European Journal of Pharmacology 734 (2014) 91–97
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Neuropharmacology and analgesia
Evidence for the participation of peripheral α5 subunit-containing GABAA receptors in GABAA agonists-induced nociception in rats Mariana Bravo-Hernández a, Luis Alberto Feria-Morales b, Jorge Elías Torres-López b,c, Claudia Cervantes-Durán a, Rodolfo Delgado-Lezama d, Vinicio Granados-Soto a, Héctor Isaac Rocha-González e,n a Neurobiology of Pain Laboratory, Departamento de Farmacobiología, Centro de Investigación y de Estudios Avanzados (Cinvestav), Sede Sur. Calzada de los Tenorios 235, Col. Granjas Coapa, 14330 México, D.F., Mexico b Centro de Investigación, División Académica de Ciencias de la Salud, Universidad Juárez Autónoma de Tabasco, 86150 Villahermosa, Tabasco, Mexico c Hospital Regional de Alta Especialidad “Dr. Juan Graham Casasús”, 86103 Villahermosa, Tabasco, Mexico d Departamento de Fisiología, Biofísica y Neurociencias, Centro de Investigación y de Estudios Avanzados, Zacatenco, México, D.F., Mexico e Sección de Estudios de Posgrado e Investigación, Escuela Superior de Medicina, Instituto Politécnico Nacional. Plan de San Luis y Díaz Mirón s/n, Col. Casco de Santo Tomas, Miguel Hidalgo, 11340 México, D.F., Mexico
art ic l e i nf o
a b s t r a c t
Article history: Received 19 December 2013 Received in revised form 11 March 2014 Accepted 22 March 2014 Available online 12 April 2014
The activation of GABAA receptor by γ-amino butyric acid (GABA) in primary afferent ﬁbers produces depolarization. In normal conditions this depolarization causes a reduction in the release of neurotransmitters. Therefore, this depolarization remains inhibitory. However, previous studies have suggested that in inﬂammatory pain, GABA shifts its signaling from inhibition to excitation by an increased GABA-induced depolarization. The contribution of peripheral α5 subunit-containing GABAA receptors to the inﬂammatory pain is unknown. The purpose of this study was to investigate the possible pronociceptive role of peripheral α5 subunit-containing GABAA receptors in the formalin test. Formalin (0.5%) injection into the dorsum of the right hind paw produced ﬂinching behavior in rats. Ipsilateral local peripheral pre-treatment ( 10 min) with exogenous GABA (0.003–0.03 mg/paw) or common GABAA receptor agonists muscimol (0.003–0.03 mg/paw), diazepam (0.017–0.056 mg/paw) or phenobarbital (1–100 mg/paw) signiﬁcantly increased 0.5% formalin-induced nociceptive behavior. The pronociceptive effects of GABA (0.03 mg/paw), muscimol (0.03 mg/paw), diazepam (0.056 mg/paw) and phenobarbital (100 mg/paw) were prevented by either the GABAA receptor antagonist bicuculline (0.01-0.1 mg/paw) or selective α5 subunit-containing GABAA receptor inverse agonist L-655,708 (0.0170.17 mg/paw). The α5 subunit-containing GABAA receptor protein was expressed in dorsal root ganglion (DRG) and dorsal spinal cord of naïve rats. The formalin injection did not modify α5 subunit-containing GABAA receptor expression. Overall, these results suggest that peripheral α5 subunit-containing GABAA receptors play a pronociceptive role in the rat formalin test. & 2014 Elsevier B.V. All rights reserved.
