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Postsynaptic mGluR mediated excitation of neurons in midbrain periaqueductal grey
Neuropharmacology 66 (2013) 348e354
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Postsynaptic mGluR mediated excitation of neurons in midbrain periaqueductal grey Adrianne R. Wilson-Poe, Vanessa A. Mitchell, Christopher W. Vaughan* Pain Management Research Institute, Level 13, Kolling Building, Kolling Institute of Medical Research, Northern Clinical School, The University of Sydney at Royal North Shore Hospital, St Leonards, NSW, Australia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 July 2011 Received in revised form 25 June 2012 Accepted 26 June 2012
Metabotropic glutamate (mGlu) receptors modulate pain from within the midbrain periaqueductal grey (PAG). In the present study, the postsynaptic mGlu receptor mediated effects on rat PAG neurons were examined using whole-cell patch-clamp recordings in brain slices. The selective group I agonist DHPG (10 mM) produced an inward current in all PAG neurons tested which was associated with a near parallel shift in the currentevoltage relationship. By contrast, the group II and III mGlu receptor agonists DCG-IV (1 mM) and L-AP4 (3 mM) produced an outward current in only 10e20% of PAG neurons tested. The DHPG induced current was concentration dependent (EC50 ¼ 1.4 mM), was reduced by the mGlu1 antagonist CPCCOEt (100 mM), and was further reduced by CPCCOEt in combination with the mGlu5 antagonist MPEP (10 mM). The glutamate transport blocker TBOA (30 mM) also produced an inward current, however, this was largely abolished by CNQX (10 mM) plus AP5 (25 mM). Slow EPSCs were evoked following train, but not single shock stimulation, which were enhanced by TBOA (30 mM). The TBOA enhancement of slow EPSCs was abolished by MPEP plus CPCCOEt. These findings indicate that endogenously released glutamate, under conditions in which neurotransmitter spill-over is enhanced, activates group I mGlu receptors to produce excitatory currents within PAG. Thus, postsynaptic group I mGlu receptors have the potential to directly modulate the analgesic, behavioural and autonomic functions of the PAG. This article is part of a Special Issue entitled ‘Metabotropic Glutamate Receptors’. Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
Keywords: Metabotropic glutamate receptors Transporter Postsynaptic Synaptic transmission Pain Midbrain periaqueductal grey
1. Introduction Metabotropic glutamate G-protein-coupled (mGlu) receptors exert a wide range of cellular actions within the central nervous system (Anwyl, 1999). Based on their sequence homology and biochemical and pharmacological profiles, mGlu receptors have been classified into three main subtypes, namely group I (mGlu1 and 5), group II (mGlu2 and 3) and group III (mGlu4, 6, 7 and 8) receptors (Pin and Duvoisin, 1995). There is strong behavioural and electrophysiological evidence that mGlu receptors are involved in nociceptive processing, although most of these studies have focussed on actions at the level of the peripheral nociceptor and the spinal cord (Neugebauer, 2002). mGlu receptors are also present in a number brain regions involved in the modulation of nociceptive information, including the amygdala and the midbrain periaqueductal grey (PAG) (Ohishi et al., 1995; Shigemoto et al., 1992; Tamaru et al., 2001).
* Corresponding author. E-mail address: [email protected] (C.W. Vaughan). URL: http://www.pmri.med.usyd.edu.au
The PAG plays a pivotal role in integrating an animal’s somatomotor, autonomic and behavioural responses to threat, stress and pain, and is a major site of the analgesic actions of opioids and cannabinoids (Fields et al., 2006; Keay and Bandler, 2001). Microinjection of mGlu receptor agonists into brain regions such as the PAG can be either antinociceptive, or pronociceptive, depending upon the mGlu receptor subtype and pain model used (Kim et al., 2002; Maione et al., 1998, 2000; Marabese et al., 2007b). This analgesia could be mediated via distinct pre- and postsynaptic mechanisms. We have previously shown that, like opioids and cannabinoids, activation of presynaptic Gi/o-coupled group II and III mGlu receptors directly inhibits GABAergic synaptic transmission within the PAG (Drew and Vaughan, 2004). In addition, activation of postsynaptic Gq-coupled group I mGlu receptors inhibits GABAergic synaptic transmission indirectly through a process of retrograde endocannabinoid signalling and presynaptic cannabinoid CB1 receptors (Drew et al., 2008; Drew and Vaughan, 2004). The postsynaptic effects of group I, II and III mGlu receptor activation on rat PAG neurons in vitro are unknown and were the subject of the present study.
