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The role of α2 adrenoceptor in mediating noradrenaline action in the ventrolateral orbital cortex on allodynia following spared nerve injury
Experimental Neurology 248 (2013) 381–386
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The role of α2 adrenoceptor in mediating noradrenaline action in the ventrolateral orbital cortex on allodynia following spared nerve injury Juan-Xia Zhu a,d,1, Feng-Yi Xu a,1, Wen-Jin Xu a, Yan Zhao b, Chao-Ling Qu a, Jing-Shi Tang a, Devin M. Barry c, Jian-Qing Du a, Fu-Quan Huo a,⁎ a
Department of Physiology and Pathophysiology, Xi'an Jiaotong University College of Medicine, Yanta Road W. 76#, Xi'an, Shaanxi 710061, PR China Department of Forensic Medicine, Xi'an Jiaotong University College of Medicine, Yanta Road W. 76#, Xi'an, Shaanxi 710061, PR China Department of Anesthesiology, Washington University School of Medicine, St. Louis, MO 63110, USA d Department of Physiology, Xi'an Medical University, Xinwang Road 1#, Xi'an, Shaanxi 710021, PR China b c
a r t i c l e
i n f o
Article history: Received 24 May 2013 Revised 5 July 2013 Accepted 11 July 2013 Available online 18 July 2013 Keywords: Noradrenaline α2 adrenoceptor GABAergic modulation Ventrolateral orbital cortex Allodynia Neuropathic pain
a b s t r a c t The present study examined the role of α2 adrenoceptor in mediating noradrenaline action in the ventrolateral orbital cortex (VLO) on allodynia induced by spared nerve injury (SNI) in the rat. The mechanical paw withdrawal threshold (PWT) was measured using von-Frey filaments. Microinjection of noradrenaline (1, 2, 4 μg in 0.5 μl) into the VLO, contralateral to the site of nerve injury, reduced allodynia; PWT increased in a dose-dependent manner. Similar to noradrenaline, microinjection of selective α2 adrenoceptor agonist clonidine into the same VLO site also reduced allodynia, and was blocked by selective α2 adrenoceptor antagonist yohimbine. Furthermore, administration of γ-aminobutyric acid A (GABAA) receptor antagonist bicuculline or picrotoxin to the VLO significantly enhanced clonidine-induced inhibition of allodynia, while GABAA receptor agonist muscimol or THIP (2,5,6,7-retrahydroisoxazolo(5,4-c)pyridine-3-ol hydrochloride) attenuated clonidine-induced inhibition. These results suggest that noradrenaline acting in the VLO can potentially reduce allodynia induced by SNI, and this effect is mediated by α2 adrenoceptor. Moreover, GABAergic disinhibition may participate in α2 receptor mediating effects in neuropathic pain in the central nervous system. © 2013 Elsevier Inc. All rights reserved.
Introduction Previous studies in our laboratory have demonstrated that electrolytic lesions of or microinjection of γ-aminobutyric acid (GABA) into the ventrolateral orbital cortex (VLO) eliminates antinociceptive effects induced by peripheral electrical stimulation, or by activation of the thalamic nucleus submedius (Sm) (Zhang et al., 1995, 1998b, 1999). However, electrically or chemically induced activation of the VLO depresses tail flick and jaw-opening reflexes. These antinociceptive effects are eliminated by lesion or functional blocking of the periaqueductal gray (PAG) (S. Zhang et al., 1997; Y.Q. Zhang et al., 1997, Zhang et al., 1998a). These data suggest that the VLO is involved in an endogenous analgesic system consisting of a spinal/medulla cord–Sm–VLO–PAG–spinal/medulla cord loop (Tang et al., 2009).
Abbreviations: Bicuculline, (+)-bicuculline, (S), 9(R); Clonidine, clonidine hydrochloride; GABA, γ-aminobutyric acid; Muscimol, muscimol hydrochloride; Noradrenaline, L(−)-norepinephrine (+)-bitartrate salt monohydrate; PAG, periaqueductal gray; PWT, paw withdrawal threshold; Sm, nucleus submedius; SNI, spared nerve injury; THIP, 2,5,6,7-retrahydroisoxazolo(5,4-c)pyridine-3-ol hydrochloride; Yohimbine, yohimbine hydrochloride; VLO, ventrolateral orbital cortex. ⁎ Corresponding author. Fax: +86 29 82656364. E-mail address: [email protected] (F.-Q. Huo). 1 These authors have contributed equally to this work. 0014-4886/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.expneurol.2013.07.004
Noradrenaline (or norepinephrine), a principal monoaminergic neurotransmitter in the central nervous system, is synthesized in distinct brainstem nuclei that project widely throughout the central nervous system and is involved in various physiological functions, including regulation of blood pressure, pain, locomotion, stress reaction, attention, arousal and cognitive processes (Arnsten et al., 1996; Berridge and Waterhouse, 2003; Drouin et al., 2002; Flavin and Winder, 2013; Hein and Kobilka, 1997; Kable et al., 2000; Maldonado, 1997; Raja, 1995; Robbins, 1984). The actions of noradrenaline are mediated by α1, α2 and β adrenoceptors (Bylund et al., 1994). α1 adrenoceptors are coupled to phospholipase C through G-protein (Gq) or they are coupled directly to Ca2+ influx and produce the excitatory effect on neurons (Summers and McMartin, 1993). In contrast, activation of α2 adrenoceptors decreases intracellular adenylyl cyclase activity through G-protein (Gi) or directly modifies activity of ion channels such as the Na+/H+ antiport, Ca2+ channels, or K+ channels (Summers and McMartin, 1993) resulting in hyperpolarization. β-adrenoceptors increase adenylyl cyclase activity through Gs-protein (Gs) and produce the excitatory effect on neurons (Summers and McMartin, 1993). Previous studies have demonstrated that α2 adrenoceptors play a key role in mediating pain regulatory effects of noradrenaline, whereas β-adrenoceptors may predominantly mediate epinephrine-induced modulation of pain (Pertovaara, 2006). Anatomic studies have indicated that the cerebral cortex including the VLO receives innervations from noradrenergic nuclei of the pons (Cooper et al., 2003)
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and α2 adrenoceptors are widely distributed throughout the brain including the VLO (Aantaa et al., 1995; Alburges et al., 1993; Buëcheler et al., 2002; Day et al., 1997; Holmberg et al., 2003; Scheinin et al., 1994; Unnerstall et al., 1984). Therefore, the aim of the present study was to examine whether noradrenaline and α2 adrenoceptors were involved in mediating the VLO-evoked inhibition effect on allodynia induced by spared nerve injury (SNI) in the rat. It is well known that activation of α2 adrenoceptors causes hyperpolarization and directly produces inhibitory action on neurons as abovementioned. How does noradrenaline via α2 adrenoceptors mediate this inhibition effect on allodynia in the VLO? We speculate that noradrenaline directly activates the α2 adrenoceptors to inhibit the inhibitory GABAergic interneurons which tonically inhibit the VLO projecting to the PAG neurons (disinhibition), such as other monoaminergic receptors (5-HT1A and D2-dopamine receptors) (Dang et al., 2010; Huo et al., 2008, 2010), leading to activation of the PAG–brainstem descending inhibitory system which depresses the nociceptive inputs at the spinal cord level. So, the mechanisms of GABAergic modulation were examined by observing the effects of GABAA receptor antagonists and agonists on α2 adrenoceptormediated anti-allodynia in the VLO of a rat model of SNI-induced neuropathic pain.