Chemical compounds studied in this article: Bicuculline (PubChem CID: 10237) Diazepam (PubChem CID: 3016) GABA (PubChem CID: 119) L-655,708 (PubChem CID: 5311203) Muscimol (PubChem CID: 4266) Phenobarbital (PubChem CID: 4763) Keywords: α5 Subunit-containing GABAA receptors Bicuculline GABA GABAA receptors Inﬂammatory pain L-655,708
1. Introduction Activation of neuronal γ-amino butyric acid (GABA) receptors typically results in hyperpolarization, and thus GABA is considered
Corresponding author. Tel.: þ 52 55 54 87 17 00x5126; fax:þ 52 55 56 65 46 23. E-mail addresses: [email protected]
(M. Bravo-Hernández), [email protected]
(L.A. Feria-Morales), [email protected]
(J.E. Torres-López), [email protected]
(C. Cervantes-Durán), [email protected]
(R. Delgado-Lezama), [email protected]
(V. Granados-Soto), [email protected]
(H.I. Rocha-González). http://dx.doi.org/10.1016/j.ejphar.2014.03.051 0014-2999/& 2014 Elsevier B.V. All rights reserved.
as the main inhibitory neurotransmitter in the central nervous system. However, there is evidence that stimulation of GABAA receptors may also lead to primary afferent depolarization (PAD) (Rudomin and Schmidt, 1999; Kullmann et al., 2005). GABA-mediated PAD produces voltage-sensitive Na þ channel inactivation, which, along with membrane voltage shunting, either suppresses or shortens action potential propagation or duration. Accordingly, Ca2 þ inﬂux and transmitter release decline (ÁlvarezLeefmans et al., 1998; Rudomin and Schmidt, 1999; Price et al., 2009). Furthermore, PAD is accompanied by low-threshold outward K þ efﬂux, which offsets this response (Gold et al., 1996). Under normal conditions, low-threshold afferent ﬁbers evoke PAD and presynaptic inhibition of nociceptive afferents reduces pain
M. Bravo-Hernández et al. / European Journal of Pharmacology 734 (2014) 91–97
sensation (Cervero and Laird, 1996; Cervero et al., 2003). However, PAD can not only cause presynaptic inhibition, but may under certain conditions also give rise to action potentials (Barron and Matthews, 1938; Willis, 1999). They occur when PAD reaches the threshold for generating spike activity. Then, these action potentials may propagate in an orthodromic and antidromic direction. The centripetally conducted action potentials excite the neurons normally driven by nociceptors and evoke pain and hyperalgesia. The centrifugally conducted action potentials release substances such as peptides in peripheral tissues (e.g., joints and skin), inducing neurogenic inﬂammation and hyperalgesia (Sluka et al., 1995; Lin et al., 1999; Willis, 1999; Zeilhofer et al., 2012a). GABAA receptors are heteropentameric ligand-gated chloride channels, most of which are composed of α, β, and γ subunits (Olsen and Sieghart, 2008). To date, 19 subunits have been cloned including α (1–6), β (1–3), γ (1–3), δ, ε, θ, λ and ρ (1–3) (Farrant and Nusser, 2005). GABAA receptors are the main sites of action for a numerous class of drugs including anxiolytics, anticonvulsants, benzodiazepines, barbiturates and alcohol. α5 Subunit-containing GABAA receptors can be localized in both synaptic and extrasynaptic sites (Serwanski et al., 2006; Hines et al., 2012). Extrasynaptic axonal GABAA receptors have a high sensitivity to GABA allowing ambient concentrations of GABA to modulate neuronal excitability in neurons of peripheral and central nervous system (Farrant and Nusser, 2005), and they are present in myelinated axons of peripheral nerves (Morris et al., 1983; Zeilhofer et al., 2012a). Previous reports have demonstrated that α5 subunitcontaining GABAA receptors are predominantly extra-synaptic (Farrant and Nusser, 2005; Delgado-Lezama et al., 2013; LoezaAlcocer et al. 2013) and they co-localize with CGRP-positive primary afferent terminals (Zeilhofer et al., 2012b) suggesting their participation in nociception. Based on these considerations, in this study we investigated the possible pronociceptive participation of the α5 subunit-containing GABAA receptors in formalininduced nociception.
