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Spinal vasopressin alleviates formalin-induced nociception by enhancing GABAA receptor function in mice
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Research article
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Spinal vasopressin alleviates formalin-induced nociception by enhancing GABAA receptor function in mice
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Fang Peng a,b , Zu-Wei Qu a , Chun-Yu Qiu a , Min Liao b,∗ , Wang-Ping Hu a,∗∗ a b
Institute of Ion Channels, Department of Pharmacology, Hubei University of Science and Technology, 88 Xianning Road, Xianning 437100, Hubei, PR China Department of Anatomy and Histology & Embryology, College of Basic Medicine, Wenzou Medical University, Wenzou 32500, Zhejiang, PR China
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h i g h l i g h t s • We have found that spinal AVP reduced formalin-induced spontaneous nociception. • Spinal AVP analgesia were mediated by V1A receptors. • Enhancing GABAergic function may be involved in the mechanism underlying spinal AVP analgesia.
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Article history: Received 5 February 2015 Received in revised form 6 March 2015 Accepted 13 March 2015 Available online xxx
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Keywords: Arginine vasopressin Vasopressin-1A receptor GABAA receptor Formalin test Pain Knock-out
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1. Introduction
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Arginine vasopressin (AVP) plays a regulatory role in nociception. Intrathecal administration of AVP displays an antinociceptive effect. However, little is understood about the mechanism underlying spinal AVP analgesia. Here, we have found that spinal AVP dose dependently reduced the second, but not first, phase of formalin-induced spontaneous nociception in mice. The AVP analgesia was completely blocked by intrathecal injected SR 49059, a vasopressin-1A (V1A ) receptor antagonist. However, spinal AVP failed to exert its antinociceptive effect on the second phase formalin-induced spontaneous nociception in V1A receptor knock-out (V1A -/-) mice. The AVP analgesia was also reversed by bicuculline, a GABAA receptor antagonist. Moreover, AVP potentiated GABA-activated currents in dorsal root ganglion neurons from wild-type littermates, but not from V1A -/- mice. Our results may reveal a novel spinal mechanism of AVP analgesia by enhancing the GABAA receptor function in the spinal cord through V1A receptors. © 2015 Published by Elsevier Ireland Ltd.
Arginine vasopressin (AVP) is involved in a wide range of physiological regulatory processes [13]. AVP has been demonstrated to play a neuromodulatory role on nociception. It is believed that AVP causes antinociception in both humans and animals [2,11]. The antinociceptive activity was observed after subcutaneous, intraperitoneal or intracerebroventricular administration of AVP [3,14]. In contrast, Brattleboro rats, which are naturally deficient in AVP, had a hyperalgesic state in flinch–jump threshold test and impaired stress analgesia [4]. Thus, AVP plays a role as an endogenous analgesic substance. Intrathecal administration of AVP displayed also an antinociceptive effect [24,27]. However,
∗ Corresponding author. ∗ ∗ Corresponding author. Tel.: +86 715 8150960; fax: +86 715 82256221. E-mail addresses: [email protected] (M. Liao), wangping [email protected] (W.-P. Hu).
another study reported that intrathecal injected AVP failed to produce hyponociception in the tail flick test [15]. The modulatory effect of AVP on nociception is mediated by binding to three distinct receptors: V1A , V1B and V2 receptors [11,13]. AVP analgesia may be mainly mediated by V1A receptors, since AVP did not alter nociceptive threshold in V1A receptor knock-out (V1A -/-) mice [16,22]. Recently, low (<500 pg) and high concentration of AVP in the blood displayed antinociceptive and pronociceptive effects, respectively. And the antinociceptive and pronociceptive effects of AVP were fully abolished in the presence of the V1A receptor antagonist [12]. To further clarify the effects of AVP in nociceptive process, we investigated again the action of spinal AVP on formalin-induced spontaneous nociception. We used also V1A -/- mice and selective V1A receptor antagonists to determine the specific receptors involved in AVP actions. Finally, we found that enhancing GABAA receptor function may be involved in the mechanism underlying spinal AVP analgesia.
http://dx.doi.org/10.1016/j.neulet.2015.03.023 0304-3940/© 2015 Published by Elsevier Ireland Ltd.
Please cite this article in press as: F. Peng, et al., Spinal vasopressin alleviates formalin-induced nociception by enhancing GABAA receptor function in mice, Neurosci. Lett. (2015), http://dx.doi.org/10.1016/j.neulet.2015.03.023
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2. Materials and methods
3. Results
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2.1. Animals
3.1. Spinal AVP dose dependently reduced formalin-induced spontaneous nociception in wild-type mice
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The experimental protocol was approved by the animal research ethics committee of Hubei University of Science and Technology. The experiments were undertaken on littermate male adult (8–12 weeks) wild-type and V1A -/- mice of C57BL/6 background as described previously [20]. All animals were kept with a 12 h light/dark cycle and with ad libitum access to food and water. 2.2. Drugs and drug administration methods
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All chemicals were purchased from Sigma–Aldrich (St. Louis, MO, USA). The drugs used were formalin, AVP, SR 49059 and bicuculline. Formalin was diluted to 5% in saline. For spinal injections, mice were briefly anesthetized with isoflurane and intrathecal (i.t) injections of drugs (5 l using a 30-gauge needle) were delivered by acute lumbar puncture between the L5 and L6 vertebrae 5 min prior to the formalin injection (by which time mice were fully recovered from the anesthetic). A characteristic tail-flick indicated successful penetration of the spinal compartment.