to mechanical stimulation (von Frey filaments) using the up–down method (Chaplan et al., 1994; Dixon, 1980). The rats were placed in a transparent plastic box (280 × 250 × 210 mm3) with a metal wire mesh floor, which allowed full access to the paws from below. Behavioral adaptation was allowed until cage exploration and major grooming activities had ceased for approximately 30 min. Ten von Frey filaments (Stoelting Company, Wood Dale, IL, USA), with approximately equal logarithmic incremental (0.17) bending force, were chosen (von Frey numbers: 3.61, 3.84, 4.08, 4.17, 4.31, 4.56, 4.74, 4.93, 5.07 and 5.18, equivalent to: 0.4, 0.6, 1.0, 1.4, 2.0, 4.0, 6.0, 8.0, 10, and 15.0 g, respectively). Starting with filament 4.31 (2.0 g), which was the middle filament in the series, von Frey filaments with different intensities were repeatedly applied over a 2-s time interval from below and perpendicular to the fourth and fifth toes of the hind paw with sufficient force to cause slight bending against the paw for approximately 6–8 s. If response to filament stimulation was positive, the next lowest force was delivered. If there was no withdrawal response (negative), the next highest force was delivered. Positive and negative responses were recorded and converted to a 50% threshold using a formula provided by Dixon (1980) and Chaplan et al. (1994). PWT measurements were performed in 10-min intervals over a 60-min observation period. If PWT was reduced to b 4.0 g, mechanical allodynia was considered to be successfully established.
Materials and methods Intracerebral microinjection of drugs Animals Male Sprague–Dawley rats (220–250 g) were provided by the Experimental Animal Center of Shaanxi Province, PR China. The experimental protocol was approved by the Institutional Animal Care Committee of Xi'an Jiaotong University, and was in accordance with ethical guidelines of the International Association for the Study of Pain (Zimmermann, 1983). All efforts were made to minimize the number of animals used and their suffering. Spared sciatic nerve injury Spared nerve injury (SNI) was performed as previously described (Arsenault and Sawynok, 2009; Decosterd and Woolf, 2000). Briefly, the rats were intraperitoneally (i.p.) anesthetized with sodium pentobarbital (50 mg/kg, SCRC, Shanghai, China), and an incision was made along the back of the thigh. The biceps femoris muscle was separated to expose the three terminal branches of the sciatic nerve. The tibial and common peroneal nerves were tightly ligated using 5.0 silk and were transected distal to the ligation, following removal of 5 mm of nerve stump. The sural nerve was left intact, and the wound was closed. Following surgery, the rats were allowed to recover for 1 week before implantation of intracerebral cannulas.
Rats were lightly anesthetized with enflurane (Baxter Caribe, Guayama, PR, USA), a very short-acting inhalation anesthetic (about 1–2 min), and a 1 μl microsyringe with the tip extending 2 mm beyond the end of the guide cannula, was inserted into the VLO through the guide cannula. Over a 60-s period, 0.5 μl of drug dissolved in saline was slowly infused at constant speed, and effects on allodynia were observed over a 60 min period following drug infusion. Experiments were performed 2 times in the same animal over a time interval of 3 days. Values (n) from each treated group were obtained from different experiments. The following drugs were used in this study: non-selective adrenoceptor agonist L-(−)-norepinephrine (+)-bitartrate salt monohydrate (noradrenaline), selective α2 adrenoceptor agonist clonidine hydrochloride (clonidine) and antagonist yohimbine hydrochloride (yohimbine), GABAA receptor antagonists (+)-bicuculline, (S), 9(R) (bicuculline) and picrotoxin and agonists muscimol hydrochloride (muscimol) and 2,5,6,7-retrahydroisoxazolo(5,4-c)pyridine-3-ol hydrochloride (THIP) (RBI/Sigma, St. Louis, MO, USA). All drugs were dissolved in physiological saline. The agonist was injected into the VLO, contralateral to the affected hind paw, and the antagonist was administered 5 min prior to the agonist. The effective drug doses were chosen according to previous studies (Dang et al., 2010; Huo et al., 2008; Ortiz et al., 2007) and our preliminary experiment. Equal volumes of saline were injected into the VLO and served as the vehicle controls.
Intracerebral guide cannula placement Histology Rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.), and the head was immobilized in a stereotaxic frame. A small craniotomy was performed just above the VLO, contralateral to the site of nerve injury. A stainless steel guide cannula (0.8 mm in diameter) was stereotaxically inserted, with the tip 2.0 mm dorsal to the VLO at the following coordinates: 3.2 mm anterior to bregma, 2.0 mm lateral, and 4.6 mm below the cortical surface (Paxinos and Watson, 1986), followed by attachment to the skull with three microscrews and dental cement. Once the rats recovered from anesthesia, they were administered sodium penicillin (0.2 million U/day for 3 days, i.p.) to prevent wound and intracerebral infections. The rats were carefully nursed and fed in clean cages.
At the end of the experiment, the drug injection sites were marked by injecting Pontamine Sky Blue dye (0.5 μl, 2% in 0.5 M sodium acetate solution). Under deep anesthesia, the rats were transcardially perfused with 0.9% normal saline, followed by 10% formalin. The brains were then removed and fixed in 10% formalin solution for 3–7 days, then placed in 30% sucrose solution/0.1 M phosphate buffer (pH 7.4) as a cryprotectant overnight. Subsequently, the brains were cut into 35-μm thick sections using a freezing microtome, and then the slices were stained with Cresyl Violet. The injection sites were histologically identified to be within the VLO for data analysis, as shown in Fig. 1.