2. Material and methods 2.1. Animals The experiments were carried out in female Wistar rats (body weight 180–200 g) of 8–10 weeks of age. Female rats were used based on the fact that previous experiments in our conditions (Wistar rats, formalin concentration 0.5% and weight range 180– 220 g) have not shown signiﬁcant differences between males and females (unpublished data). Other authors have found differences only with other rat strains, greater weight or different formalin concentrations (Gaumond et al., 2002). Animals were obtained from our own breeding facilities and had free access to food and drinking water before the experiments. All experiments followed the Guidelines on Ethical Standards for Investigation of Experimental Pain in Animals (Zimmermann, 1983) and were approved by our local Ethics Committee. In addition, every effort was made to minimize pain and suffering in animals and the number of rats used was the minimal required to obtain signiﬁcant statistical power. 2.2. Induction and measurement of nociceptive activity Nociception was assessed with the formalin test (Dubuisson and Dennis, 1977; Wheeler-Aceto and Cowan, 1991). The rats were placed in open acrylic observation chambers for 30 min to allow them to acclimatize to their surroundings. Then, they were removed for formalin administration. Rats were gently retrained while the dorsum of the hind paw was injected with 50 mL of
diluted 0.5% formalin with a 30-gauge needle. The rats were returned to the chambers and the nociceptive behavior was assessed immediately after formalin administration. Mirrors were placed in each chamber to enable unhindered observation. Nociceptive behavior was quantiﬁed as the numbers of ﬂinches of the injected paw during 1-min periods every 5 min, up to 60 min after injection. Flinching was readily discriminated and was characterized as rapid and brief withdrawal, or as ﬂexing of the injected paw. We decided to evaluate ﬂinching because it is a simple and reliable parameter of pain behavior and one producing high scores (Wheeler-Aceto and Cowan, 1991). It is known that formalininduced nociceptive behavior occurs in two phases (WheelerAceto and Cowan, 1991; Granados-Soto et al., 2010; CervantesDurán et al., 2012). The initial acute phase (0–10 min) was followed by a relatively short quiescent period, which was then followed by a prolonged tonic response (15–60 min). At the end of the experiment, the rats were sacriﬁced in a CO2 chamber. 2.3. Western blot Rats were sacriﬁced by decapitation. The lumbar segment of the spinal cord as well as ipsilateral and contralateral dorsal root ganglia (L4–L6) were excised, placed on ice-cold isotonic saline solution and cleaned from the surrounding tissue. The dorsal and ventral parts of the spinal cord were gently marked unilaterally by a scalpel incision to enable the ipsilateral (injured) and contralateral (uninjured) sides to be identiﬁed. Excised tissues were dropped into liquid nitrogen for 1 min and then stored in a freezer ( 70 1C). Tissues were homogenized in ice-cold lysis buffer (150 mM NaCl, 50 mM Tris–HCl, 5 mM EDTA, 1 mM PMSF, 10 μg/mL aprotinin, 10 μg/mL leupeptin, 10 μg/mL pepstatin A and 0.1% Triton X-100 during 30 min at 4 1C). After that, they were centrifuged and the supernatant fraction was used to measure protein concentration by the Bradford's method (BioRad, Hercules, CA). Sixty micrograms of protein were resolved by denaturing by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene diﬂuoride membranes (PVDF). The membranes were blocked with 5% non-fat milk in phosphatebuffered saline at pH 7.4 (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 2 mM KH2PO4) with Tween 0.05% and they were incubated with