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2.3. Formalin-induced spontaneous nociceptive behavior
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Mice were gently restrained, and then 20 l of 5% formalin was injected subcutaneously into the plantar surface of one hind paw using a micro syringe with a 30-gauge needle. The total time spent in spontaneous nociceptive behavior (licking and lifting of the injected paw) was recorded in 5-min intervals for 1 h, starting immediately after formalin injection. The first phase was defined as 0–10 min and the second phase as 10–60 min. All behavioral experiments were performed by an observer blinded for the animal treatment. 2.4. Isolation of the DRG neurons and electrophysiological recordings
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The mouse DRG neurons were isolated as described previously [20]. Dissociated neurons were placed into a 35 mm petri dish and kept for at least another 60 min before electrophysiological recordings. The neurons selected for electrophysiological experiment were 15–35 m in diameter. Whole-cell patch clamp recording was carried out using a MultiClamp-700B amplifier and Digidata-1440A A/D converter (Axon Instruments, Foster city, CA, USA). Recording micropipettes were filled with internal solution containing KCl 140 mM, MgCl2 2.5 mM, HEPES 10 mM, EGTA 11 mM and ATP 5 mM and the pH was adjusted to 7.2 with KOH. Cells were bathed in an external solution containing NaCl 150 mM, KCl 5 mM, CaCl2 2.5 mM, MgCl2 2 mM, HEPES 10 mM and d-glucose 10 mM and the osmolarity was adjusted to 330 mOsm/L with sucrose and pH to 7.4. The resistance of the recording pipette was in the range of 3–6 M. A small patch of membrane underneath the tip of the pipette was aspirated to form a gigaseal and then negative pressure was applied to rupture it. The adjustment of capacitance compensation and series resistance compensation was done before recording the membrane currents. The application of each drug was driven by gravity and controlled by the corresponding valve.
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2.5. Data analysis
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Data were statistically compared using the Student’s t-test or analysis of variance (ANOVA), followed by Bonferroni’s post hoc test. Data are expressed as mean ± standard error of the mean (SEM).
Injection of 5% formalin subcutaneously in the hind paw of a mouse resulted in intense spontaneous licking or lifting of the injected paw with a classic biphasic response in the present study. The first phase began and lasted for 10 min, and then the second phase lasted from 10 to 60 min. As illustrated in Fig. 1, intrathecal administration a high dose of AVP (3 or 10 ng) significantly decreased the second (10–60 min), but not the first (0–10 min), phase of formalin-induced nociception as compared with the vehicle control (P < 0.05 or 0.01, one way ANOVA followed by post hoc Bonferroni’s test, n = 10-13). On the other hand, lower dose of AVP (1 ng) did not alter either the first or second phase of formalininduced nociception. To verify whether the effect of AVP was mediated via its receptors, SR 49059, a vasopressin-1A (V1A ) receptor antagonist, was administered intrathecally. The antinociceptive effect of AVP on the second phase of formalin-induced nociception failed to display when SR 49059 (10 ng) was co-injected together with 10 ng AVP (P < 0.01, compared with 10 ng AVP column, one way ANOVA followed by post hoc Bonferroni’s test, n = 12). However, intrathecal administration of 10 ng SR 49059 alone had no effect on formalin-evoked nociceptive behavior (Fig. 1). Together, spinal AVP dose dependently reduced the second, but not first, phase of formalin-induced spontaneous nociception, which was blocked by V1A receptor antagonist. 3.2. AVP had no effect on formalin-induced spontaneous nociception in V1A receptor knock-out (V1A -/-) mice To further address the finding that the AVP analgesia on formalin-induced spontaneous nociception was mediated by V1A receptors, we investigated the roles of AVP in specific V1A -/- mice. Similar to that observed in wild-type mice, injection of 5% formalin subcutaneously also induced spontaneous biphasic nociceptive
Nociceptive behavior time (s)
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Fig. 1. Spinal AVP dose dependently reduced formalin-induced spontaneous nociception in wild-type mice. Intrathecal (i.t.) administration of AVP dose dependently (1–10 ng) decreased spontaneous paw-licking or lifting time during the second (10–60 min), but not the first (0–10 min), phase of the formalin test. The antinociceptive effect of 10 ng AVP on the second phase of formalin-induced nociception was blocked by co-treated 10 ng SR 49059, a V1A receptor antagonist. SR 49059 alone had no effect on nociceptive behavior. * P < 0.05, ** P < 0.01, compared with vehicle control; ## P < 0.01, compared with 10 ng AVP column (one way ANOVA followed by post hoc Bonferroni’s test). Data are expressed as the total time spent in spontaneous licking or lifting of the injected paw after subcutaneous formalin (5%, 20 l) injection into the hind paw. AVP or its vehicle was injection i.t. 5 min before the injection of formalin. Each column represents the mean with S.E.M. of 10–13 mice.
