Potassium channels underlie postsynaptic but not presynaptic GABAB receptor-mediated inhibition on ventrolateral periaqueductal gray neurons

Brain Research Bulletin 88 (2012) 529–533

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Potassium channels underlie postsynaptic but not presynaptic GABAB receptor-mediated inhibition on ventrolateral periaqueductal gray neurons Zhi-Liang Liu a, Hongyu Ma b,∗, Ru-Xiang Xu a, Yi-Wu Dai a, Hong-Tian Zhang a, Xue-Qin Yao a, Kun Yang c,d,∗∗ a

Bayi Brain Hospital, The Military General Hospital of Beijing PLA, Beijing 100700, China Clinical Laboratory Center, Air Force General Hospital, Beijing 100142, China c Department of Anatomy and K.K. Leung Brain Research Centre, The Fourth Military Medical University, Xi’an 710032, China d Department of Neurology, University of Maryland School of Medicine, Baltimore, MD 21201, USA b

a r t i c l e

i n f o

Article history: Received 11 May 2012 Accepted 15 May 2012 Available online 23 May 2012 Keywords: Periaqueductal gray K+ channels GABAB receptors Pain

a b s t r a c t ␥-Aminobutyric acid (GABA) is the principle inhibitory neurotransmitter in adult mammalian brain. GABA receptors B subtype (GABAB Rs) are abundantly expressed at presynaptic and postsynaptic neuronal structures in the rat ventrolateral periaqueductal gray (PAG), an area related to pain regulation. Activation of GABAB Rs by baclofen, a selective agonist, induces presynaptic inhibition by decreasing presynaptic glutamate release. At the same time, baclofen induces a postsynaptic inhibitory membrane current or potential. We here report that in the ventrolateral PAG, the postsynaptic inhibition is mediated by activation of G protein-coupled inwardly rectifying K+ (GIRK) channels. Blockade of K+ channels largely prevents postsynaptic action of baclofen. In contrast, presynaptic inhibition of baclofen is insensitive to K+ channel blockade. The data indicate that potassium channels play different roles in GABAB R-mediated presynaptic and postsynaptic inhibition on PAG neurons. © 2012 Elsevier Inc. All rights reserved.

1. Introduction ␥-Aminobutyric acid (GABA) is the principle inhibitory neurotransmitter in adult mammalian central nervous system. Two kinds of GABA receptors have been identified in the pain circuit including periaqueductal gray (PAG) in the midbrain: ionotropic GABAA receptors (GABAA Rs), which are channel complexes mainly permeable to chloride anions, and metabotropic GABAB receptors

Abbreviations: aCSF, artificial cerebrospinal fluid; AMPA, ␣-amino-3-hydroxy5-methyl-4-isoxazole propionic acid; AP-5, D-2-amino-5-phosphonopentanoic acid; CGP52432, 3-[[[(3,4-dichlorophenyl)methyl]amino]propyl] (diethoxymethyl) phosphinic acid; DNQX, 6,7-dinitroquinoxaline-2,3(1H,4H)-dione; EPSCs, excitatory postsynaptic currents; GABA, ␥-aminobutyric acid; GDP-␤-S, guanosine 5 -[␤-thio]diphosphate trilithium salt; GIRK channels, G protein-coupled inwardly rectifying K+ channels; GPCRs, G protein-coupled receptors; K–S test, Kolmogorov–Smirnov test; PAG, periaqueductal gray; TTX, tedrodotoxin. ∗ Corresponding author at: Clinical Laboratory Center, Air Force General Hospital, 30 Fucheng Road, Beijing 100142, China. Tel.: +86 10 6692 8545; fax: +86 10 6692 8559. ∗∗ Corresponding author at: Department of Neurology, University of Maryland School of Medicine, 655 West Baltimore Street, Baltimore, MD 21201, USA. Tel.: +1 410 706 2346; fax: +1 410 706 0186. E-mail addresses: [email protected] (H. Ma), [email protected] (K. Yang). 0361-9230/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.brainresbull.2012.05.010

