by [6.5e13.1] pA, P¼0.16; Fig. 5aec). co-application of the NMDA receptor and sigma-1 receptor DHEAS enhances pain transmission in spinal cord - e219

Fig. 3. DHEAS facilitates excitatory synaptic transmission mainly via the presynaptic mechanism in a sigma-1 receptor-, NMDA receptor-, þ and extracellular Ca2 -dependent manner. (a) Representative traces of mEPSCs in the spinal dorsal horn. Neurones in the spinal dorsal horn were voltage clamped at 70 mV. Downward arrows indicate outtakes of the top trace shown on an expanded timescale. Heavy horizontal bars show periods of drug application. (b, c) Bath-applied DHEAS (100 mM, 5 min) increased the frequency and amplitude of mEPSCs. Changes in frequency (b) and amplitude (c) of mEPSCs before and after DHEAS administration (n¼9). (d) In the presence of AP5 (50 mM), an NMDA receptor antagonist, DHEAS increased mEPSC frequency. (e) DHEAS had no effect on mEPSC amplitude (n¼9). (f) In the presence of BD1047 (100 mM), a sigma-1 receptor antagonist, DHEAS increased mEPSC frequency. (g) DHEAS had no effect on mEPSC þ amplitude (n¼14). (h) Under a Ca2 -free Krebs solution environment, DHEAS had no effect on mEPSC frequency and amplitude (n¼7). Boxes show inter-quartile range (IQR), middle lines show medians, and whiskers show the range of values. DHEAS, dehydroepiandrosterone sulphate; mEPSCs, miniature excitatory postsynaptic currents; NMDA, N-methyl-D-aspartate. e220 - Yamamoto et al.

Fig. 4. DHEAS did not affect evoked excitatory synaptic currents in the spinal dorsal horn. (a) Representative traces of Ad fibre-evoked monosynaptic EPSCs recorded during baseline and 5 min after DHEAS (100 mM) superfusion to spinal cord slices. (b) DHEAS had no effect on the amplitude of Ad fibre-evoked monosynaptic EPSCs (n¼5). (c) Representative traces of C fibre-evoked monosynaptic EPSCs recorded during baseline and 5 min after DHEAS superfusion. (e) DHEAS had no effect on the amplitude of C fibre-evoked monosynaptic EPSCs (n¼5). All recordings were performed at a holding potential of e70 mV. Boxes show inter-quartile range (IQR), middle lines show medians, and whiskers show the range of values. DHEAS, dehydroepiandrosterone sulphate; EPSCs, excitatory postsynaptic currents.

antagonists. Moreover, DHEAS did not affect the amplitude of show that direct inhibition of GABAA receptors by DHEAS þ mEPSCs in a Ca2 -free environment, and mEPSC frequency was should elicit postsynaptic neurone excitability. Our results are strongly suppressed and summation of mEPSCs did not occur. also compatible with evidence showing that muscimol, a 2þ NMDA receptors are known to allow permeation of Ca ,and GABAA receptor agonist, attenuates the hyperalgesic effects of þ this Ca2 is thought to be crucial to synaptic plasticity by both DHEAS.24 pre- and postsynaptic mechanisms. Hence, facilitation of mEPSC The nociceptive properties of DHEAS in the spinal dorsal þ frequency by DHEAS is thought to be Ca2 -independent because horn have been explained by alterations of NMDA receptor of NMDA receptor activation. function via the sigma-1 receptor. Activation of the sigma-1 DHEAS is known to be hyperalgesic when intrathe- receptor is known to initiate activation of protein kinase C cally22,24,26 and intraplantarly35 administered. A previous (PKC) that phosphorylates the GluN1 subunit and enhances study showed that DHEAS, but not its non-sulphated form cell surface expression of NMDA receptors,38 and is involved in DHEA, produced mechanical allodynia in a sigma-1- and the pathophysiology of hypersensitivity.39 Our ﬁndings are

GABAA-receptor-dependent manner. The hyperalgesic effect partially compatible with those of previous studies. The of DHEAS administered intrathecally is caused by post- amplitude of mEPSCs was inhibited by co-application of a synaptic NMDA receptor activation via sigma-1 receptor- sigma-1 receptor and NMDA receptor antagonist. However, mediated phosphorylation of the GluN122,26 subunit and direct the frequency of mEPSCs was not affected by either NMDA or 24,34,36 inhibition of GABAA receptors. Similar to our results, sigma-1 receptor antagonists. Our results suggest that DHEAS another study showed that DHEAS potentiated both synaptic predominantly enhances nociceptive synaptic transmission at potentials and cell membrane excitability.37 These studies presynaptic sites. Previous studies have shown that DHEAS DHEAS enhances pain transmission in spinal cord - e221