Mechanical paw withdrawal threshold measurement
Data analysis
One week after intracerebral guide cannula placement (i.e. 2 weeks after SNI), paw withdrawal threshold (PWT) was measured in response
All values are expressed as mean ± SEM. Differences in total observation time, as well as at each time point, were statistically tested
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indicated that there was significant difference between treatments (F(5, 145) = 22.511, P b 0.001), across time (F(5, 145) = 45.701, P b 0.001), and their interaction (F(15, 145) = 12.467, P b 0.001) (Fig. 3A). The detailed comparisons at individual time points between various groups are shown in Fig. 3A. Microinjection of selective α2 adrenoceptor antagonist yohimbine alone into the VLO did not influence allodynia induced by SNI. However, when yohimbine association with clonidine was applied, the clonidineinduced inhibition of allodynia was eliminated; PWT reduced to the saline control level (Figs. 3B and D). Influence of microinjection of GAGAA agonists and antagonists on clonidine-induced inhibition of allodynia
Fig. 1. Photomicrograph example of an injection site in the ventrolateral orbital cortex (VLO). Arrow points to injection site within VLO. AI, agranular insular cortex; Cl, claustrum; fmi, forceps minor of the corpus callosum; Fr, frontal cortex, LO, lateral orbital cortex; VLO, ventrolateral orbital cortex. Scale bar = 1000 μm.
between the different groups using two-way repeated measure analysis of variance (two-way RM ANOVA), followed by a Bonferroni post hoc analysis for multiple comparisons (Sigmastat 2.03). Linear regression was used to analyze the relationship between drug dose and effect. P b 0.05 was considered to be statistically significant. Results As reported previously (Dang et al., 2010; Decosterd and Woolf, 2000), the PWT dramatically decreased to 1.91 ± 0.23 g (n = 8) two weeks after rat SNI compared to 17.78 ± 1.15 g (n = 8) in control animals (i.e. with no SNI). Microinjection of saline into the VLO had no effect on SNI-induced allodynia; PWT (1.93 ± 0.21 g, n = 8) was maintained at pre-injection control level in the entire observation period, which was used as the vehicle control to analyze the effect of drug injection in the following experiments. Inhibitory effect of microinjection of noradrenalin into the VLO on SNI-induced allodynia Microinjection of noradrenaline (1, 2, 4 μg, respectively) into the VLO reduced SNI-induced allodynia as PWT was increased in a dosedependent manner (r = 0.973, P = 0.027, Figs. 2A and C). As shown in Fig. 2A, time course curves of PWT for saline and different doses of noradrenaline treated groups were different between treatments (F(3, 140) = 106.698, P b 0.001), across times (F(5, 140) = 100.357, P b 0.001) and treatment × time interaction (F(15, 140) = 26.630, P b 0.001). The detailed comparisons at individual time points between various groups also are shown in Fig. 2A. Pretreatment with α2 adrenoceptor antagonist yohimbine (4 μg), 5min prior to noradrenaline injection, blocked noradrenaline-induced inhibition of allodynia as PWT was reduced to the saline control level, as shown in Figs. 2B and D. The same dose of yohimbine applied alone to the VLO had no effect on allodynia induced by SNI as PWT was maintained at the control level (Figs. 2B and D). Inhibitory effect of microinjection of clonidine into the VLO on SNI-induced allodynia Microinjection of a selective α2 adrenoceptor agonist clonidine (1, 2, 4 μg, in 0.5 μl) into the VLO mimicked the noradrenaline-induced inhibition of SNI-induced allodynia; PWT increased in a dose-dependent manner (r = 0.983, P = 0.017, Figs. 3A and C). Statistical analysis
Microinjection of a low dose of selective GABAA receptor antagonist bicuculline (100 ng) or picrotoxin (100 ng) (Dang et al., 2010) into the VLO, 5-min prior to 2 μg clonidine injection, significantly enhanced clonidine-induced inhibition of SNI-induced allodynia; PWT increased from 2.75 ± 0.19 g to 4.65 ± 0.21 g and 3.75 ± 0.24 g (P b 0.001), respectively (Figs. 4A and C), during the 60-min observation period. The detailed comparisons at individual time points in various groups are shown in Fig. 4A. In contrast, administration of selective GABAA receptor agonists muscimol (250 ng) or THIP (1 μg) (Dang et al., 2010) into the VLO, 5min prior to 4 μg clonidine injection, significantly attenuated clonidine-induced inhibition of SNI-induced allodynia; PWT decreased from 4.37 ± 0.26 g to 2.65 ± 0.09 g and 2.46 ± 0.21 (P b 0.001), respectively, during the 60-min observation period (Figs. 4B and D). The detailed comparisons at individual time points in different groups are shown in Fig. 4B. Discussion Role of noradrenaline and α2-adrenoceptor in the VLO nociceptive modulation The results of the present study demonstrate that noradrenaline microinjection into the VLO region reduces SNI-induced allodynia in a dose-dependent manner and this inhibitory effect can be antagonized by pre-microinjection of the selective α2 adrenoceptor antagonist yohimbine into the same VLO site. The anti-allodynic effect of noradrenaline was mimicked by microinjection of the selective α2 adrenoceptor agonist clonidine into the same VLO area, and was also blocked by yohimbine. These results suggest that the VLO noradrenergic system plays a role in anti-allodynia of the neuropathic pain state, which is mediated by α2 adrenoceptors. Previous studies indicated that activation of α2 adrenoceptor subtype produces antinociception in the spinal cord, the rostral ventromedial medulla, the locus coeruleus, the dorsal raphe nucleus, the nucleus raphe magnus, the PAG, the amygdala, and the caudate putamen (Alojado et al., 1994; Danzebrink and Gebhart, 1990; FleetwoodWalker et al., 1985; Guo et al., 1996; Hammond et al., 1980; Haws et al., 1990; Ortiz et al., 2007; Zhang et al., 2010). Results in the present study provide novel evidence that the α2 adrenoceptor is involved in descending antinociception at the cerebral cortex level (VLO). In addition, the results of the present study show that α2 adrenergic antagonist yohimbine applied alone to the VLO did not influence the allodynia induced by SNI, suggesting that this receptor subtype in the VLO lacks a tonic inhibition action. Interaction between GABA and α2 adrenoceptors in VLO nociceptive modulation Our previous studies have demonstrated that the VLO, as a higher center, is involved in an endogenous analgesic system consisting of a spinal cord–Sm–VLO–PAG–spinal cord loop (Tang et al., 2009), and that VLO
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Fig. 2. Microinjection of noradrenalin into the VLO reduced SNI-induced allodynia. (A) Time course curves showing the inhibitory effect of different doses of noradrenaline (NE) on allodynia induced by SNI. (B) Time course curves showing the blocking effect of yohimbine on NE-induced inhibition of allodynia. (C) Bar graphs showing the inhibitory effect of different NE doses on allodynia during the 60-min observation period. (D) Bar graph showing the blocking effect of yohimbine on NE-induced inhibition of allodynia during the 60-min observation period. *P b 0.05 and ***P b 0.001, compared with saline group; #P b 0.05 ###P b 0.001, compared with 1 μg NE group; ΔP b 0.05 and ΔΔΔP b 0.001, compared with 2 μg NE group; Φ P b 0.05 and ΦΦΦP b 0.001, compared with 4 μg NE group (two-way RM-ANOVA with a Bonferroni post hoc).