(GABAB Rs), which are linked to intracellular G protein-coupled receptors [2,3,14]. We have demonstrated distribution and function of GABAB Rs in the spinal dorsal horn and ventrolateral part of PAG, both of which are crucial for nociceptive information transmission and modulation [22,25,26]. Morphological studies show that GABAB Rs are distributed throughout presynaptic and postsynaptic neuronal structures [13,25,26]. Electrophysiological studies support the morphological data by showing that activation of GABAB R results in both presynaptic and postsynaptic inhibition. Baclofen, a specific GABAB R agonist, depresses either spontaneous or evoked neurotransmitter (glutamate or GABA) release presynaptically [19,22]. Postsynaptically, baclofen induces a slow inhibitory membrane current (potential) [5,22]. Many postsynaptic inhibitions by G protein-coupled receptors (GPCRs) are mediated by K+ conductances [12,15,19]. It is possible that presynaptic inhibition of synaptic transmission is due to modulation of the conductances because GABAB R-positive structures are found both presynaptic and postsynaptic. Studies on hippocampus CA1 neurons, however, suggested that presynaptic and postsynaptic GABAB R action mediated by baclofen is different [12,18]. Thus it raises the question of whether the action of baclofen on presynaptic and postsynaptic of PAG neurons is similar. In the present study, we compared the action of baclofen on presynaptic and postsynaptic sites of PAG neurons with whole-cell recordings on acute midbrain slices, with special reference to role of K+ conductances.

Z.-L. Liu et al. / Brain Research Bulletin 88 (2012) 529–533

2. Materials and methods 2.1. Slice preparation All experiments were approved by the Animal Use and Care Committee of the institutes. The methods for slice preparation have been described in detail previously with modification [22,23]. Briefly, adult (5–8 wks old) male Sprague–Dawley rats were anesthetized with urethane (1.5 g kg−1 bodyweight, i.p.), decapitated and the whole brain was quickly removed and immersed in cold (0–3 ◦ C) “dissection solution” containing (in mM): KCl 5, NaH2 PO4 1.2, CaCl2 0.5, MgSO4 10, NaHCO3 26, glucose 10, sucrose 212.7, gassed with 95% O2 /5% CO2 . Transverse slices of 400 ␮m thickness were excised from midbrain containing PAG by a vibratome slicer (DTK-1000, Dosaka EM, Kyoto, Japan or Vibratome Series 1000, Technical Products International Inc., Saint Louis, USA), and kept in oxygenated artificial cerebrospinal fluid (aCSF) containing (in mM): NaCl 124, KCl 3.6, CaCl2 2.5, NaH2 PO4 1.2, MgCl2 1.2, NaHCO3 25, and glucose 11 (pH 7.2, mOsm 300–305) at ∼36 ◦ C, for 30–40 min before recordings. 2.2. Whole-cell recordings and data analysis Ventrolateral PAG neurons under direct visual observation by video camera were recorded at room temperature in a recording chamber with volume of ∼1 ml. All signals were amplified by an AxoPatch 200B, controlled by Clampex 9.0 or Clampex 10.2 (Molecular Devices) run on a personal computer (Windows XP). aCSF was perfused by a gravity-driven system at 8–10 ml/min to the recording chamber. Slices were perfused by aCSF and drugs were contained in the aCSF at certain concentrations unless otherwise stated. In some cases, postsynaptic action was studied by blocking presynaptic effect using an antagonist cocktail containing tedrodotoxin (TTX, Na+ channel blocker; 0.5 ␮M), DNQX (selective non-NMDA receptor antagonist; 10 ␮M), AP-5 (NMDA receptor antagonist; 50 ␮M), picrotoxin (GABAA R antagonist; 100 ␮M) and Cd2+ (voltage-dependent calcium channel blocker; 200 ␮M). Presynaptic focal-evoked glutamate release was stimulated by using a monopolar electrode pre-located 50–200 ␮m from the recorded neurons. To isolate presynaptic action, in some experiments, postsynaptic action was blocked by intracellular dialysis GDP-␤-S (1 mM). Two kinds of internal solution were used in the present work. For postsynaptic action study, in most cases potassium-based internal solution was used (in mM): K-gluconate 135, KCl 5, CaCl2 0.5, MgCl2 2, EGTA 5, Hepes 5, Mg-ATP 3.6; pH 7.2 modulated by KOH and mOsm ∼285. For presynaptic action study, pipette solution was (in mM): Cs-methanesulfonate 115, CsCl 20, Hepes 10, MgCl2 2.5, Na2 -ATP 4, Na3 -GTP 0.4, Na-phosphocreatine 10, EGTA 0.6, GDP-␤-S 1; pH 7.2 modulated by CsOH and mOsm ∼285. All drugs were obtained from Tocris Bioscience, Sigma or Ascent Scientific. Data were analyzed offline by Clampfit 9.0 or AxoGraph 4.0 (both from Molecular Devices). All values are expressed as mean ± S.E.M. Kolmogorov–Smirnov test (K–S test) was used to compare cumulative distribution of frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) for the same neuron before and with drug treatment, as shown before [24]. Paired or unpaired Student’s t-test was used to compare two groups and two-way ANOVA for comparing three groups. Significant difference was judged as P < 0.05.