Please cite this article in press as: F. Peng, et al., Spinal vasopressin alleviates formalin-induced nociception by enhancing GABAA receptor function in mice, Neurosci. Lett. (2015), http://dx.doi.org/10.1016/j.neulet.2015.03.023
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Fig. 2. AVP had no effect on formalin-induced spontaneous nociception in V1A -/- mice. A. V1A -/- mice displayed more nociceptive behavior than their wild-type littermates during the second, but not the first, phases of formalin test. * P < 0.01. B. Unlike the antinociceptive effect of AVP in WT littermates, AVP at dose of 10 ng failed to decrease the second phase of formalin-induced nociception in V1A -/- mice. Each column represents the mean with S.E.M. of 12 mice.
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behavior in V1A -/- mice. V1A -/- mice displayed more nociceptive behavior than their wild-type littermates during the second phases of formalin test, although no significant phenotype differences were detected in the first phases of formalin-induced nociception (Fig. 2A). Unlike the antinociceptive effect of AVP in wild-type littermates, AVP at dose of 10 ng failed to decrease the second phase of formalin-induced nociception in V1 A -/- mice (Fig. 2B). After intrathecal administration of 10 ng AVP, the nociceptive behavior time during the second phase of formalin test was 285.2 ± 11.2 s, which was not significantly different from 297.2 ± 16.5 s of vehicle treatment in V1A -/- mice (P > 0.1, unpaired t-test, n = 12). These results suggest that AVP-induced analgesic effect on formalininduced spontaneous nociception was absent in V1A -/- mice, but present in wild-type littermates. 3.3. The antinociceptive effect of AVP was reversed by co-treated GABAA receptor antagonist To assess whether the antinociceptive effect of AVP on formalininduced spontaneous nociception was involved endogenous pain modulatory system, we treated intrathecally bicuculline, a GABAA receptor antagonist. As illustrated in Fig. 3, bicuculline at dose of 1 g alone did not affect the first and second phases of the formalininduced nociception. Interestingly, the nociceptive behavior time during the second phase of formalin test significantly prolonged when bicuculline (1 g) was co-injected together with 10 ng AVP, compared with the injection of 10 ng AVP alone (192.4 ± 14.1 s vs. 127.6 ± 13.5 s, P < 0.01, one way ANOVA followed by post hoc Bonferroni’s test, n = 12) (Fig. 3). These results indicated that the antinociceptive effect of AVP was reversed by GABAA receptor antagonist. 3.4. AVP potentiated GABA-activated currents in wild-type littermates, but not in V1A -/- mice
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Fig. 3. The antinociceptive effect of AVP was reversed by co-treated bicuculline. The antinociceptive effect of AVP (10 ng) on the second phase formalin-induced nociception was reversed by intrathecal co-application of AVP and bicuculline, a GABAA receptor antagonist. Intrathecal administration of 1 g bicuculline alone did not affect the first and second phases of the formalin-induced nociception. ** P < 0.01 (one way ANOVA followed by post hoc Bonferroni’s test). Each column represents the mean with S.E.M. of 12 mice.
applied. However, the EC50 values of both curves had no statistical difference (51.3 ± 0.7 M vs. 50.8 ± 0.9 M). In addition, the AVP potentiation of IGABA was completely blocked by co-treatment of AVP and SR 49059 (3 × 10−6 M), a selective V1A receptor antagonist (Fig. 4A). A similar application of AVP (10−6 M) failed to increase IGABA in twelve DRG neurons tested from V1A -/- mice (Fig. 4A and C). These results indicated that AVP potentiated IGABA in wild-type littermates, but not in V1A -/- mice. 4. Discussion
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To further study the mechanism of antihyperalgesic action of AVP, we next investigated whether AVP modulated GABAA receptor-mediated currents. Extracellular application of GABA (10−4 M for 10 s) evoked a rapid inward current (IGABA ) in dissociated DRG neurons from wild-type and V1A -/- mice. Application of AVP (10−6 M) 60 s prior to GABA enhanced IGABA in a DRG neurons tested from wild-type mouse (Fig. 4A). Fig. 4B shows pretreatment of AVP (10−6 M) shifted the concentration-response curve for GABA upwards, as indicated by an increase of 56.9 ± 11.1% in the maximal current response to GABA when AVP was pre-
This study demonstrates that spinal AVP can exert an antinociceptive effect on the second phase of formalin-induced spontaneous nociception. Effects of AVP were mediated by V1A receptor and completely absent in V1A -/- mice. The AVP analgesia was also reversed by GABAA receptor antagonist, while GABAactivated currents were potentiated by AVP in mouse DRG neurons. Previous studies demonstrated that the plasma AVP levels and AVP mRNA expression in CNS were increased after subcutaneous injection of formalin into the bilateral hindpaws [23]. However, the
Please cite this article in press as: F. Peng, et al., Spinal vasopressin alleviates formalin-induced nociception by enhancing GABAA receptor function in mice, Neurosci. Lett. (2015), http://dx.doi.org/10.1016/j.neulet.2015.03.023
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Fig. 4. AVP potentiated GABA-activated currents in wild-type littermates, but not in V1A -/- mice. Representative current traces in A shows that pre-application of AVP (10−6 M) for 60 sec increased GABA-activated currents (IGABA , 10−4 M) in a DRG neuron from wild-type mice, but not in a DRG neuron from V1A -/- mice. The potentiation of IGABA by AVP pre-applied alone was abolished by the co-application of AVP and SR 49059 (3 × 10−6 M), a selective V1 A receptor antagonist. Neurons with membrane potential clamped at −60 mV. B. AVP pretreatment shifted the concentration-response curve for GABA upwards in wild-type mice. Each point represents the mean ± SEM of 10–12 neurons. Summary data in C show that pre-application of AVP failed to increase IGABA in twelve DRG neurons from V1A -/- mice.