can be activated to produce descending antinociception by ascending noxious inputs relayed by the Sm and also can be blocked by lesion or inhibition of the PAG (S. Zhang et al., 1997; Y.Q. Zhang et al., 1997; Zhang
et al., 1998a,b). Therefore, it is reasonable that the anti-allodynia effect elicited by noradrenaline or α2 adrenoceptor agonist injection into the VLO is due to activation of the VLO neurons projecting to the PAG, leading
Fig. 3. Microinjection of clonidine into the VLO reduced SNI-induced allodynia. (A) Time course curves showing the inhibitory effect of different doses of clonidine on allodynia induced by SNI. (B) Time course curves showing the blocking effect of yohimbine on clonidine-induced inhibition of allodynia. (C) Bar graphs showing the inhibitory effect of different doses of clonidine on allodynia during the 60-min observation period. (D) Bar graph showing the blocking effect of yohimbine on clonidine-induced inhibition of allodynia during the 60-min observation period. *P b 0.05, compared with saline group; #P b 0.05, compared with 1 μg clonidine group; ΔP b 0.05 and ΔΔΔP b 0.001, compared with 2 μg clonidine group; ΦP b 0.05 and ΦΦΦ P b 0.001, compared with 4 μg clonidine group (two-way RM-ANOVA with a Bonferroni post hoc).
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to activation the PAG–brainstem descending inhibitory system which depresses the nociceptive transmission at the spinal cord level. However, as is well known, the α2 adrenoceptor is an inhibitory G-protein couple receptor, and activation of this receptor inhibits the neuronal activity through cell membrane hyperpolarization (Summers and McMartin, 1993). We speculate that, as with 5-HT1A and D2-dopamine receptors (Dang et al., 2010; Huo et al., 2008, 2010), the excitatory effect on the VLO neurons induced by α2 adrenoceptor activation may result from inhibiting the inhibitory action of GABAergic interneurons on output neurons projecting to PAG (disinhibition). This disinhibition leads to activation of the PAG–brainstem descending inhibitory system and reduction of the nociceptive information transmission at the spinal cord level. Anatomic studies have indicated that the cortex, including the VLO, receives innervations from noradrenergic nuclei of the pons (Cooper et al., 2003) and the α2 adrenoceptors are expressed sparsely in the brain including the VLO (Alburges et al., 1993; Day et al., 1997; Holmberg et al., 2003; Scheinin et al., 1994; Unnerstall et al., 1984). Morphological studies have established that GABAergic immunoreactive neurons and GABAA receptors are distributed in the frontal cortex including the VLO (Esclapez et al., 1987; Huo et al., 2005, 2009; Pirker et al., 2000; Princivalle et al., 2001). VLO neurons projecting to PAG also express GABAA receptors and make symmetrical (presumably inhibitory) synapses with GABAergic terminals (Huo et al., 2009). Therefore, a triple arrangement, consisting of noradrenergic afferent terminals, GABAergic interneurons, and output neurons, in addition to their receptors, exists within the VLO. This arrangement provides a morphological foundation for GABAergic modulation. In addition, a recent study in our laboratory (Dang et al., 2010) has found that microinjection of the GABAA receptor antagonist bicuculline or picrotoxin into the VLO depresses SNI-induced allodynia in a dose-dependent manner, suggesting that the GABAA receptor may exert a tonic inhibitory influence on VLO neurons projecting
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to PAG, and its blockage results in enhancement of the activity of the VLO–PAG brainstem descending inhibitory system and depression of the nociceptive inputs at the spinal cord level. The results in the present study further revealed that a low dose of GABAA receptor antagonist bicuculline or picrotoxin administered into the VLO, which did not influence the PWT (Dang et al., 2010), significantly enhanced α2 adrenoceptor activation-induced inhibition of the allodynia, while GABAA receptor agonist muscimol or THIP applied to the VLO reversed the α2 adrenoceptor activation-evoked anti-allodynia. Therefore, this study provides behavioral evidence for GABAergic modulation of the VLO α2 adrenoceptor activation-induced descending antinociception in the neuropathic pain state. It is possible that local application of noradrenaline or α2 adrenoceptor agonist into the VLO or stimulationevoked endogenous noradrenaline release from noradrenergic terminals directly activates the α2 adrenergic receptors and inhibits the inhibitory GABAergic interneurons that tonically inhibit the VLO output neurons projecting to the PAG (disinhibition), leading to activation of the PAG– brainstem descending inhibitory system which depresses the nociceptive inputs at the spinal cord level. Future investigation using morphological and electrophysiological approaches will be necessary to support our proposed model. To combine our previous findings (Dang et al., 2010; Huo et al., 2008, 2010; Tang et al., 2009; Zhao et al., 2006), VLO is an important source of descending inhibition of nociceptive and peripheral neuropathic pain in the rodent; the VLO descending projection neurons can be modulated by several different neurotransmitters (such as, noradrenaline, morphine, 5-HT, and dopamine) acting at a variety of corresponding receptors (e.g. α2 adrenoceptor, μ-opioid receptor, 5-HT1A receptor and D-2 dopamine receptor) and at least one common mechanism in VLO appears to be the inhibition of a tonic GABAergic inhibition of the projection neurons (i.e. disinhibition) by several different pathways and transmitters.
Fig. 4. GABAA agonist microinjection in the VLO enhanced clonidine-induced inhibition of allodynia, whereas GABAA antagonist reduced inhibition of allodynia. (A) Time course curves showing the enhanced effect of smaller dose of bicuculline (Bic, 100 ng) and picrotoxin (Pic, 100 ng) on clonidine (2 μg)-induced inhibition of allodynia induced by SNI. (B) Time course curves showing the attenuated effect of muscimol (250 ng) and THIP (1 μg) on clonidine (4 μg)-induced inhibition of allodynia. (C) Bar graphs showing the enhanced effect of bicuculline and picrotoxin on clonidine-induced inhibition of allodynia during the 60-min observation period. (D) Bar graph showing the attenuated effect of muscimol and THIP on clonidine-induced inhibition of allodynia during the 60-min observation period. *P b 0.05 and ***P b 0.001, compared with saline group; #P b 0.05 and ###P b 0.001, compared with clonidine group (two-way RM-ANOVA with a Bonferroni post hoc).