A

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Fig. 1. Activation of GABAB Rs by baclofen induces an outward membrane current in PAG neurons. (A) Representative traces under voltage-clamp recording showing baclofen-induced slow inhibitory postsynaptic current in one neuron (upper) and in another neuron with GDP-␤-S intracellular dialysis (intra. GDP-␤-S; bottom). Both were recorded in the presence of TTX (0.5 ␮M), DNQX (10 ␮M), AP-5 (50 ␮M) and picrotoxin (100 ␮M). (B) Bar graphs showing the mean amplitude of baclofeninduced membrane currents under various conditions with the agonist cocktail as shown in (A). Ctrl., control; w/o TTX, without TTX; GDP-␤-S, dialysis by internal solution with GDP-␤-S (1 mM) included; Ba2+ , aCSF with Ba2+ (500 ␮M); CGP, CGP52432 (1 ␮M). **P < 0.01, significant difference from control (ctrl).

(n = 10; P < 0.05 compared to that of control, unpaired t-test), indicating baclofen-induced postsynaptic membrane current is mediated by intracellular G protein-coupled GABAB Rs.

3. Results 3.1. Activation of GABAB Rs by baclofen induces an inhibitory membrane current In voltage-clamp recording with K-gluconate pipette (holding potential = −70 mV), in aCSF with 0.5 ␮M TTX, baclofen application in the bath (10 ␮M, 1 min) induced an outward membrane current (n = 10; data not shown, see a similar results in [22]). In the presence of agonist cocktail to block synaptic transmission (see Section 2), baclofen (10 ␮M, 1 min) induced an outward current, with an average peak amplitude of 25 ± 2 pA (Fig. 1A, n = 21). When TTX was absent, the baclofen-induced outward current amplitude was 27 ± 3 mV, not significantly different from that with TTX (n = 11, P > 0.05, unpaired t-test) (Fig. 1B). Baclofen-activated membrane current was largely attenuated by intracellular dialysis of GDP-␤-S (1 mM), a non-hydrolysable analog of GDP that competitively inhibits G-proteins (6 ± 1 pA, n = 12; P < 0.05, unpaired t-test) (Fig. 1A and B). Pretreatment of slices by bath application of Ba2+ (500 ␮M), a K+ channel blocker, also significantly reduced the baclofen-induced membrane current to 10 ± 1 pA (n = 12; P < 0.05 compared to that of control, unpaired t-test). The baclofen-induced membrane current was 5 ± 3 pA following pretreatment of a specific GABAB R antagonist, CGP52432 (1 ␮M)