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role of upregulated AVP after the formalin test has not been determined. The formalin test for nociception is commonly used to assess the response of animal to pain, and this model is thought to resemble clinical pain [1]. The formalin injection produces a biphasic behavior response. The first phase starts immediately after injection and lasted for 10 min, and then the second phase lasted from 10 to 60 min. The first phase is probably attributable to formalin direct stimulation of nociceptors, while the second phase involves central sensitization within the dorsal horn [25]. The present study demonstrated that spinal administration of AVP significantly reduced the second phase, but not the first phase, of the formalin-induced nociception in a dose dependent manner, consistent with previous studies [24]. AVP may not act on the nociceptive perception, since nociception during the first phase was not affected. The inhibition of the second phase of the formalin-induced nociception indicated that spinal AVP may attenuate the spinal central sensitization. In rodents, AVP analgesia is mediated by binding to V1A , V1B or V2 receptors [11,13]. In the present study, the analgesic effect of AVP failed to display when intrathecal administration of AVP together with SR 49059, a V1A receptor antagonist. Further, the spinal AVP-induced analgesia on the second phase of formalin-induced nociception was absent in V1A -/- mice. These results suggested that spinal AVP acted via V1A receptors to produce its analgesic effect in formalin-induced spontaneous nociception. Anatomical evidence demonstrated that V1A receptor is only weakly expressed in the spinal cord of adult rats [26]. In contrast, V1A receptor mRNA is reported to be abundantly expressed in small and medium diameter DRGs in mice [22]. Thus, we speculated that spinal AVP may exert its analgesic effect via V1A receptors expressed in the central terminal of primary afferent neurons. We observed that AVP analgesia on the second phase of formalin-induced nociception was significantly reversed by bicuculline, a GABAA receptor antagonist, indicating involvement of
spinal GABAergic system. GABAA receptors are enriched in the spinal dorsal horn, wherein they are localized on the terminals of small and large diameter primary afferent fibers [6,18]. It has been established that some primary sensory afferents may activate spinal interneurons and release GABA [17]. GABA activates GABAA receptors located in primary afferent terminals and then modulates the afferent input from DRG neurons into nociceptive-specific projection neurons (presynaptic inhibition) [8,9]. Indeed, the activation of spinal GABAergic circuits attenuates nociceptive process [28]. For example, intrathecal application of muscimol exhibits an antinociceptive effect in formalin test [7]. Consistent with our previous study in rats [19], AVP potentiated GABAA receptor-mediated currents in isolated mouse DRG neurons, which was completely blocked by SR 49059, a V1A receptor antagonist. AVP failed to increase GABA-activated currents in DRG neurons from V1A -/- mice, further supporting the conclusion that the enhancing GABAA receptor function by AVP was specifically mediated via V1 A receptors. Activation of V1A receptors elevate intracellular calcium and activate protein kinase C (PKC). Previous report indicated that GABAA receptor functions are attenuated by the activation of PKC [5]. However, membrane depolarization potentiates GABAergic IPSCs’ amplitude through the mechanism of Ca2+ release from internal stores in Purkinje cells of the rat [10]. Elevating intracellular calcium by activation of V1 A receptors may thus play a role in AVPinduced potentiation of GABAA receptor functions, which needs to further be verified. In developing hypoglossal motoneurons, AVP increases also spontaneous GABAergic IPSCs’ amplitude through V1A receptors [21]. Our results may reveal a novel mechanism of AVP analgesia by enhancing GABAA receptor function in primary sensory neurons via V1A receptors. If AVP increases the GABA response at the central terminal of primary afferent neurons, as it does at the soma membrane, this would enhance the GABA-induced depolarization.
Please cite this article in press as: F. Peng, et al., Spinal vasopressin alleviates formalin-induced nociception by enhancing GABAA receptor function in mice, Neurosci. Lett. (2015), http://dx.doi.org/10.1016/j.neulet.2015.03.023
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When the enhanced depolarization reached threshold value, GABA produced an excitatory effect. AVP seemed to increase the shunting effect by potentiating GABA-mediated current. When the enhanced depolarization did not reach threshold value, AVP amplified GABAA receptor-mediated presynaptic inhibition. After formalin injection, whether presynaptic inhibition switches to excitation or remains inhibitory needs further study. The current behavioral results showed that spinal AVP was analgesic on formalin-induced nociception and its effect was reversed by bicuculline after formalin injection. In conclusion, the present study demonstrates that spinal AVP significantly reduced pain responses in the second phase of the formalin test via V1A receptors. Enhancing GABAergic inhibition may be involved in the mechanism underlying the spinal effect of AVP. Conflicts of interest We have no conflict-of-interest to declare. Acknowledgements
We thank Dr. Shuang-Bao Hu (National Institute of Mental Health, Bethesda, Maryland) for providing V1aR knockout mice. 291 Q3 This work was supported by the National Natural Science Foun292 dation of China (No. 81171039, No. 31471062), Program for New 293 Century Excellent Talents in University (NCET-11-0967). 294 290
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Research article
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Spinal vasopressin alleviates formalin-induced nociception by enhancing GABAA receptor function in mice
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Fang Peng a,b , Zu-Wei Qu a , Chun-Yu Qiu a , Min Liao b,∗ , Wang-Ping Hu a,∗∗ a b
Institute of Ion Channels, Department of Pharmacology, Hubei University of Science and Technology, 88 Xianning Road, Xianning 437100, Hubei, PR China Department of Anatomy and Histology & Embryology, College of Basic Medicine, Wenzou Medical University, Wenzou 32500, Zhejiang, PR China
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h i g h l i g h t s • We have found that spinal AVP reduced formalin-induced spontaneous nociception. • Spinal AVP analgesia were mediated by V1A receptors. • Enhancing GABAergic function may be involved in the mechanism underlying spinal AVP analgesia.