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The role of α2 adrenoceptor in mediating noradrenaline action in the ventrolateral orbital cortex on allodynia following spared nerve injury Juan-Xia Zhu a,d,1, Feng-Yi Xu a,1, Wen-Jin Xu a, Yan Zhao b, Chao-Ling Qu a, Jing-Shi Tang a, Devin M. Barry c, Jian-Qing Du a, Fu-Quan Huo a,⁎ a
Department of Physiology and Pathophysiology, Xi'an Jiaotong University College of Medicine, Yanta Road W. 76#, Xi'an, Shaanxi 710061, PR China Department of Forensic Medicine, Xi'an Jiaotong University College of Medicine, Yanta Road W. 76#, Xi'an, Shaanxi 710061, PR China Department of Anesthesiology, Washington University School of Medicine, St. Louis, MO 63110, USA d Department of Physiology, Xi'an Medical University, Xinwang Road 1#, Xi'an, Shaanxi 710021, PR China b c
a r t i c l e
i n f o
Article history: Received 24 May 2013 Revised 5 July 2013 Accepted 11 July 2013 Available online 18 July 2013 Keywords: Noradrenaline α2 adrenoceptor GABAergic modulation Ventrolateral orbital cortex Allodynia Neuropathic pain
a b s t r a c t The present study examined the role of α2 adrenoceptor in mediating noradrenaline action in the ventrolateral orbital cortex (VLO) on allodynia induced by spared nerve injury (SNI) in the rat. The mechanical paw withdrawal threshold (PWT) was measured using von-Frey filaments. Microinjection of noradrenaline (1, 2, 4 μg in 0.5 μl) into the VLO, contralateral to the site of nerve injury, reduced allodynia; PWT increased in a dose-dependent manner. Similar to noradrenaline, microinjection of selective α2 adrenoceptor agonist clonidine into the same VLO site also reduced allodynia, and was blocked by selective α2 adrenoceptor antagonist yohimbine. Furthermore, administration of γ-aminobutyric acid A (GABAA) receptor antagonist bicuculline or picrotoxin to the VLO significantly enhanced clonidine-induced inhibition of allodynia, while GABAA receptor agonist muscimol or THIP (2,5,6,7-retrahydroisoxazolo(5,4-c)pyridine-3-ol hydrochloride) attenuated clonidine-induced inhibition. These results suggest that noradrenaline acting in the VLO can potentially reduce allodynia induced by SNI, and this effect is mediated by α2 adrenoceptor. Moreover, GABAergic disinhibition may participate in α2 receptor mediating effects in neuropathic pain in the central nervous system. © 2013 Elsevier Inc. All rights reserved.
Introduction Previous studies in our laboratory have demonstrated that electrolytic lesions of or microinjection of γ-aminobutyric acid (GABA) into the ventrolateral orbital cortex (VLO) eliminates antinociceptive effects induced by peripheral electrical stimulation, or by activation of the thalamic nucleus submedius (Sm) (Zhang et al., 1995, 1998b, 1999). However, electrically or chemically induced activation of the VLO depresses tail flick and jaw-opening reflexes. These antinociceptive effects are eliminated by lesion or functional blocking of the periaqueductal gray (PAG) (S. Zhang et al., 1997; Y.Q. Zhang et al., 1997, Zhang et al., 1998a). These data suggest that the VLO is involved in an endogenous analgesic system consisting of a spinal/medulla cord–Sm–VLO–PAG–spinal/medulla cord loop (Tang et al., 2009).
Abbreviations: Bicuculline, (+)-bicuculline, (S), 9(R); Clonidine, clonidine hydrochloride; GABA, γ-aminobutyric acid; Muscimol, muscimol hydrochloride; Noradrenaline, L(−)-norepinephrine (+)-bitartrate salt monohydrate; PAG, periaqueductal gray; PWT, paw withdrawal threshold; Sm, nucleus submedius; SNI, spared nerve injury; THIP, 2,5,6,7-retrahydroisoxazolo(5,4-c)pyridine-3-ol hydrochloride; Yohimbine, yohimbine hydrochloride; VLO, ventrolateral orbital cortex. ⁎ Corresponding author. Fax: +86 29 82656364. E-mail address: [email protected] (F.-Q. Huo). 1 These authors have contributed equally to this work. 0014-4886/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.expneurol.2013.07.004
Noradrenaline (or norepinephrine), a principal monoaminergic neurotransmitter in the central nervous system, is synthesized in distinct brainstem nuclei that project widely throughout the central nervous system and is involved in various physiological functions, including regulation of blood pressure, pain, locomotion, stress reaction, attention, arousal and cognitive processes (Arnsten et al., 1996; Berridge and Waterhouse, 2003; Drouin et al., 2002; Flavin and Winder, 2013; Hein and Kobilka, 1997; Kable et al., 2000; Maldonado, 1997; Raja, 1995; Robbins, 1984). The actions of noradrenaline are mediated by α1, α2 and β adrenoceptors (Bylund et al., 1994). α1 adrenoceptors are coupled to phospholipase C through G-protein (Gq) or they are coupled directly to Ca2+ influx and produce the excitatory effect on neurons (Summers and McMartin, 1993). In contrast, activation of α2 adrenoceptors decreases intracellular adenylyl cyclase activity through G-protein (Gi) or directly modifies activity of ion channels such as the Na+/H+ antiport, Ca2+ channels, or K+ channels (Summers and McMartin, 1993) resulting in hyperpolarization. β-adrenoceptors increase adenylyl cyclase activity through Gs-protein (Gs) and produce the excitatory effect on neurons (Summers and McMartin, 1993). Previous studies have demonstrated that α2 adrenoceptors play a key role in mediating pain regulatory effects of noradrenaline, whereas β-adrenoceptors may predominantly mediate epinephrine-induced modulation of pain (Pertovaara, 2006). Anatomic studies have indicated that the cerebral cortex including the VLO receives innervations from noradrenergic nuclei of the pons (Cooper et al., 2003)
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and α2 adrenoceptors are widely distributed throughout the brain including the VLO (Aantaa et al., 1995; Alburges et al., 1993; Buëcheler et al., 2002; Day et al., 1997; Holmberg et al., 2003; Scheinin et al., 1994; Unnerstall et al., 1984). Therefore, the aim of the present study was to examine whether noradrenaline and α2 adrenoceptors were involved in mediating the VLO-evoked inhibition effect on allodynia induced by spared nerve injury (SNI) in the rat. It is well known that activation of α2 adrenoceptors causes hyperpolarization and directly produces inhibitory action on neurons as abovementioned. How does noradrenaline via α2 adrenoceptors mediate this inhibition effect on allodynia in the VLO? We speculate that noradrenaline directly activates the α2 adrenoceptors to inhibit the inhibitory GABAergic interneurons which tonically inhibit the VLO projecting to the PAG neurons (disinhibition), such as other monoaminergic receptors (5-HT1A and D2-dopamine receptors) (Dang et al., 2010; Huo et al., 2008, 2010), leading to activation of the PAG–brainstem descending inhibitory system which depresses the nociceptive inputs at the spinal cord level. So, the mechanisms of GABAergic modulation were examined by observing the effects of GABAA receptor antagonists and agonists on α2 adrenoceptormediated anti-allodynia in the VLO of a rat model of SNI-induced neuropathic pain.