goat anti-α5 subunit-containing GABAA receptors (1:500, Santa Cruz Biotechnology Inc, Santa Cruz, CA). Horseradish peroxidase-conjugated secondary antibody (1:2000, Invitrogen, Life Technologies, Carlsbad, CA) was applied for detecting the primary antibody. The signal of the α5 subunit-containing GABAA receptors was detected using an enhanced chemiluminescence detection system according to the manufacturer´s instructions (Millipore, Billerica, MA). Blots were stripped and incubated with a monoclonal antibody directed against β-actin, which was used as an internal control to normalize α5 subunit-containing GABAA receptors protein expression levels. 2.4. Drugs GABA (γ-amino butyric acid), muscimol (5-(aminomethyl)-2,3dihydro-1,2-oxazol-3-one), diazepam (7-chloro-1-methyl-5-phenyl2,3-dihydro-1H-1,4-benzodiazepin-2-one), phenobarbital (5-ethyl5-phenyl-1,3-diazinane-2,4,6-trione), bicuculline ((6R)-6-[(5S)6-methyl-7,8-dihydro-5H-[1,3]dioxolo[4,5-g]isoquinolin-5-yl]-6Hfuro[4,3-g][1,3]benzodioxol-8-one), muscimol (3-hydroxy-5-aminomethyl-isoxazole) and L-655,708 (ethyl (S)-11,12,13,13a-tetrahydro7-methoxy-9-oxo-9H-imidazo[1,5-a]pyrrolo[2,1-c][1,4]benzodiazepine1-carboxylate) were purchased from Sigma-Aldrich (St. Louis, MO). All of them were diluted in 20% dimethyl sulfoxide (DMSO). Formalin was diluted in saline.
M. Bravo-Hernández et al. / European Journal of Pharmacology 734 (2014) 91–97
Table 1 Binding afﬁnity (pKi) or efﬁcacy (EC50) constants of the compounds used in the present study at GABAA and α5GABAA receptor subtypes. DRUGS
All the experiments were analyzed by one-way analysis of variance (ANOVA) followed by the Tukey's test for post-hoc comparison. P values less than 0.05 (Po0.05) were considered as signiﬁcant.
Agonist GABA Muscimol Diazepam Phenobarbital
7.6a (pKi) 8.1a (pKi) 9.8c,d (pKi) 300e (EC50 [μM])
8.0a (pKi) 8.5a (pKi) 32f (EC50 [nM]) 300e (EC50 [μM])
Antagonist Bicuculline L-655,708
5.7a (pKi) 7.0b (pKi)
5.3a (pKi) 9.3b (pKi)
Ebert et al. (1997). Quirk et al. (1996). c Berezhnoy et al. (2004). d Gu et al. (1993). e Rho et al. (1996). f Ramerstorfer et al. (2010). b
The GABAergic agonists and antagonists were selected based on their selectivity for the GABAA receptor (Table 1) and in pilot studies in our laboratory. 2.5. Experimental design The rats received a subcutaneous (s.c.) injection (50 μL) of vehicle or increasing doses of GABAergic receptor agonists as follows: GABA (0.003–0.03 μg/paw), muscimol (0.003–0.03 μg/paw), diazepam (0.017–0.056 μg/paw) and phenobarbital (1–100 μg/paw) 10 min before 0.5% formalin injection into the dorsal surface of the right hind paw. To determine whether the drugs acted locally, these were injected individually into the left paw (contralateral) 10 min before 0.5% formalin injection into the right paw and the nociceptive behavior was assessed. In order to assess the participation of peripheral GABAA receptors as well as the α5 subunit-containing GABAA receptors in the pronociceptive effects observed with GABA, muscimol, diazepam or phenobarbital, the maximal effective dose of each agonist was coadministered with a per se ineffective dose of the selective GABAA receptor antagonist bicuculline (0.01-0.1 μg/paw) or the selective α5 subunit-containing GABAA receptor antagonist L-655,708 (0.017-0.17 μg/paw, Quirk et al., 1996) 10 min before 0.5% formalin injection into the dorsal surface of the right hind paw. Western blot analysis was used to detect α5 subunit-containing GABAA receptor protein levels in the dorsal part of the spinal cord and L4-L6 DRG. The rats were injected with 0.5% formalin into the dorsum of the right hind paw and then were sacriﬁced by decapitation with a guillotine at 1 h post formalin injection.