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Article history: Received 5 February 2015 Received in revised form 6 March 2015 Accepted 13 March 2015 Available online xxx
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Keywords: Arginine vasopressin Vasopressin-1A receptor GABAA receptor Formalin test Pain Knock-out
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1. Introduction
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Arginine vasopressin (AVP) plays a regulatory role in nociception. Intrathecal administration of AVP displays an antinociceptive effect. However, little is understood about the mechanism underlying spinal AVP analgesia. Here, we have found that spinal AVP dose dependently reduced the second, but not first, phase of formalin-induced spontaneous nociception in mice. The AVP analgesia was completely blocked by intrathecal injected SR 49059, a vasopressin-1A (V1A ) receptor antagonist. However, spinal AVP failed to exert its antinociceptive effect on the second phase formalin-induced spontaneous nociception in V1A receptor knock-out (V1A -/-) mice. The AVP analgesia was also reversed by bicuculline, a GABAA receptor antagonist. Moreover, AVP potentiated GABA-activated currents in dorsal root ganglion neurons from wild-type littermates, but not from V1A -/- mice. Our results may reveal a novel spinal mechanism of AVP analgesia by enhancing the GABAA receptor function in the spinal cord through V1A receptors. © 2015 Published by Elsevier Ireland Ltd.
Arginine vasopressin (AVP) is involved in a wide range of physiological regulatory processes [13]. AVP has been demonstrated to play a neuromodulatory role on nociception. It is believed that AVP causes antinociception in both humans and animals [2,11]. The antinociceptive activity was observed after subcutaneous, intraperitoneal or intracerebroventricular administration of AVP [3,14]. In contrast, Brattleboro rats, which are naturally deficient in AVP, had a hyperalgesic state in flinch–jump threshold test and impaired stress analgesia [4]. Thus, AVP plays a role as an endogenous analgesic substance. Intrathecal administration of AVP displayed also an antinociceptive effect [24,27]. However,
∗ Corresponding author. ∗ ∗ Corresponding author. Tel.: +86 715 8150960; fax: +86 715 82256221. E-mail addresses: [email protected] (M. Liao), wangping [email protected] (W.-P. Hu).
another study reported that intrathecal injected AVP failed to produce hyponociception in the tail flick test [15]. The modulatory effect of AVP on nociception is mediated by binding to three distinct receptors: V1A , V1B and V2 receptors [11,13]. AVP analgesia may be mainly mediated by V1A receptors, since AVP did not alter nociceptive threshold in V1A receptor knock-out (V1A -/-) mice [16,22]. Recently, low (<500 pg) and high concentration of AVP in the blood displayed antinociceptive and pronociceptive effects, respectively. And the antinociceptive and pronociceptive effects of AVP were fully abolished in the presence of the V1A receptor antagonist [12]. To further clarify the effects of AVP in nociceptive process, we investigated again the action of spinal AVP on formalin-induced spontaneous nociception. We used also V1A -/- mice and selective V1A receptor antagonists to determine the specific receptors involved in AVP actions. Finally, we found that enhancing GABAA receptor function may be involved in the mechanism underlying spinal AVP analgesia.
http://dx.doi.org/10.1016/j.neulet.2015.03.023 0304-3940/© 2015 Published by Elsevier Ireland Ltd.
Please cite this article in press as: F. Peng, et al., Spinal vasopressin alleviates formalin-induced nociception by enhancing GABAA receptor function in mice, Neurosci. Lett. (2015), http://dx.doi.org/10.1016/j.neulet.2015.03.023
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2. Materials and methods
3. Results
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2.1. Animals
3.1. Spinal AVP dose dependently reduced formalin-induced spontaneous nociception in wild-type mice
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The experimental protocol was approved by the animal research ethics committee of Hubei University of Science and Technology. The experiments were undertaken on littermate male adult (8–12 weeks) wild-type and V1A -/- mice of C57BL/6 background as described previously [20]. All animals were kept with a 12 h light/dark cycle and with ad libitum access to food and water. 2.2. Drugs and drug administration methods
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All chemicals were purchased from Sigma–Aldrich (St. Louis, MO, USA). The drugs used were formalin, AVP, SR 49059 and bicuculline. Formalin was diluted to 5% in saline. For spinal injections, mice were briefly anesthetized with isoflurane and intrathecal (i.t) injections of drugs (5 l using a 30-gauge needle) were delivered by acute lumbar puncture between the L5 and L6 vertebrae 5 min prior to the formalin injection (by which time mice were fully recovered from the anesthetic). A characteristic tail-flick indicated successful penetration of the spinal compartment.