to mechanical stimulation (von Frey filaments) using the up–down method (Chaplan et al., 1994; Dixon, 1980). The rats were placed in a transparent plastic box (280 × 250 × 210 mm3) with a metal wire mesh floor, which allowed full access to the paws from below. Behavioral adaptation was allowed until cage exploration and major grooming activities had ceased for approximately 30 min. Ten von Frey filaments (Stoelting Company, Wood Dale, IL, USA), with approximately equal logarithmic incremental (0.17) bending force, were chosen (von Frey numbers: 3.61, 3.84, 4.08, 4.17, 4.31, 4.56, 4.74, 4.93, 5.07 and 5.18, equivalent to: 0.4, 0.6, 1.0, 1.4, 2.0, 4.0, 6.0, 8.0, 10, and 15.0 g, respectively). Starting with filament 4.31 (2.0 g), which was the middle filament in the series, von Frey filaments with different intensities were repeatedly applied over a 2-s time interval from below and perpendicular to the fourth and fifth toes of the hind paw with sufficient force to cause slight bending against the paw for approximately 6–8 s. If response to filament stimulation was positive, the next lowest force was delivered. If there was no withdrawal response (negative), the next highest force was delivered. Positive and negative responses were recorded and converted to a 50% threshold using a formula provided by Dixon (1980) and Chaplan et al. (1994). PWT measurements were performed in 10-min intervals over a 60-min observation period. If PWT was reduced to b 4.0 g, mechanical allodynia was considered to be successfully established.
Materials and methods Intracerebral microinjection of drugs Animals Male Sprague–Dawley rats (220–250 g) were provided by the Experimental Animal Center of Shaanxi Province, PR China. The experimental protocol was approved by the Institutional Animal Care Committee of Xi'an Jiaotong University, and was in accordance with ethical guidelines of the International Association for the Study of Pain (Zimmermann, 1983). All efforts were made to minimize the number of animals used and their suffering. Spared sciatic nerve injury Spared nerve injury (SNI) was performed as previously described (Arsenault and Sawynok, 2009; Decosterd and Woolf, 2000). Briefly, the rats were intraperitoneally (i.p.) anesthetized with sodium pentobarbital (50 mg/kg, SCRC, Shanghai, China), and an incision was made along the back of the thigh. The biceps femoris muscle was separated to expose the three terminal branches of the sciatic nerve. The tibial and common peroneal nerves were tightly ligated using 5.0 silk and were transected distal to the ligation, following removal of 5 mm of nerve stump. The sural nerve was left intact, and the wound was closed. Following surgery, the rats were allowed to recover for 1 week before implantation of intracerebral cannulas.
Rats were lightly anesthetized with enflurane (Baxter Caribe, Guayama, PR, USA), a very short-acting inhalation anesthetic (about 1–2 min), and a 1 μl microsyringe with the tip extending 2 mm beyond the end of the guide cannula, was inserted into the VLO through the guide cannula. Over a 60-s period, 0.5 μl of drug dissolved in saline was slowly infused at constant speed, and effects on allodynia were observed over a 60 min period following drug infusion. Experiments were performed 2 times in the same animal over a time interval of 3 days. Values (n) from each treated group were obtained from different experiments. The following drugs were used in this study: non-selective adrenoceptor agonist L-(−)-norepinephrine (+)-bitartrate salt monohydrate (noradrenaline), selective α2 adrenoceptor agonist clonidine hydrochloride (clonidine) and antagonist yohimbine hydrochloride (yohimbine), GABAA receptor antagonists (+)-bicuculline, (S), 9(R) (bicuculline) and picrotoxin and agonists muscimol hydrochloride (muscimol) and 2,5,6,7-retrahydroisoxazolo(5,4-c)pyridine-3-ol hydrochloride (THIP) (RBI/Sigma, St. Louis, MO, USA). All drugs were dissolved in physiological saline. The agonist was injected into the VLO, contralateral to the affected hind paw, and the antagonist was administered 5 min prior to the agonist. The effective drug doses were chosen according to previous studies (Dang et al., 2010; Huo et al., 2008; Ortiz et al., 2007) and our preliminary experiment. Equal volumes of saline were injected into the VLO and served as the vehicle controls.
Intracerebral guide cannula placement Histology Rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.), and the head was immobilized in a stereotaxic frame. A small craniotomy was performed just above the VLO, contralateral to the site of nerve injury. A stainless steel guide cannula (0.8 mm in diameter) was stereotaxically inserted, with the tip 2.0 mm dorsal to the VLO at the following coordinates: 3.2 mm anterior to bregma, 2.0 mm lateral, and 4.6 mm below the cortical surface (Paxinos and Watson, 1986), followed by attachment to the skull with three microscrews and dental cement. Once the rats recovered from anesthesia, they were administered sodium penicillin (0.2 million U/day for 3 days, i.p.) to prevent wound and intracerebral infections. The rats were carefully nursed and fed in clean cages.
At the end of the experiment, the drug injection sites were marked by injecting Pontamine Sky Blue dye (0.5 μl, 2% in 0.5 M sodium acetate solution). Under deep anesthesia, the rats were transcardially perfused with 0.9% normal saline, followed by 10% formalin. The brains were then removed and fixed in 10% formalin solution for 3–7 days, then placed in 30% sucrose solution/0.1 M phosphate buffer (pH 7.4) as a cryprotectant overnight. Subsequently, the brains were cut into 35-μm thick sections using a freezing microtome, and then the slices were stained with Cresyl Violet. The injection sites were histologically identified to be within the VLO for data analysis, as shown in Fig. 1.