3. Results 3.1. Formalin-induced nociception Subcutaneous formalin injection into the right hind paw produced a typical pattern of ﬂinching behavior characterized by a biphasic time course (Fig. 1) separated by a relative pain-free interval (Dubuisson and Dennis, 1977). Phase 1 began immediately after formalin administration and declined gradually in 10 min approximately. Phase 2 began about 15 min after formalin administration and lasted about 1 h as previously reported (Rocha-González et al., 2005; Castañeda-Corral et al., 2011; Cervantes-Durán et al., 2012).
3.2. Pronociceptive effect of GABAA receptor agonists Local peripheral ipsilateral, but not contralateral, pre-treatment ( 10 min) with GABA, muscimol, diazepam and phenobarbital signiﬁcantly (P o0.05) increased 0.5% formalin-induced nociception during phase 2 (Fig. 1). Furthermore, GABA, muscimol, diazepam and phenobarbital produced a signiﬁcant (P o0.05) and dose-dependent pronociceptive effect (Fig. 2). Since the drugs had an effect only on phase 2, phase 1 was not considered in the subsequent experiments.
3.3. Effect of bicuculline and L-655,708 on the pronociceptive effects of GABAA receptor agonists Local peripheral ipsilateral pre-treatment ( 10 min) with GABA (0.03 μg/paw), muscimol (0.03 μg/paw), diazepam (0.056 μg/paw) and phenobarbital (100 μg/paw) signiﬁcantly (Po0.05) increased 0.5% formalin-induced pronociceptive effect. Bicuculline (0.010.1 μg/paw, Fig. 3) or L-655,708 (0.017-0.17 μg/paw, Fig. 4) completely prevented (Po0.05) the pronociceptive effect of each drug. It must be emphasized that the greatest doses of bicuculline or L-655,708 used in this protocol did not modify per se formalininduced nociceptive behavior.
2.6. Data analysis and statistics Behavioral data are presented as mean7S.E.M. for 6 animals. Curves were constructed by plotting the number of ﬂinches as a function of time. The area under the number of ﬂinches against the time (AUC), an expression of the duration and intensity of the effect, was calculated by trapezoidal rule. Dose–response curves for each compound tested were established based on the percent of maximum possible effect (expressed as % of nociception) calculated by the AUC of phase 1 and phase 2 of each individual rat: % Nociception ¼ ðPost compoundn100Þ=vehicleÞ Western blot data are presented as mean7S.E.M. for 3 animals. Data were normalized against β-actin.
Fig. 1. Time course of ﬂinching behavior observed after the local peripheral pretreatment with GABAA receptor agonists GABA and muscimol in rats submitted to the 0.5% formalin test. Data are expressed as the mean number of ﬂinches per min7 S.E.M. for six animals. *Signiﬁcantly different (Po 0.05) from the group treated with 0.5% formalin (0.5% F), as determined by repeated measures two-way analysis of variance followed by the Tukey’s test.
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Fig. 4. Effect of the local peripheral pre-treatment with the selective α5 subunitcontaining GABAA receptor inverse agonist L-655,708 on the pronociceptive effect induced by local peripheral GABA, muscimol, diazepam or phenobarbital during phase 2 of the 0.5% formalin test (0.5% F). Data are expressed as % of nociception. Bars are the mean 7 S.E.M. for 6 animals.nSigniﬁcantly different (Po 0.05) from the vehicle group (Veh) and #signiﬁcantly different (Po 0.05) from its respective agonist group, as determined by one-way analysis of variance followed by the Tukey´s test. Vehicle: 20% dimethyl sulfoxide þ 0.5% F; GABA: 0.03 μg/paw; Muscimol: 0.03 μg/paw; Diazepam: 0.056 μg/paw; Phenobarbital: 100 μg/paw; L-655,708 (L-655): 0.017–0.17 μg/paw. CL: contralateral.