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2.3. Formalin-induced spontaneous nociceptive behavior
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Mice were gently restrained, and then 20 l of 5% formalin was injected subcutaneously into the plantar surface of one hind paw using a micro syringe with a 30-gauge needle. The total time spent in spontaneous nociceptive behavior (licking and lifting of the injected paw) was recorded in 5-min intervals for 1 h, starting immediately after formalin injection. The first phase was defined as 0–10 min and the second phase as 10–60 min. All behavioral experiments were performed by an observer blinded for the animal treatment. 2.4. Isolation of the DRG neurons and electrophysiological recordings
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The mouse DRG neurons were isolated as described previously [20]. Dissociated neurons were placed into a 35 mm petri dish and kept for at least another 60 min before electrophysiological recordings. The neurons selected for electrophysiological experiment were 15–35 m in diameter. Whole-cell patch clamp recording was carried out using a MultiClamp-700B amplifier and Digidata-1440A A/D converter (Axon Instruments, Foster city, CA, USA). Recording micropipettes were filled with internal solution containing KCl 140 mM, MgCl2 2.5 mM, HEPES 10 mM, EGTA 11 mM and ATP 5 mM and the pH was adjusted to 7.2 with KOH. Cells were bathed in an external solution containing NaCl 150 mM, KCl 5 mM, CaCl2 2.5 mM, MgCl2 2 mM, HEPES 10 mM and d-glucose 10 mM and the osmolarity was adjusted to 330 mOsm/L with sucrose and pH to 7.4. The resistance of the recording pipette was in the range of 3–6 M. A small patch of membrane underneath the tip of the pipette was aspirated to form a gigaseal and then negative pressure was applied to rupture it. The adjustment of capacitance compensation and series resistance compensation was done before recording the membrane currents. The application of each drug was driven by gravity and controlled by the corresponding valve.
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2.5. Data analysis
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Data were statistically compared using the Student’s t-test or analysis of variance (ANOVA), followed by Bonferroni’s post hoc test. Data are expressed as mean ± standard error of the mean (SEM).
Injection of 5% formalin subcutaneously in the hind paw of a mouse resulted in intense spontaneous licking or lifting of the injected paw with a classic biphasic response in the present study. The first phase began and lasted for 10 min, and then the second phase lasted from 10 to 60 min. As illustrated in Fig. 1, intrathecal administration a high dose of AVP (3 or 10 ng) significantly decreased the second (10–60 min), but not the first (0–10 min), phase of formalin-induced nociception as compared with the vehicle control (P < 0.05 or 0.01, one way ANOVA followed by post hoc Bonferroni’s test, n = 10-13). On the other hand, lower dose of AVP (1 ng) did not alter either the first or second phase of formalininduced nociception. To verify whether the effect of AVP was mediated via its receptors, SR 49059, a vasopressin-1A (V1A ) receptor antagonist, was administered intrathecally. The antinociceptive effect of AVP on the second phase of formalin-induced nociception failed to display when SR 49059 (10 ng) was co-injected together with 10 ng AVP (P < 0.01, compared with 10 ng AVP column, one way ANOVA followed by post hoc Bonferroni’s test, n = 12). However, intrathecal administration of 10 ng SR 49059 alone had no effect on formalin-evoked nociceptive behavior (Fig. 1). Together, spinal AVP dose dependently reduced the second, but not first, phase of formalin-induced spontaneous nociception, which was blocked by V1A receptor antagonist. 3.2. AVP had no effect on formalin-induced spontaneous nociception in V1A receptor knock-out (V1A -/-) mice To further address the finding that the AVP analgesia on formalin-induced spontaneous nociception was mediated by V1A receptors, we investigated the roles of AVP in specific V1A -/- mice. Similar to that observed in wild-type mice, injection of 5% formalin subcutaneously also induced spontaneous biphasic nociceptive
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Fig. 1. Spinal AVP dose dependently reduced formalin-induced spontaneous nociception in wild-type mice. Intrathecal (i.t.) administration of AVP dose dependently (1–10 ng) decreased spontaneous paw-licking or lifting time during the second (10–60 min), but not the first (0–10 min), phase of the formalin test. The antinociceptive effect of 10 ng AVP on the second phase of formalin-induced nociception was blocked by co-treated 10 ng SR 49059, a V1A receptor antagonist. SR 49059 alone had no effect on nociceptive behavior. * P < 0.05, ** P < 0.01, compared with vehicle control; ## P < 0.01, compared with 10 ng AVP column (one way ANOVA followed by post hoc Bonferroni’s test). Data are expressed as the total time spent in spontaneous licking or lifting of the injected paw after subcutaneous formalin (5%, 20 l) injection into the hind paw. AVP or its vehicle was injection i.t. 5 min before the injection of formalin. Each column represents the mean with S.E.M. of 10–13 mice.
Please cite this article in press as: F. Peng, et al., Spinal vasopressin alleviates formalin-induced nociception by enhancing GABAA receptor function in mice, Neurosci. Lett. (2015), http://dx.doi.org/10.1016/j.neulet.2015.03.023
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Fig. 2. AVP had no effect on formalin-induced spontaneous nociception in V1A -/- mice. A. V1A -/- mice displayed more nociceptive behavior than their wild-type littermates during the second, but not the first, phases of formalin test. * P < 0.01. B. Unlike the antinociceptive effect of AVP in WT littermates, AVP at dose of 10 ng failed to decrease the second phase of formalin-induced nociception in V1A -/- mice. Each column represents the mean with S.E.M. of 12 mice.