3.2. Baclofen decreases postsynaptic membrane resistance and induces GIRK current Under current-clamp recordings, baclofen (10 ␮M, 1 min) application with aCSF induced membrane hyperpolarization with a decrease of the membrane resistance to 76 ± 10% of the control (n = 9, P < 0.05, paired t-test), which was measured from middle of the depolarization current injection (Fig. 2A1). However, intracellular dialysis of GDP-␤-S (1 mM) largely prevented the membrane conductance increase (92 ± 7% of the control, n = 5, P > 0.05, paired t-test) (Fig. 2A2). In voltage-clamp recording mode with synaptic transmission being blocked, on the other hand, current responses to series hyperpolarizing voltage pulses (600 ms duration, from −130 mV to −40 mV with 10 mV increment) were increased with baclofen application. The “net” baclofen action was obtained by subtracting control currents from that in baclofen (Fig. 2B1). Amplitude of “net” current at −120 mV was −65 ± 15 pA (n = 11). Ba2+ (500 ␮M) pre-treatment or GDP-␤-S dialysis largely prevented a baclofen-induced “net” effect at the same holding potential (−120 mV) to 8 ± 5 pA (n = 5) and 12 ± 6 pA (n = 6), respectively (P < 0.05 in both groups compared to the control, unpaired t-test) (Fig. 2B2 and B3), suggesting that the baclofen-induced current was mediated by postsynaptic K+ conductances and/or GPCRs.

Z.-L. Liu et al. / Brain Research Bulletin 88 (2012) 529–533

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Fig. 2. GABAB R agonist baclofen decreases postsynaptic membrane input resistance and activates G protein-coupled inwardly rectifying K+ (GIRK) current. (A1) Upper: membrane conductance was monitored by 300 ms hyperpolarizing current (0.1 nA) every 5 s. The membrane hyperpolarization was compensated by a current injection to compare the membrane conductance change. Bottom: expanded traces from chart recording. Hyperpolarizing protocol is shown upon expanded traces. (A2) GDP-␤-S dialysis from recording pipette largely prevented baclofen-induced membrane conductance change. Note that action potentials were truncated in both chart recordings in A1 and A2. (B1) Current response in 3.6 mM extracellular K+ aCSF ([K+ ]o 3.6 mM). The current traces were evoked by voltage steps from −130 mV to −40 mV (600 ms, 10 mV increment) in the presence of TTX (0.5 ␮M), DNQX (10 ␮M), AP-5 (50 ␮M), and picrotoxin (100 ␮M). The “net” baclofen-induced current (right) is shown by subtracting control (left) from that in baclofen (middle). (B2) and (B3) “Net” currents in Ba2+ (500 ␮M) and with GDP-␤-S (1 mM) pipette (intra. GDP-␤-S), respectively. Calibrations under B1 (right) also apply to B2 and B3. (C) Left: current response in [K+ ]o 3.6 mM aCSF in control and with baclofen (10 ␮M). Right: “net” baclofen-induced current (subtraction of control from baclofen) and the “net” current in Ba2+ (500 ␮M; baclofen + Ba2+ ). The current traces were evoked by voltage ramps from −120 mV to −20 mV in the presence of TTX (0.5 ␮M), DNQX (10 ␮M), AP-5 (50 ␮M), picrotoxin (100 ␮M) and Cd2+ (200 ␮M).