Fig. 2. Dose-response curves obtained after local peripheral pre-treatment with the GABAA receptor agonists GABA, muscimol, diazepam and phenobarbital during phase 1 (A) and 2 (B) of the 0.5% formalin test. Data are expressed as % pronociception versus dose. Data are the means 7S.E.M. of at least six animals. n Signiﬁcantly different (Po 0.05) from the group treated with 20% DMSO, as determined by one-way analysis of variance followed by the Tukey’s test. CL: contralateral, Veh: vehicle.
interest to determine the α5 subunit-containing GABAA receptors level in the DRG and spinal cord of naïve and formalin-treated rats. Western blot analysis revealed a band of about 55 KDa corresponding to the molecular weight expected for the α5 subunit-containing GABAA receptor protein in both tissues in naïve animals. Formalin injection did not modify the expression of the 55 kDa α5 subunit-containing GABAA receptor protein, as compared to the naïve group, in the dorsal part of the spinal cord nor DRG at 1 h postinjection (Fig. 5).
Fig. 3. Effect of the local peripheral pre-treatment with the GABAA receptor antagonist bicuculline on the pronociceptive effect induced by local peripheral GABA, muscimol, diazepamal during phase 2 of the 0.5% formalin test (0.5% F). Data are expressed as % of nociception. Bars are the mean7S.E.M. for 6 animals. nSigniﬁcantly different (Po0.05) from the vehicle group (Veh) and #signiﬁcantly different (Po0.05) from its respective agonist group, as determined by one-way analysis of variance followed by the Tukey’s test. Vehicle: 20% dimethyl sulfoxideþ0.5% F; GABA: 0.03 μg/paw; Muscimol: 0.03 μg/paw; Diazepam: 0.056 μg/paw; Phenobarbital: 100 μg/paw; Bicuculline (Bic): 0.01–0.1 μg/paw. CL: contralateral.
3.4. Expression of and spinal cord
α5 subunit-containing GABAA receptors in DRG
Based on the results obtained with the α5 subunit-containing GABAA receptor inverse agonist L-655,708, it was considered of
We have observed that local peripheral injection of the GABAA receptor agonists GABA, muscimol, diazepam and phenobarbital are able to increase 0.5% formalin-induced nociception during phase 2. Our study agrees with previous observations showing that local peripheral injection of muscimol enhances formalininduced nociceptive behaviors (Carlton et al., 1999), and exacerbates inﬂammation-induced decrease in nociceptive threshold (Anseloni and Gold, 2008) in the rat. Furthermore, there is evidence that barbiturates and benzodiazepines produce hyperalgesia in intact or formalin-treated rats (Carlsson and Jurna, 1986; Carmody et al., 1986; Franklin and Abbott, 1993; Tatsuo et al., 1997). In support of our data, electrophysiological studies suggest that GABA and muscimol, injected intravenously, enhance the excitability of primary afferents and amplitude of dorsal root reﬂexes (Polc, 1979), and increase electrical excitability in a subset (40%) of C-ﬁbers in human sural nerve fascicles (Carr et al., 2010). Furthermore, these drugs, superfused over exposed tail skin, evoke excitation of nociceptive afferents (Ault and Hildebrand, 1994). In addition, GABA produces membrane depolarization in DRG preparations and increases current in neurons freshly isolated from rat DRG (Si et al., 1997). In a similar way, muscimol causes an increase in the frequency of small amplitude spontaneous dorsal root action potentials (Bagust and Willis, 2002), depolarizes glycinergic pre-synaptic nerve terminals (Jang et al., 2002) and increases the frequency of spontaneous excitatory postsynaptic currents in rats (Jang et al., 2006). Taken together, data suggest
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Fig. 5. Representative blots obtained from α5 subunit-containing GABAA receptors in ipsilateral (ID) and contralateral (CD) dorsal spinal cord (A), as well as in ipsilateral (IL) and contralateral (CL) dorsal root ganglia L4–L6 (DRG) (B) from naïve rats (N) and 1 h after 0.5% formalin (0.5% F) injection. Bar graphs show the α5 subunit-containing GABAA receptor protein expression (C–D). Data were normalized against β-actin and are expressed as the mean 7 S.E.M. of three independent rats. nThere is no signiﬁcant differences (Po 0.05) from the naïve group, as determined by student’s t-test.