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behavior in V1A -/- mice. V1A -/- mice displayed more nociceptive behavior than their wild-type littermates during the second phases of formalin test, although no significant phenotype differences were detected in the first phases of formalin-induced nociception (Fig. 2A). Unlike the antinociceptive effect of AVP in wild-type littermates, AVP at dose of 10 ng failed to decrease the second phase of formalin-induced nociception in V1 A -/- mice (Fig. 2B). After intrathecal administration of 10 ng AVP, the nociceptive behavior time during the second phase of formalin test was 285.2 ± 11.2 s, which was not significantly different from 297.2 ± 16.5 s of vehicle treatment in V1A -/- mice (P > 0.1, unpaired t-test, n = 12). These results suggest that AVP-induced analgesic effect on formalininduced spontaneous nociception was absent in V1A -/- mice, but present in wild-type littermates. 3.3. The antinociceptive effect of AVP was reversed by co-treated GABAA receptor antagonist To assess whether the antinociceptive effect of AVP on formalininduced spontaneous nociception was involved endogenous pain modulatory system, we treated intrathecally bicuculline, a GABAA receptor antagonist. As illustrated in Fig. 3, bicuculline at dose of 1 g alone did not affect the first and second phases of the formalininduced nociception. Interestingly, the nociceptive behavior time during the second phase of formalin test significantly prolonged when bicuculline (1 g) was co-injected together with 10 ng AVP, compared with the injection of 10 ng AVP alone (192.4 ± 14.1 s vs. 127.6 ± 13.5 s, P < 0.01, one way ANOVA followed by post hoc Bonferroni’s test, n = 12) (Fig. 3). These results indicated that the antinociceptive effect of AVP was reversed by GABAA receptor antagonist. 3.4. AVP potentiated GABA-activated currents in wild-type littermates, but not in V1A -/- mice
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Fig. 3. The antinociceptive effect of AVP was reversed by co-treated bicuculline. The antinociceptive effect of AVP (10 ng) on the second phase formalin-induced nociception was reversed by intrathecal co-application of AVP and bicuculline, a GABAA receptor antagonist. Intrathecal administration of 1 g bicuculline alone did not affect the first and second phases of the formalin-induced nociception. ** P < 0.01 (one way ANOVA followed by post hoc Bonferroni’s test). Each column represents the mean with S.E.M. of 12 mice.
applied. However, the EC50 values of both curves had no statistical difference (51.3 ± 0.7 M vs. 50.8 ± 0.9 M). In addition, the AVP potentiation of IGABA was completely blocked by co-treatment of AVP and SR 49059 (3 × 10−6 M), a selective V1A receptor antagonist (Fig. 4A). A similar application of AVP (10−6 M) failed to increase IGABA in twelve DRG neurons tested from V1A -/- mice (Fig. 4A and C). These results indicated that AVP potentiated IGABA in wild-type littermates, but not in V1A -/- mice. 4. Discussion
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To further study the mechanism of antihyperalgesic action of AVP, we next investigated whether AVP modulated GABAA receptor-mediated currents. Extracellular application of GABA (10−4 M for 10 s) evoked a rapid inward current (IGABA ) in dissociated DRG neurons from wild-type and V1A -/- mice. Application of AVP (10−6 M) 60 s prior to GABA enhanced IGABA in a DRG neurons tested from wild-type mouse (Fig. 4A). Fig. 4B shows pretreatment of AVP (10−6 M) shifted the concentration-response curve for GABA upwards, as indicated by an increase of 56.9 ± 11.1% in the maximal current response to GABA when AVP was pre-
This study demonstrates that spinal AVP can exert an antinociceptive effect on the second phase of formalin-induced spontaneous nociception. Effects of AVP were mediated by V1A receptor and completely absent in V1A -/- mice. The AVP analgesia was also reversed by GABAA receptor antagonist, while GABAactivated currents were potentiated by AVP in mouse DRG neurons. Previous studies demonstrated that the plasma AVP levels and AVP mRNA expression in CNS were increased after subcutaneous injection of formalin into the bilateral hindpaws [23]. However, the
Please cite this article in press as: F. Peng, et al., Spinal vasopressin alleviates formalin-induced nociception by enhancing GABAA receptor function in mice, Neurosci. Lett. (2015), http://dx.doi.org/10.1016/j.neulet.2015.03.023
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Fig. 4. AVP potentiated GABA-activated currents in wild-type littermates, but not in V1A -/- mice. Representative current traces in A shows that pre-application of AVP (10−6 M) for 60 sec increased GABA-activated currents (IGABA , 10−4 M) in a DRG neuron from wild-type mice, but not in a DRG neuron from V1A -/- mice. The potentiation of IGABA by AVP pre-applied alone was abolished by the co-application of AVP and SR 49059 (3 × 10−6 M), a selective V1 A receptor antagonist. Neurons with membrane potential clamped at −60 mV. B. AVP pretreatment shifted the concentration-response curve for GABA upwards in wild-type mice. Each point represents the mean ± SEM of 10–12 neurons. Summary data in C show that pre-application of AVP failed to increase IGABA in twelve DRG neurons from V1A -/- mice.