To further address the mechanism of baclofen postsynaptic action, we next employed voltage ramp protocol (from −120 mV to −30 mV with 600 ms duration) to evoked K+ current in the presence of agonist cocktail using K+ -based internal solution. Voltage ramps-induced current in the presence of baclofen (10 ␮M) was subtracted by that in control, revealing an inward rectifying component of baclofen-induced current (Fig. 2C). The reversal potential was −92.3 ± 2.5 mV (n = 8), i.e., close to the K+ equilibrium potential as calculated by the Nernst equation (EK = −93.1 mV). The average amplitude of baclofen-induced current was −55 ± 10 pA at −110 mV (n = 9); this was greater than that with baclofen in the presence of Ba2+ (−7 ± 4 pA, n = 5, P < 0.05, unpaired t-test: Fig. 3B) or current obtained with a GDP-␤-S dialysis pipette (−11 ± 6 pA, n = 4, P < 0.05, unpaired t-test). All these results are consistent with the idea that baclofen-induced postsynaptic action is mediated by activation of an inwardly rectifying K+ current. Based on the similarities in the above properties, we proposed that baclofen increases a postsynaptic GIRK current in PAG neurons.

3.3. Presynaptic inhibition by baclofen is not mediated by K+ conductance Finally, the contribution of K+ conductances at presynaptic terminals to the baclofen action was tested by using Ba2+ or Cs+ as blockers for K+ channels. Under the condition of postsynaptic GABAB Rs being blocked by GDP-␤-S dialysis in Cs+ -based internal solution [22], the miniature glutamate release (recorded as mEPSCs under voltage-clamp at −70 mV [23]) were readily recorded in all neurons tested (n = 12). In control condition (with “normal” aCSF), application of baclofen (10 ␮M) to the bath solution reduced the frequency of mEPSCs significantly from 3.4 ± 0.5 Hz to 50 ± 9% of the control (n = 12, P < 0.05, paired t-test), while the amplitude were not altered significantly (from 15.7 ± 3.2 pA to 93 ± 4% of the control, n = 12; data not shown, see [22] for a similar result). In the presence of Ba2+ (500 ␮M), baclofen (10 ␮M) treatment reduced the frequency of mEPSCs to 61 ± 6% of the value (n = 7, P < 0.05, paired t-test) (Fig. 3A1 and A2). This inhibition magnitude, however, was not significantly different than that without Ba2+ (Fig. 3A2). In

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potential-independent release (recorded as mEPSCs) nor action potential-dependent release (recorded as eEPSCs) was modulated by presynaptic K+ channel alteration.

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Fig. 3. Presynaptic K+ channel blockade has only minimal effects on baclofeninduced presynaptic inhibition on glutamate release. (A1) Representative traces of mEPSCs recorded in the presence of Ba2+ (500 ␮M in aCSF) before (control), during (baclofen; 10 ␮M) and after washout of baclofen. (A2) Bar graphs showing the pooled data of frequency and amplitude of mEPSCs under different conditions. All recordings were carried out in the presence of TTX (0.5 ␮M) and picrotoxin (100 ␮M) at a holding potential of −70 mV. (B1) Representative traces showing eEPSCs amplitudes inhibited under different conditions. Each line is an average of 5 consecutive recording at 0.1 Hz of presynaptic stimulation. (B2) Bar graphs show no significant difference in magnitude of baclofen inhibition on eEPSCs under different conditions (normal aCSF, 500 ␮M Ba2+ or 3 mM Cs+ ). *P < 0.05, **P < 0.01, significant difference from control aCSF; n.s., not significant.

the presence of Ba2+ , baclofen did not significantly change the average amplitude (90 ± 9% of the that before baclofen treatment, n = 7, P > 0.05, paired t-test), indicating presynaptic Ba2+ -sensitive K+ channels were not involved in the baclofen inhibition on miniature glutamate release. For the evoked presynaptic glutamate release [recorded as evoked EPSCs (eEPSCs) at holding potential of −70 mV], blockade of K+ channels resulted in a slight enhancement of eEPSCs amplitude. Following the same experimental design as that for mEPSCs, we first determined the magnitude of inhibition by baclofen on eEPSCs amplitude alone, and then assessed the effect of baclofen in the presence of either Ba2+ (500 ␮M) or Cs+ (3 mM). Baclofen (10 ␮M) depressed the amplitude of eEPSCs to 46 ± 6% of the control in “normal” aCSF (n = 10), to 56 ± 11% in the presence of Ba2+ (n = 5) and to 43 ± 12% in the presence of Cs+ (n = 4). The baclofen-induced inhibition magnitude was not significantly difference in three groups (Fig. 3B, P > 0.05, ANOVA). Taken together, the results suggest that baclofen-induced inhibition of neither action