that GABAA receptor agonists produce nociceptive and excitatory effects at the periphery. The peripheral pronociceptive effects of the GABAA receptor agonists were fully prevented by the ipsilateral, but not contralateral, local peripheral administration of the GABAA receptor antagonist bicuculline, indicating that the pronociceptive effects may be related to peripheral GABAA receptors. Of note, the pronociceptive effects of the GABAA receptor agonists were also prevented by the selective α5 subunit-containing GABAA receptor inverse agonist L-655,708, suggesting that peripheral α5 subunitcontaining GABAA receptors participate in the pronociceptive effects of GABA, muscimol, diazepam and phenobarbital in the formalin test. Our study agrees with a recent report, which demonstrates that systemic administration of the selective α5 subunit-containing GABAA receptors inverse agonist (α5IA-II) attenuates formalin-induced nociception (Munro et al., 2011). As far as we can determine, it is the ﬁrst report showing that peripheral α5 subunit-containing GABAA receptors participate in GABA-induced pronociception in the formalin test. Accordingly, we have provided evidence that extrasynaptic α5 subunitcontaining GABAA receptors mediate a tonic state of excitability of primary afferents in the turtle, and that blockade of the α5 subunit-containing GABAA receptors with L-655,708 depresses the dorsal root reﬂex without affecting the phasic increase in excitability of primary afferents (Loeza-Alcocer et al., 2013). The pronociceptive effect of GABAA receptor agonists might result from activation of peripheral α5 subunit-containing GABAA receptors, which in turn could depolarize peripheral primary afferent terminals leading to generation of action potentials in C and Aδ ﬁbers, and consequent increasing of formalin-induced nociception. In support of this, there is evidence that GABA and muscimol increase the amplitude of depolarizing responses as well as the amplitude of chloride current in DRG (Akasu, 1988). Furthermore, these drugs have higher afﬁnity for the α5 subunit-containing GABAA receptors than for other α subunit-containing GABAA receptors (Table 1).
GABAA receptor agonists may also act at peripheral α5 subunitcontaining GABAA receptors facilitating neuropeptide release from primary afferent terminals as suggested previously for the GABAA receptors (Lao and Marvizon, 2005). A mixture of prostaglandin E2, bradykinin, and histamine potentiated GABA-induced currents in a subpopulation of medium-to-large diameter capsaicin insensitive DRG neurons (Lee and Gold, 2012). Since many of these inﬂammatory mediators are released during the nociceptive process induced by formalin (Parada et al., 2001; Oliveira-Fusaro et al., 2012), it is likely that the observed effect of GABAA receptor agonists may also result from this effect. Admittedly, the source of GABA at the periphery is unknown. However, GABA has been identiﬁed in human peripheral blood monocyte-derived macrophages (Stuckey et al., 2005), macrophages of inﬂamed skin of psoriatic patients (Nigam et al., 2010) as well as in dermal ﬁbroblasts (Ito et al., 2007). Additionally, macrophages also produce ß-alanine, an agonist of the GABAA receptors (Jones et al., 1998). Moreover, GABA might be glutamate-containing primary afferent ﬁbers. This amino acid is present in more than 90% of peptidergic afferent primary ﬁbers (Battaglia and Rustioni, 1988) and is converted by glutamic acid decarboxylase into GABA (Malcangio and Bowery, 1996). Thus, after inﬂammatory injury produced by formalin, GABA could be released by macrophages or ﬁbroblasts in the micro-enviroment of the peripheral afferent terminal. In this site, GABA could activate α5 subunit-containing GABAA receptors present at the peripheral afferent terminal, generating action potentials analogous to the dorsal root reﬂexes that arise following activation of GABAA receptors on central primary afferent terminals, and the subsequent nociception. The expression of α5 subunit-containing GABAA receptors was observed in the spinal cord and DRG of naïve and formalin-treated rats. This is the ﬁrst report about the protein expression of the α5 subunit-containing GABAA receptors in rats. However, our data agree with previous studies showing the mRNA for the α5 subunitcontaining GABAA receptors is expressed in DRG of naïve and axotomized rats (Ma et al., 1993; Maddox et al., 2004; Yang et al., 2004).