0028-3908/$ e see front matter Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuropharm.2012.06.057
A.R. Wilson-Poe et al. / Neuropharmacology 66 (2013) 348e354 2. Materials and methods 2.1. Animals and slice preparation Experiments were carried out on male and female Sprague-Dawley rats (15e24 days old), following the guidelines of the National Health and Medical Research Council ‘Australian code of practice for the care and use of animals for scientific purposes’ and with the approval of the Royal North Shore Hospital Animal Care and Ethics Committee. Animals were deeply anaesthetized with isoflurane, decapitated and coronal midbrain slices (300 mm) containing PAG were cut using a vibratome (VT1000S, Leica Microsystems, Nussloch, Germany) in ice-cold artificial cerebrospinal fluid (ACSF), of the following composition: (in mM): NaCl 126, KCl 2.5, NaH2PO4 1.4, MgCl2 1.2, CaCl2 2.4, glucose 11, NaHCO3 25, as described previously (Drew et al., 2005). The slices were maintained at 34 C in a submerged chamber containing ACSF equilibrated with 95% O2 and 5% CO2. Individual slices were then transferred to a chamber and superfused continuously (1.8 ml min1) with ACSF at 34 C. 2.2. Drug solutions DL-2-Amino-5-phosphonovaleric acid (AP5), ()-baclofen, 7-(Hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt), (2S,3S,4R)-3(Carboxymethyl)-4-isopropylpyrrolidine-2-carboxylic acid (dihydrokainic acid, DHK), (RS)-3,5-dihydroxyphenylglycine (DHPG), picrotoxin and strychnine hydrochloride were obtained from Sigma (Sydney, Australia); L-(þ)-2-amino-4phosphonobutyric acid (L-AP4), (2S)-3-[[(1S)-1-(3,4-Dichlorophenyl)ethyl]amino-2hydroxypropyl](phenylmethyl)phosphinic acid hydrochloride (CGP55845), 3-[2-[4-(4-Fluorobenzoyl)-1-piperidinyl]ethyl]-2,4[1H,3H]-quinazolinedione tartrate 0 0 0 0 (ketanserin), (2S,2 R,3 R)-2-(2 ,3 -dicarboxycyclopropyl)glycine (DCG-IV), and DLthreo-b-benzyloxyaspartic acid (TBOA) from Tocris Cookson (Bristol, UK); 6-Cyano-7nitroquinoxaline-2,3-dione disodium (CNQX), 2-Methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP), SR95531, (S)-N-tert-Butyl-3-(4-(2-methoxyphenyl)-piperazin1-yl)-2-phenylpropanamide dihydrochloride (WAY100135) and tetrodotoxin (TTX) from Abcam Biochemicals (Cambridge, U.K.). Stock solutions of all drugs were made in distilled water, except MPEP and TBOA which were made in dimethylsulfoxide. All agents were diluted to working concentrations in ACSF (solvent 0.03% v/v) immediately before use and applied by superfusion. 2.3. Electrophysiology and analysis
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neurons, using concentrations which we have previously shown to produce maximal presynaptic inhibition within PAG (Drew and Vaughan, 2004). DHPG (10 mM) produced an inward current in all neurons tested throughout the ventrolateral, lateral and dorsolateral PAG (Fig. 1A, B, mean current ¼ 27 3 pA, p < 0.0001, n ¼ 25). By contrast, DCG-IV (1 mM) and L-AP4 (3 mM) had no effect in most neurons, producing an outward current in 10% and 18% of PAG neurons, respectively (Fig. 1A, B, n ¼ 2/20 and 4/22). When averaged across all neurons, DCG-IV and L-AP4 did not produce a significant change in membrane current (Fig. 1B, mean currents ¼ 3 2 pA and 1 1 pA, p ¼ 0.2 and 0.4). Subsequent application of the GABAB agonist baclofen (10 mM) produced