4. Discussion The principal result in this study is that activation of GABAB Rs by a specific agonist, baclofen, on rat PAG neurons, the postsynaptic action is mediated by GIRK channels while the presynaptic inhibition is not sensitive to K+ channel alteration, indicating that different mechanisms underlie the presynaptic and postsynaptic inhibition mediated by GABAB Rs. Consistent with previous reports, in the present study, we observed that with synaptic transmission blockade, baclofen induced an outward (inhibitory) membrane current. This postsynaptic current was significantly decreased by intracellular GDP-␤-S dialysis (which inhibited intracellular Gi/Go protein), or barium (which blocked K+ channels) pretreatment in external solution, or in the presence of a specific GABAB R antagonist, CGP52432. Activation of GABAB Rs by baclofen also decreased membrane input resistance. Furthermore, the “net” current of baclofen showed a characteristic of inward rectifying. The results lead to a conclusion that baclofen’s postsynaptic action on PAG neurons is mediated by activating G protein-coupled, barium-sensitive GIRK current. For the study of neurotransmitter release, a change in mEPSCs amplitude indicates a postsynaptic responsiveness alteration while a change in mEPSCs frequency indicates a presynaptic release change [23]. In the present study, postsynaptic GABAB Rs effect has been inhibited by GDP-␤-S or Cs+ -based internal solution through the pipette, thus a change in the frequency of the mEPSCs indicates baclofen inhibition on presynaptic release but not postsynaptic responsiveness to glutamate. Furthermore, K–S test showed that frequency but not amplitude distribution altered to baclofen treatment, indicating a presynaptic baclofen action [24]. Blocking presynaptic K+ conductances by Ba2+ or Cs+ did not affect evoked glutamate release. We conclude that baclofen inhibits presynaptic spontaneous miniature release and evoked release by a way other than K+ conductance alteration. Several possibilities might underlie the difference of presynaptic and postsynaptic action of GABAB Rs in the present study. Firstly, although morphological data show that GABAB Rs are expressed both presynaptic and postsynaptic structures, the distribution of GABAB R subtypes at presynaptic and postsynaptic sites may vary [6,7]. GABAB Rs are constituted of subunits GABAB 1a, GABAB 1b and GABAB 2 [3,17]. The cellular and subcellular localization of different GABAB R subunits was documented [6,17]. GABAB 1a-containing heterodimers predominantly control presynaptic inhibition, while GABAB 1b subunits mainly mediated postsynaptic action [7]. Secondly, GABAB Rs on the same neuron exert distinct inhibition depending on the location of the receptors, as suggested in hippocampus [12] and septal nucleus [21]. The lack of Ba2+ -sensitive K+ conductance involvement in presynaptic inhibition could be due to the lack of K+ channels at the synapse or to the failure of K+ conductance at presynaptic to alter glutamate release. Further experiments are required to elucidate the mechanisms of presynaptic and postsynaptic modulation. It is worth noting that in our present study for presynaptic baclofen action, while K+ channel blockers (Ba2+ or Cs+ ) did not affect baclofen inhibition on mEPSCs or eEPSCs, we cannot rule out the possibility that baclofen activates presynaptic GPCRs. This is because modulation of presynaptic GPCRs is suggested as fundamental to the control of neurotransmitter release [17]. For example, in cerebellum, baclofen mediates presynaptic inhibition on presynaptic GPCRs while its inhibition on miniature GABA release remains unaffected [8]. Nevertheless, our data gave strong