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Our study is also in accord with data showing the α5 subunitcontaining GABAA receptors immunoreactivity in laminae I–V and within primary afferent terminals (Knabl et al., 2008; Paul et al., 2012). Taken together, data suggest that α5 subunit-containing GABAA receptors are present in DRG, an important site for nociceptive processing. A previous study reported that β2/β3 and α1 subunits of the GABAA receptors are present on 10–14% of the unmyelinated primary afferent axons in the glabrous skin of the cat paw (Carlton et al., 1999). Others have reported the presence of α2 subunits of the GABAA receptors (Persohn et al., 1991; Witschi et al., 2011). Our study extends these observations by demonstrating the presence of α5 subunit-containing GABAA receptors in DRG and dorsal spinal cord. Besides the pronociceptive effect of local peripheral GABAA agonists, other authors have reported that peripheral muscimol (at low doses) attenuates formalin- or prostaglandin E2-induced nociception (Carlton et al., 1999; Reis et al., 2007). In addition, local peripheral phenobarbital reverses formalin-induced secondary hyperalgesia (da Motta et al., 2004). Systemic administration of diazepam or muscimol and other GABAA receptor agonists reduces behaviors in several nociceptive paradigms including tail ﬂick, tail immersion, hot plate, paw pressure, inﬂammation, writhing and grid shock tests. Furthermore, these drugs produce antinociception after systemic, spinal and supra-spinal administration (Sawynok, 1987; Munro et al., 2008; Hansen et al., 2012). This discrepancy can be explained by the fact that primary afferent neurons have the chloride equilibrium potential that is more positive than the axonal membrane potential (Gallagher et al., 1978; Álvarez-Leefmans et al., 1988,1998). Thus, activation of peripheral GABAA receptors, by low doses of GABAA receptor agonists, in these cells leads to depolarization and shunting of central primary afferent terminals resulting in pre-synaptic inhibition, and a reduction in the amount of neurotransmitter released from these terminals (Eccles et al., 1963; Carlton et al., 1999; Willis, 1999; Cervero et al., 2003). As the concentration of GABA or GABAA receptor agonists increases, stimulation of GABAA receptors on primary afferents would depolarize the peripheral terminals even more, and thus would cause the appearance of action potentials and nociception (Eccles et al., 1963; Willis, 1999; Cervero et al., 2003). Here we propose that α5 subunit-containing GABAA receptors could be responsible for such effects. 4.1. Conclusion L-655,708 prevented the pronociceptive effect of GABAA receptor agonists in the rat formalin test. In addition, peripheral α5 subunit-containing GABAA receptors were found at sites related to nociceptive processing. These results suggest that peripheral α5 subunit-containing GABAA receptors contribute to formalininduced inﬂammatory pain.
Acknowledgments Authors greatly appreciate the technical assistance of Guadalupe C. Vidal Cantú. This work was part of the M.Sc. dissertation of Luis Alberto Feria-Morales. Mariana Bravo-Hernández and Claudia Cervantes-Durán are Conacyt fellows. Partially supported by Conacyt CB-2012/179294 (VG-S), Conacyt 154880 (HIR-G) and SIP-20140107 (HIR-G) grants. References Akasu, T., 1988. 5-Hydroxytryptamine facilitates GABA-induced depolarization in bullfrog primary afferent neurons. Neurosci. Lett. 92, 270–274.
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