Fig. 5. DHEAS suppressed inhibitory synaptic transmission in a GABAA and sigma-1 receptor-dependent manner. (a) Representative traces of mIPSCs in spinal dorsal horn. Neurones in the spinal dorsal horn were voltage clamped at 0 mV. Downward arrows indicate outtakes of the top trace shown on an expanded timescale. Heavy horizontal bars show periods of drug application. (b, c) Bath-applied DHEAS (100 mM, 5 min) decreased mIPSC frequency. However, DHEAS had no effect on amplitude. Change in the frequency (b) and amplitude (c) of mEPSCs before and after DHEAS administration (n¼7). (d, e) In the presence of bicuculline (10 mM), a GABAA receptor antagonist, DHEAS had no effect on glycinergic mIPSC frequency and amplitude (n¼14). (f, g) In the presence of strychnine (2 mM), a glycine receptor antagonist, DHEAS decreased GABAergic mIPSC frequency and amplitude (n¼11). (h) In the presence of BD1047 (100 mM), a sigma-1 receptor antagonist, DHEAS had no effect on mIPSC frequency (n¼7). (i) BD1047 suppressed the amplitude of mIPSCs. Boxes show inter-quartile range (IQR), middle lines show medians, and whiskers show the range of values. DHEAS, dehydroepiandrosterone sulphate; GABAA, gamma- aminobutyric acid A; mEPSCs, miniature excitatory postsynaptic currents. e222 - Yamamoto et al.

þ increased glutamate release from presynaptic terminals12,40 Ca2 -permeable ion channels, such as P2X purinergic re- and potentiated excitatory synaptic transmission17 via ceptors,5,43 which may account for these discrepancies. sigma-1 receptor activation. Moreover, presynaptic NMDA Dorsal root-evoked EPSCs in the spinal dorsal horn were receptor activation is thought to be associated with the path- not influenced by DHEAS. During evoked EPSCs in the dorsal þ ophysiology of neuropathic pain.41 However, we also found horn, glutamate release is thought to be elicited by Ca2 via þ þ that extracellular Ca2 , but not NMDA receptor or sigma-1 voltage-gated Ca2 channels (VGCCs), and its amplitude þ receptor activation, is crucial for presynaptic facilitation of should be mainly determined by presynaptic Ca2 influx glutamate release by DHEAS. Although our findings were not (which is equal to VGCC activity) or the conductance and compatible with those of previous studies,12,17,40 DHEAS is also amount of surface expression of postsynaptic AMPA receptors. þ known to activate the Ca2 -activated K channel (KCa)42 and Our results indicated that both presynaptic VGCCs and

Fig. 6. Model of the spinal dorsal horn circuit underlying the mechanism of DHEAS-produced hyperalgesia. DHEAS potentiates glutamate release from the presynaptic site to the recorded substantia gelatinosa (SG) neurone via neither sigma-1 receptor- nor NMDA-receptor-

dependent manners and also inhibits postsynaptic GABAA receptors. This increases neuronal excitability of SG neurones to elicit me-

chanical hyperalgesia and spontaneous pain behaviours. SG, substantia gelatinosa; DHEAS, dehydroepiandrosterone sulphate; GABAA, gamma-aminobutyric acid A; NMDA, N-methyl-D-aspartate. DHEAS enhances pain transmission in spinal cord - e223