Mechanical paw withdrawal threshold measurement
Data analysis
One week after intracerebral guide cannula placement (i.e. 2 weeks after SNI), paw withdrawal threshold (PWT) was measured in response
All values are expressed as mean ± SEM. Differences in total observation time, as well as at each time point, were statistically tested
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indicated that there was significant difference between treatments (F(5, 145) = 22.511, P b 0.001), across time (F(5, 145) = 45.701, P b 0.001), and their interaction (F(15, 145) = 12.467, P b 0.001) (Fig. 3A). The detailed comparisons at individual time points between various groups are shown in Fig. 3A. Microinjection of selective α2 adrenoceptor antagonist yohimbine alone into the VLO did not influence allodynia induced by SNI. However, when yohimbine association with clonidine was applied, the clonidineinduced inhibition of allodynia was eliminated; PWT reduced to the saline control level (Figs. 3B and D). Influence of microinjection of GAGAA agonists and antagonists on clonidine-induced inhibition of allodynia
Fig. 1. Photomicrograph example of an injection site in the ventrolateral orbital cortex (VLO). Arrow points to injection site within VLO. AI, agranular insular cortex; Cl, claustrum; fmi, forceps minor of the corpus callosum; Fr, frontal cortex, LO, lateral orbital cortex; VLO, ventrolateral orbital cortex. Scale bar = 1000 μm.
between the different groups using two-way repeated measure analysis of variance (two-way RM ANOVA), followed by a Bonferroni post hoc analysis for multiple comparisons (Sigmastat 2.03). Linear regression was used to analyze the relationship between drug dose and effect. P b 0.05 was considered to be statistically significant. Results As reported previously (Dang et al., 2010; Decosterd and Woolf, 2000), the PWT dramatically decreased to 1.91 ± 0.23 g (n = 8) two weeks after rat SNI compared to 17.78 ± 1.15 g (n = 8) in control animals (i.e. with no SNI). Microinjection of saline into the VLO had no effect on SNI-induced allodynia; PWT (1.93 ± 0.21 g, n = 8) was maintained at pre-injection control level in the entire observation period, which was used as the vehicle control to analyze the effect of drug injection in the following experiments. Inhibitory effect of microinjection of noradrenalin into the VLO on SNI-induced allodynia Microinjection of noradrenaline (1, 2, 4 μg, respectively) into the VLO reduced SNI-induced allodynia as PWT was increased in a dosedependent manner (r = 0.973, P = 0.027, Figs. 2A and C). As shown in Fig. 2A, time course curves of PWT for saline and different doses of noradrenaline treated groups were different between treatments (F(3, 140) = 106.698, P b 0.001), across times (F(5, 140) = 100.357, P b 0.001) and treatment × time interaction (F(15, 140) = 26.630, P b 0.001). The detailed comparisons at individual time points between various groups also are shown in Fig. 2A. Pretreatment with α2 adrenoceptor antagonist yohimbine (4 μg), 5min prior to noradrenaline injection, blocked noradrenaline-induced inhibition of allodynia as PWT was reduced to the saline control level, as shown in Figs. 2B and D. The same dose of yohimbine applied alone to the VLO had no effect on allodynia induced by SNI as PWT was maintained at the control level (Figs. 2B and D). Inhibitory effect of microinjection of clonidine into the VLO on SNI-induced allodynia Microinjection of a selective α2 adrenoceptor agonist clonidine (1, 2, 4 μg, in 0.5 μl) into the VLO mimicked the noradrenaline-induced inhibition of SNI-induced allodynia; PWT increased in a dose-dependent manner (r = 0.983, P = 0.017, Figs. 3A and C). Statistical analysis
Microinjection of a low dose of selective GABAA receptor antagonist bicuculline (100 ng) or picrotoxin (100 ng) (Dang et al., 2010) into the VLO, 5-min prior to 2 μg clonidine injection, significantly enhanced clonidine-induced inhibition of SNI-induced allodynia; PWT increased from 2.75 ± 0.19 g to 4.65 ± 0.21 g and 3.75 ± 0.24 g (P b 0.001), respectively (Figs. 4A and C), during the 60-min observation period. The detailed comparisons at individual time points in various groups are shown in Fig. 4A. In contrast, administration of selective GABAA receptor agonists muscimol (250 ng) or THIP (1 μg) (Dang et al., 2010) into the VLO, 5min prior to 4 μg clonidine injection, significantly attenuated clonidine-induced inhibition of SNI-induced allodynia; PWT decreased from 4.37 ± 0.26 g to 2.65 ± 0.09 g and 2.46 ± 0.21 (P b 0.001), respectively, during the 60-min observation period (Figs. 4B and D). The detailed comparisons at individual time points in different groups are shown in Fig. 4B. Discussion Role of noradrenaline and α2-adrenoceptor in the VLO nociceptive modulation The results of the present study demonstrate that noradrenaline microinjection into the VLO region reduces SNI-induced allodynia in a dose-dependent manner and this inhibitory effect can be antagonized by pre-microinjection of the selective α2 adrenoceptor antagonist yohimbine into the same VLO site. The anti-allodynic effect of noradrenaline was mimicked by microinjection of the selective α2 adrenoceptor agonist clonidine into the same VLO area, and was also blocked by yohimbine. These results suggest that the VLO noradrenergic system plays a role in anti-allodynia of the neuropathic pain state, which is mediated by α2 adrenoceptors. Previous studies indicated that activation of α2 adrenoceptor subtype produces antinociception in the spinal cord, the rostral ventromedial medulla, the locus coeruleus, the dorsal raphe nucleus, the nucleus raphe magnus, the PAG, the amygdala, and the caudate putamen (Alojado et al., 1994; Danzebrink and Gebhart, 1990; FleetwoodWalker et al., 1985; Guo et al., 1996; Hammond et al., 1980; Haws et al., 1990; Ortiz et al., 2007; Zhang et al., 2010). Results in the present study provide novel evidence that the α2 adrenoceptor is involved in descending antinociception at the cerebral cortex level (VLO). In addition, the results of the present study show that α2 adrenergic antagonist yohimbine applied alone to the VLO did not influence the allodynia induced by SNI, suggesting that this receptor subtype in the VLO lacks a tonic inhibition action. Interaction between GABA and α2 adrenoceptors in VLO nociceptive modulation Our previous studies have demonstrated that the VLO, as a higher center, is involved in an endogenous analgesic system consisting of a spinal cord–Sm–VLO–PAG–spinal cord loop (Tang et al., 2009), and that VLO
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Fig. 2. Microinjection of noradrenalin into the VLO reduced SNI-induced allodynia. (A) Time course curves showing the inhibitory effect of different doses of noradrenaline (NE) on allodynia induced by SNI. (B) Time course curves showing the blocking effect of yohimbine on NE-induced inhibition of allodynia. (C) Bar graphs showing the inhibitory effect of different NE doses on allodynia during the 60-min observation period. (D) Bar graph showing the blocking effect of yohimbine on NE-induced inhibition of allodynia during the 60-min observation period. *P b 0.05 and ***P b 0.001, compared with saline group; #P b 0.05 ###P b 0.001, compared with 1 μg NE group; ΔP b 0.05 and ΔΔΔP b 0.001, compared with 2 μg NE group; Φ P b 0.05 and ΦΦΦP b 0.001, compared with 4 μg NE group (two-way RM-ANOVA with a Bonferroni post hoc).