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evidence that presynaptic barium-sensitive or cesium-sensitive K+ conductances do not affect baclofen inhibition upon presynaptic glutamate release. Although we have no direct evidence in the present study, baclofen depression on eEPSCs may be due to the inhibition of presynaptic calcium channels, as suggested in other brain structures [2,3]. PAG in the midbrain is divided into four longitudinal columns along the rostrocaudal extent, where ventrolateral part is recognized as pivotal for nociceptive information modulation [14]. PAG is thought to be one of origination for “descending” pain modulation system and sends multiple excitatory and inhibitory fibers to the target structures [4,10,11,14]. For example, baclofen modulates fibers originating from PAG project to the spinal cord dorsal horn and thus modulates nociceptive information transmission [22,25]. It is predicable that activation of GABAB Rs in PAG modulates pain signals. In behavior study, Levy and Proudfit [9] demonstrated that microinjection of baclofen in PAG produced analgesia. The present study further shows that baclofen differentially regulates presynaptic and postsynaptic inhibition. Since baclofen holds therapeutic promise for pain treatment at different levels of pain circuit [1,9,16,20], better understanding of the molecular mechanisms of GABAB R-mediated inhibition at PAG may lead to more effective pain treatment. Conflict of interest The authors declare no conflict of interest associated with the present study. Acknowledgments The authors thank Curtis Gallagher for critical reading of the manuscript. This study was supported in part by the National Natural Science Foundation of China (No. 30000052) to K.Y. References [1] G. Bonanno, A. Fassio, R. Sala, G. Schmid, M. Raiteri, GABAB receptors as potential targets for drugs able to prevent excessive excitatory amino acid transmission in the spinal cord, European Journal of Pharmacology 362 (2008) 143–148. [2] N.G. Bowery, GABAB receptor: a site of therapeutic benefit, Current Opinion in Pharmacology 6 (2006) 37–43. [3] N.G. Bowery, B. Bettler, W. Froestl, J.P. Gallagher, F. Marshall, M. Raiteri, T.I. Bonner, S.J. Enna, International Union of Pharmacology. XXXIII. Mammalian ␥-aminobutyric acidB receptors: structure and function, Pharmacological Reviews 54 (2002) 247–264. [4] T. Chen, R. Hui, X.-L. Wang, T. Zhang, Y.-X. Dong, Y.-Q. Li, Origins of endomorphin-immunoreactive fibers and terminals in different columns of the periaqueductal gray in the rat, Journal of Comparative Neurology 509 (2008) 72–87. [5] B. Chieng, M.J. Christie, Hyperpolarization by GABAB receptor agonists in midbrain periaqueductal gray neurones in vitro, British Journal of Pharmacology 116 (1995) 1583–1588.