postsynaptic AMPA receptors were not modulated by DHEAS rats and facilitated excitatory synaptic transmission and in- via sigma-1 receptor activation. Although sigma-1 receptor hibition of inhibitory synaptic transmission in the spinal activation decreases VGCC current44 and expression of AMPA dorsal horn. Synthetic enzymes of neurosteroids are abundant receptors,45 these previous studies were carried out using in the nervous system, and some of them, including DHEAS, isolated cardiac or sympathetic neurones and cultured cortical facilitate synaptic transmission. Our results suggest that neurones, and the properties of the expressed receptors and DHEAS may be a potential therapeutic target for prevention of intracellular signal transduction mechanisms may differ from plastic changes of nociceptive transmission in the spinal cord. the sensory nervous system. This may explain why the results of this study and previous studies are inconsistent. Authors’ contributions During inhibitory synaptic transmission, DHEAS can 34,36 46,47 Conception and design of study: GY, MI, YK. inhibit both GABAA receptors and glycine receptors ; Conduct of experiments: GY, MS. however, the effects of DHEAS on inhibitory neurotransmitter Analysis of data: GY. release are still unclear. This study showed that DHEAS sup- Drafting of the manuscript: GY, YK, TK, HB. pressed GABAA-receptor-mediated mIPSCs via sigma-1 acti- Editing and revision of the manuscript: YK. vation. Previous studies showed that 10 mM of DHEAS All authors read and approved the final version of the suppressed spontaneous IPSCs in midbrain neurones, and manuscript. sigma-1 receptor activation suppressed the frequency of mIPSCs in medullary dorsal horn neurones48; our results were compatible with these findings. However, our results also Declaration of interest indicate that DHEAS suppressed GABAergic mIPSCs, not glycinergic mIPSCs. The frequency of glycinergic mIPSCs The authors declare that they have no conflicts of interest. increased after administration of pregnenolone sulphate, another neuroactive steroid that activates the sigma-1 recep- Funding tor.6 In that study, the frequency of glycinergic mIPSCs was not sigma-1 receptor-dependent, and the authors suggested that Japan Society for the Promotion of Science, Tokyo, Japan transient receptor potential (TRP) channel-mediated intracel- (26861224 and 16K20082 to GY; 24592330 to MI; 18H02897 to þ lular Ca2 may be involved. Involvement of a TRP channel for YK). neurotransmitter release by DHEAS is a likely mechanism 2þ because intracellular Ca elevation is crucial for enhancing Acknowledgements both excitatory and inhibitory neurotransmitter release from þ presynaptic terminals. Our results also indicate that a Ca2 - We thank Editage (www.editage.jp) for English language free environment strongly suppressed the frequency of editing. mEPSCs induced by administration of DHEAS. These studies have several limitations. First, experiments References were performed only in male rats. However, DHEAS is a type of androgen and it may affect sexual cycle in female rats. we 1. Baulieu EE. Neurosteroids: of the nervous system, by the want to exclude the possibility of sexual cycle-modified nervous system, for the nervous system. Recent Prog Horm þ behavior in this study. Second, Ca2 influx and neurotrans- Res 1997; 52:1e32 mitter release were not measured directly. However, many 2. Zheng P. Neuroactive steroid regulation of neurotrans- studies have performed previously clearly showed that elec- mitter release in the CNS: action, mechanism and possible trophysiological assessment was used for evaluating neuro- significance. Prog Neurobiol 2009; 89: 134e52 þ transmitter release and influence of extracellular Ca2 3. Abdrachmanova G, Chodounska H, Vyklicky Jr L. Effects of concentration for neurotransmitter release, and our data were steroids on NMDA receptors and excitatory synaptic also showed that DHEAS increases release of excitatory transmission in neonatal motoneurons in rat spinal cord þ neurotransmitter (glutamate) in extracellular Ca2 -dependent slices. Eur J Neurosci 2001; 14: 495e502 manner. Third, pharmacological concentration of DHEAS were 4. Cheng ZX, Lan DM, Wu PY, et al. Neurosteroid dehydro- used, which may not translate to physiological conditions. epiandrosterone sulphate inhibits persistent sodium cur- Further study should be needed to clarify the physiological rents in rat medial prefrontal cortex via activation of role of DHEAS in neurotransmission of pain. sigma-1 receptors. Exp Neurol 2008; 210: 128e36 Considering these findings, we propose the following 5. De Roo M, Rodeau JL, Schlichter R. Dehydroepiandroster- model circuit for the underlying mechanism of DHEAS in the one potentiates native ionotropic ATP receptors contain- spinal dorsal horn (Fig. 6): 1) DHEAS inhibits GABAA receptors ing the P2X2 subunit in rat sensory neurones. J Physiol located on postsynaptic sites of the recorded SG neurones and 2003; 552:59e71 inhibits presynaptic GABA release on GABAergic neurones via 6. Hong JS, Cho JH, Choi IS, Lee MG, Jang IS. Pregnenolone sigma-1 receptors, and 2) DHEAS leads to increased glutamate sulfate modulates glycinergic transmission in rat medul- release from presynaptic excitatory interneurones to SG neu- lary dorsal horn neurons. Eur J Pharmacol 2013; 712:30e8 rones via indirect activation of neither NMDA nor sigma-1 7. Krzascik P, Zajda ME, Majewska MD. The neurosteroid receptors. We propose that these mechanisms could produce dehydroepiandrosterone sulfate, but not androsterone, hyperalgesia. enhances the antidepressant effect of cocaine examined in the forced swim testdpossible role of serotonergic neurotransmission. Horm Behav 2015; 70:64e72 Conclusions 8. Sliwinski A, Monnet FP, Schumacher M, Morin-Surun MP. We showed that intrathecally administered DHEAS induced Pregnenolone sulfate enhances long-term potentiation in mechanical hyperalgesia and spontaneous pain behaviours in CA1 in rat hippocampus slices through the modulation of e224 - Yamamoto et al.

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