an outward current in all of these neurons which was reversed by the GABAB antagonist CGP55845 (1 mM) (Fig. 1A, 33 4 pA, n ¼ 25). The inward current produced by DHPG (10 mM) was associated with a near parallel inward shift in the currentevoltage relationship (Fig. 1C). The mean slope conductance in these neurons was 1.4 0.3 nS and 2.5 0.7 nS in the absence and 1.6 0.4 nS and 2.3 0.6 nS in the presence of DHPG, when measured between 60/90 mV and 110/130 mV (n ¼ 6). By contrast, subsequent application of baclofen (10 mM) produced an outward current which reversed polarity at 110 4 mV (Fig. 1C, n ¼ 6). In these neurons, baclofen (10 mM) increased the slope conductance to 1.9 0.4 nS and 3.3 0.7 nS, when measured between 60/ 90 mV and 110/130 mV (n ¼ 6). 3.2. DHPG acts via postsynaptic mGlu1 and mGlu5 receptors DHPG produced an inward current over concentrations ranging from 0.3 to 30 mM (n ¼ 4e14). The inward current produced by
PAG neurons were visualized using infrared Dodt contrast gradient optics on an upright microscope (BX51; Olympus, Tokyo, Japan). Whole-cell voltage-clamp recordings at 60 mV (liquid junction potential corrected) were made using an Axopatch 200B (Molecular Devices, Sunnyvale, USA) with an internal solution comprising (mM): K-gluconate 95, KCl 30, NaCl 15, MgCl2 2, HEPES 10, EGTA 11, MgATP 2, NaGTP 0.3 and QX-314 3; with pH of 7.3 and osmolality of 280e285 mosmol l1. Series resistance (<25 MU) was compensated by 80% and continuously monitored during experiments. In some experiments EPSCs (excitatory postsynaptic currents) were electrically evoked via a unipolar glass electrode (tip diameter 5e20 mm) placed 50e200 mm from the recording electrode (10e55 V, 100e200 ms). Single and train stimuli (2e20 per train, rate ¼ 100 s1) were delivered every 12 s. Recordings of postsynaptic currents and evoked EPSCs were filtered (0.5, 2 kHz low-pass filter) and sampled (1, 10 kHz) for analysis (Axograph X, Axograph Scientific Software, Sydney, Australia). Neurons were considered to respond to an agonist if it produced a current of greater than 5 pA (approximately 1 standard deviation of the noise level) and reversed upon washout. Agonist induced currents were measured as the difference between the peak current during agonist application compared to that immediately prior to application. The total charge transfer of evoked EPSCs was measured as the area of the EPSC relative to the baseline over a 10 ms period prior to stimulation. The time constant of the evoked EPSC decay phase was fit to an exponential using the least square method based. The EPSCs had mixed decay phase kinetics, with some being best fit by one and others by two exponentials; this was decided by the fit with the lowest sums of squared errors. The tau values (decay time constant) presented are the weighted average. All numerical data are expressed as mean SEM. Statistical comparisons of mean drug effects were made using paired Student’s t-test, and comparisons between multiple treatment groups with a one-way ANOVA (using Dunnett’s, or the NewmaneKeuls corrections for post-hoc comparisons), or two-way ANOVA (using a Bonferroni correction for post-hoc comparisons). Comparisons of proportions were made using Chi-squared, or Fisher’s exact tests. Differences were considered significant if p < 0.05.