can be activated to produce descending antinociception by ascending noxious inputs relayed by the Sm and also can be blocked by lesion or inhibition of the PAG (S. Zhang et al., 1997; Y.Q. Zhang et al., 1997; Zhang
et al., 1998a,b). Therefore, it is reasonable that the anti-allodynia effect elicited by noradrenaline or α2 adrenoceptor agonist injection into the VLO is due to activation of the VLO neurons projecting to the PAG, leading
Fig. 3. Microinjection of clonidine into the VLO reduced SNI-induced allodynia. (A) Time course curves showing the inhibitory effect of different doses of clonidine on allodynia induced by SNI. (B) Time course curves showing the blocking effect of yohimbine on clonidine-induced inhibition of allodynia. (C) Bar graphs showing the inhibitory effect of different doses of clonidine on allodynia during the 60-min observation period. (D) Bar graph showing the blocking effect of yohimbine on clonidine-induced inhibition of allodynia during the 60-min observation period. *P b 0.05, compared with saline group; #P b 0.05, compared with 1 μg clonidine group; ΔP b 0.05 and ΔΔΔP b 0.001, compared with 2 μg clonidine group; ΦP b 0.05 and ΦΦΦ P b 0.001, compared with 4 μg clonidine group (two-way RM-ANOVA with a Bonferroni post hoc).
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to activation the PAG–brainstem descending inhibitory system which depresses the nociceptive transmission at the spinal cord level. However, as is well known, the α2 adrenoceptor is an inhibitory G-protein couple receptor, and activation of this receptor inhibits the neuronal activity through cell membrane hyperpolarization (Summers and McMartin, 1993). We speculate that, as with 5-HT1A and D2-dopamine receptors (Dang et al., 2010; Huo et al., 2008, 2010), the excitatory effect on the VLO neurons induced by α2 adrenoceptor activation may result from inhibiting the inhibitory action of GABAergic interneurons on output neurons projecting to PAG (disinhibition). This disinhibition leads to activation of the PAG–brainstem descending inhibitory system and reduction of the nociceptive information transmission at the spinal cord level. Anatomic studies have indicated that the cortex, including the VLO, receives innervations from noradrenergic nuclei of the pons (Cooper et al., 2003) and the α2 adrenoceptors are expressed sparsely in the brain including the VLO (Alburges et al., 1993; Day et al., 1997; Holmberg et al., 2003; Scheinin et al., 1994; Unnerstall et al., 1984). Morphological studies have established that GABAergic immunoreactive neurons and GABAA receptors are distributed in the frontal cortex including the VLO (Esclapez et al., 1987; Huo et al., 2005, 2009; Pirker et al., 2000; Princivalle et al., 2001). VLO neurons projecting to PAG also express GABAA receptors and make symmetrical (presumably inhibitory) synapses with GABAergic terminals (Huo et al., 2009). Therefore, a triple arrangement, consisting of noradrenergic afferent terminals, GABAergic interneurons, and output neurons, in addition to their receptors, exists within the VLO. This arrangement provides a morphological foundation for GABAergic modulation. In addition, a recent study in our laboratory (Dang et al., 2010) has found that microinjection of the GABAA receptor antagonist bicuculline or picrotoxin into the VLO depresses SNI-induced allodynia in a dose-dependent manner, suggesting that the GABAA receptor may exert a tonic inhibitory influence on VLO neurons projecting
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to PAG, and its blockage results in enhancement of the activity of the VLO–PAG brainstem descending inhibitory system and depression of the nociceptive inputs at the spinal cord level. The results in the present study further revealed that a low dose of GABAA receptor antagonist bicuculline or picrotoxin administered into the VLO, which did not influence the PWT (Dang et al., 2010), significantly enhanced α2 adrenoceptor activation-induced inhibition of the allodynia, while GABAA receptor agonist muscimol or THIP applied to the VLO reversed the α2 adrenoceptor activation-evoked anti-allodynia. Therefore, this study provides behavioral evidence for GABAergic modulation of the VLO α2 adrenoceptor activation-induced descending antinociception in the neuropathic pain state. It is possible that local application of noradrenaline or α2 adrenoceptor agonist into the VLO or stimulationevoked endogenous noradrenaline release from noradrenergic terminals directly activates the α2 adrenergic receptors and inhibits the inhibitory GABAergic interneurons that tonically inhibit the VLO output neurons projecting to the PAG (disinhibition), leading to activation of the PAG– brainstem descending inhibitory system which depresses the nociceptive inputs at the spinal cord level. Future investigation using morphological and electrophysiological approaches will be necessary to support our proposed model. To combine our previous findings (Dang et al., 2010; Huo et al., 2008, 2010; Tang et al., 2009; Zhao et al., 2006), VLO is an important source of descending inhibition of nociceptive and peripheral neuropathic pain in the rodent; the VLO descending projection neurons can be modulated by several different neurotransmitters (such as, noradrenaline, morphine, 5-HT, and dopamine) acting at a variety of corresponding receptors (e.g. α2 adrenoceptor, μ-opioid receptor, 5-HT1A receptor and D-2 dopamine receptor) and at least one common mechanism in VLO appears to be the inhibition of a tonic GABAergic inhibition of the projection neurons (i.e. disinhibition) by several different pathways and transmitters.
Fig. 4. GABAA agonist microinjection in the VLO enhanced clonidine-induced inhibition of allodynia, whereas GABAA antagonist reduced inhibition of allodynia. (A) Time course curves showing the enhanced effect of smaller dose of bicuculline (Bic, 100 ng) and picrotoxin (Pic, 100 ng) on clonidine (2 μg)-induced inhibition of allodynia induced by SNI. (B) Time course curves showing the attenuated effect of muscimol (250 ng) and THIP (1 μg) on clonidine (4 μg)-induced inhibition of allodynia. (C) Bar graphs showing the enhanced effect of bicuculline and picrotoxin on clonidine-induced inhibition of allodynia during the 60-min observation period. (D) Bar graph showing the attenuated effect of muscimol and THIP on clonidine-induced inhibition of allodynia during the 60-min observation period. *P b 0.05 and ***P b 0.001, compared with saline group; #P b 0.05 and ###P b 0.001, compared with clonidine group (two-way RM-ANOVA with a Bonferroni post hoc).
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