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[6] M.P. Engle, M.A. Merrill, B.M. de Prado, D.L. Hammond, Spinal nerve ligation decreases ␥-aminobutyric acidB receptors on specific populations of immunohistochemically identified neurons in L5 dorsal root ganglion of the rat, Journal of Comparative Neurology 520 (2012) 1663–1677. [7] C. Goudet, V. Magnaghi, M. Landry, F. Nagy, R.W. Gereau IV, J.P. Pin, Metabotropic receptors for glutamate and GABA in pain, Brain Research Reviews 60 (2009) 43–56. [8] V.L. Harvey, G.J. Stephens, Mechanism of GABAB receptor-mediated inhibition of spontaneous GABA release onto cerebellar Purkinje cells, European Journal of Neuroscience 20 (2004) 684–700. [9] R.A. Levy, H.K. Proudfit, Analgesia produced by microinjection of baclofen and morphine at brain stem sites, European Journal of Pharmacology 57 (1979) 43–55. [10] Y.-Q. Li, Z.-R. Rao, J.-W. Shi, Collateral projections from the midbrain periaqueductal gray to the nucleus raphe magnus and nucleus accumbens in the rat. A fluorescent retrograde double-labelling study, Neuroscience Letters 117 (1990) 285–288. [11] Y.-Q. Li, S.-L. Zeng, Z.-R. Rao, J.-W. Shi, Serotonin-, substance P- and tyrosine hydroxylase-like immunoreactive neurons projecting from the midbrain periaqueductal gray to the nucleus tractus solitarii in the rat, Neuroscience Letters 134 (1992) 175–179. [12] C. Lüscher, L.Y. Jan, M. Stoffel, R.C. Malenka, R.A. Nicoll, G protein-coupled inwardly rectifying K+ channels (GIRKs) mediate postsynaptic but not presynaptic transmitter actions in hippocampal neurons, Neuron 19 (1997) 687–695. [13] M. Margeta-Mitrovic, I. Mitrovic, R.C. Riley, L.Y. Jan, A.I. Basbaum, Immunohistochemical localization of GABAB receptors in the rat central nervous system, Journal of Comparative Neurology 405 (1999) 299–321. [14] M.J. Millan, Descending control of pain, Progress in Neurobiology 66 (2002) 355–474. [15] T. Nakatsuka, T. Fujita, K. Inoue, E. Kumamoto, Activation of GIRK channels in substantia gelatinosa neurones of the adult rat spinal cord: a possible involvement of somatostatin, Journal of Physiology 586 (2008) 2511–2522. [16] G.W. Price, G.P. Wilkin, M.J. Turnbull, N.G. Bowery, Are baclofen-sensitive GABAB receptors present on primary afferent terminals of the spinal cord, Nature 307 (1984) 71–74. [17] G.J. Stephens, G-protein-coupled-receptor-mediated presynaptic inhibition in the cerebellum, Trends in Pharmacological Sciences 30 (2009) 421–430. [18] S.M. Thompson, B.H. Gahwiler, Comparison of the actions of baclofen at preand postsynaptic receptor, Journal of Physiology 451 (1992) 329–345. [19] C.W. Vaughan, S.L. Ingram, M.A. Connor, M.J. Christie, How opioids inhibit GABA-mediated neurotransmission, Nature 390 (1997) 611–614. [20] P.R. Wilson, T.L. Yaksh, Baclofen is antinociceptive in the spinal intrathecal space of animals, European Journal of Pharmacology 51 (1978) 323–330. [21] K. Yamada, B. Yu, J.P. Gallagher, Different subtypes of GABAB receptors are present at pre- and postsynaptic sites within the rat dorsolateral septal nucleus, Journal of Neurophysiology 81 (1999) 2875–2883. [22] K. Yang, H. Furue, E. Kumamoto, Y.-X. Dong, M. Yoshimura, Pre- and postsynaptic inhibition mediated by GABAB receptors in rat ventrolateral periaqueductal gray neurons, Biochemical and Biophysical Research Communications 302 (2003) 233–237. [23] K. Yang, Y.-Q. Li, E. Kumamoto, H. Furue, M. Yoshimura, Voltage-clamp recordings of postsynaptic currents in substantia gelatinosa neurons in vitro and its applications to assess synaptic transmission, Brain Research Protocols 7 (2001) 235–240. [24] K. Yang, H. Ma, Blockade of GABAB receptors facilitates evoked neurotransmitter release at spinal dorsal horn synapse, Neuroscience 193 (2011) 411–420. [25] K. Yang, W.-L. Ma, Y.-P. Feng, Y.-X. Dong, Y.-Q. Li, Origins of GABAB receptor-like immunoreactive terminals in the rat spinal dorsal horn, Brain Research Bulletin 58 (2002) 499–507. [26] K. Yang, D. Wang, Y.-Q. Li, Distribution and depression of the GABAB receptor in the spinal dorsal horn of adult rat, Brain Research Bulletin 55 (2001) 279–285.