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role of upregulated AVP after the formalin test has not been determined. The formalin test for nociception is commonly used to assess the response of animal to pain, and this model is thought to resemble clinical pain [1]. The formalin injection produces a biphasic behavior response. The first phase starts immediately after injection and lasted for 10 min, and then the second phase lasted from 10 to 60 min. The first phase is probably attributable to formalin direct stimulation of nociceptors, while the second phase involves central sensitization within the dorsal horn [25]. The present study demonstrated that spinal administration of AVP significantly reduced the second phase, but not the first phase, of the formalin-induced nociception in a dose dependent manner, consistent with previous studies [24]. AVP may not act on the nociceptive perception, since nociception during the first phase was not affected. The inhibition of the second phase of the formalin-induced nociception indicated that spinal AVP may attenuate the spinal central sensitization. In rodents, AVP analgesia is mediated by binding to V1A , V1B or V2 receptors [11,13]. In the present study, the analgesic effect of AVP failed to display when intrathecal administration of AVP together with SR 49059, a V1A receptor antagonist. Further, the spinal AVP-induced analgesia on the second phase of formalin-induced nociception was absent in V1A -/- mice. These results suggested that spinal AVP acted via V1A receptors to produce its analgesic effect in formalin-induced spontaneous nociception. Anatomical evidence demonstrated that V1A receptor is only weakly expressed in the spinal cord of adult rats [26]. In contrast, V1A receptor mRNA is reported to be abundantly expressed in small and medium diameter DRGs in mice [22]. Thus, we speculated that spinal AVP may exert its analgesic effect via V1A receptors expressed in the central terminal of primary afferent neurons. We observed that AVP analgesia on the second phase of formalin-induced nociception was significantly reversed by bicuculline, a GABAA receptor antagonist, indicating involvement of
spinal GABAergic system. GABAA receptors are enriched in the spinal dorsal horn, wherein they are localized on the terminals of small and large diameter primary afferent fibers [6,18]. It has been established that some primary sensory afferents may activate spinal interneurons and release GABA [17]. GABA activates GABAA receptors located in primary afferent terminals and then modulates the afferent input from DRG neurons into nociceptive-specific projection neurons (presynaptic inhibition) [8,9]. Indeed, the activation of spinal GABAergic circuits attenuates nociceptive process [28]. For example, intrathecal application of muscimol exhibits an antinociceptive effect in formalin test [7]. Consistent with our previous study in rats [19], AVP potentiated GABAA receptor-mediated currents in isolated mouse DRG neurons, which was completely blocked by SR 49059, a V1A receptor antagonist. AVP failed to increase GABA-activated currents in DRG neurons from V1A -/- mice, further supporting the conclusion that the enhancing GABAA receptor function by AVP was specifically mediated via V1 A receptors. Activation of V1A receptors elevate intracellular calcium and activate protein kinase C (PKC). Previous report indicated that GABAA receptor functions are attenuated by the activation of PKC [5]. However, membrane depolarization potentiates GABAergic IPSCs’ amplitude through the mechanism of Ca2+ release from internal stores in Purkinje cells of the rat [10]. Elevating intracellular calcium by activation of V1 A receptors may thus play a role in AVPinduced potentiation of GABAA receptor functions, which needs to further be verified. In developing hypoglossal motoneurons, AVP increases also spontaneous GABAergic IPSCs’ amplitude through V1A receptors [21]. Our results may reveal a novel mechanism of AVP analgesia by enhancing GABAA receptor function in primary sensory neurons via V1A receptors. If AVP increases the GABA response at the central terminal of primary afferent neurons, as it does at the soma membrane, this would enhance the GABA-induced depolarization.
Please cite this article in press as: F. Peng, et al., Spinal vasopressin alleviates formalin-induced nociception by enhancing GABAA receptor function in mice, Neurosci. Lett. (2015), http://dx.doi.org/10.1016/j.neulet.2015.03.023
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When the enhanced depolarization reached threshold value, GABA produced an excitatory effect. AVP seemed to increase the shunting effect by potentiating GABA-mediated current. When the enhanced depolarization did not reach threshold value, AVP amplified GABAA receptor-mediated presynaptic inhibition. After formalin injection, whether presynaptic inhibition switches to excitation or remains inhibitory needs further study. The current behavioral results showed that spinal AVP was analgesic on formalin-induced nociception and its effect was reversed by bicuculline after formalin injection. In conclusion, the present study demonstrates that spinal AVP significantly reduced pain responses in the second phase of the formalin test via V1A receptors. Enhancing GABAergic inhibition may be involved in the mechanism underlying the spinal effect of AVP. Conflicts of interest We have no conflict-of-interest to declare. Acknowledgements
We thank Dr. Shuang-Bao Hu (National Institute of Mental Health, Bethesda, Maryland) for providing V1aR knockout mice. 291 Q3 This work was supported by the National Natural Science Foun292 dation of China (No. 81171039, No. 31471062), Program for New 293 Century Excellent Talents in University (NCET-11-0967). 294 290
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Please cite this article in press as: F. Peng, et al., Spinal vasopressin alleviates formalin-induced nociception by enhancing GABAA receptor function in mice, Neurosci. Lett. (2015), http://dx.doi.org/10.1016/j.neulet.2015.03.023
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