3. Results 3.1. Group I, but not group II and III mGlu receptor activation produces an inward current in most PAG neurons We first examined the postsynaptic effects of the group I, II and III mGlu receptor agonists DHPG, DCG-IV and L-AP4 on PAG
Fig. 1. The group I mGlu receptor agonist DHGP produces an inward current in all PAG neurons. (A) Current trace of a PAG neuron during superfusion of DCG-IV (1 mM), L-AP4 (3 mM), DHPG (10 mM), baclofen (10 mM) and CGP55845 (CGP, 1 mM). (B) Scatter plot of the inward currents produced by DHPG, DCG-IV and L-AP4, with the bars indicating the mean current. (C) Currentevoltage relationship for a PAG neuron before (Pre), during DHPG, then during baclofen and following washout of baclofen (Wash). Membrane currents were evoked by voltage steps in 10 mV increments from 60 mV to 130 mV (250 ms duration). In (B) responders are shown as filled circles and non-responders as open circles; *** denotes p < 0.0001. (A) and (C) are from different neurons.
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DHPG was concentration dependent, with an EC50 of 1.4 mM (90% confidence interval 0.8e2.4 mM) and a Hill slope of 1.2 0.2 (Fig. 2A). The DHPG (10 mM) induced current was observed in the combined presence of TTX (500 nM), the non-NMDA and NMDA antagonists CNQX (5 mM) and AP5 (50 mM), the GlyR antagonist strychnine (3 mM), and the GABAA antagonist SR95531 (10 mM) (mean current ¼ 24 6 pA, n ¼ 5). We next examined whether the inward current produced by DHPG was mediated by group I, II, or III mGlu receptors. To do this, DHPG (10 mM) was applied twice at a 10e12 min interval with, or without addition of mGlu receptor antagonists after the first application of DHPG (Fig. 2C, D). The current produced by the first and second applications of DHPG differed significantly across treatment groups (Fig. 2B, p ¼ 0.03, F4,26 ¼ 4.2). When no antagonist was added, DHPG produced inward currents which were similar during the first and second applications (Fig. 2B, C, n ¼ 6, p > 0.05, DHPG 2:1 ¼ 81 10%). Addition of LY341495 (100 mM), at a concentration which antagonises group I, II and III mGlu receptors (Drew et al., 2008), reduced the DHPG induced inward current (Fig. 2B, p < 0.01, n ¼ 4, DHPG 2:1 ¼ 14 9%). Addition of LY341495 (0.3 mM), at a concentration which antagonises only group II and III mGlu receptors (Drew et al., 2008), had no effect on the DHPG induced inward current (Fig. 2B, p > 0.05, n ¼ 4, DHPG 2:1 ¼ 98 9%). While addition of the mGlu5 antagonist MPEP (10 mM) had no effect on the DHPG induced inward current (Fig. 2B, p > 0.05, n ¼ 7, DHPG 2:1 ¼ 88 15%), addition of the mGlu1 antagonist CPCCOEt (100 mM) reduced the inward current produced by DHPG (Fig. 2B, p < 0.01, n ¼ 6, DHPG 2:1 ¼ 61 11%). Addition of
both MPEP and CPCCOEt reduced the inward current produced by DHPG (p < 0.0001, n ¼ 8, DHPG 2:1 ¼ 26 7%) to a greater extent than by MPEP, or CPCCOEt alone (p < 0.05) (Fig. 2B, D). Subsequent application of baclofen (10 mM) produced an outward current in these neurons which was similar in the absence and presence of LY341495, MPEP and CPCCOEt (Fig. 2C, D, p > 0.05). 3.3. Basal endogenously released glutamate acts mainly via ionotropic glutamate receptors We next examined whether endogenously released glutamate activates mGlu receptors. Application of the glutamate transport blocker TBOA (30e100 mM) produced a slowly developing inward current which gradually reversed following washout in all neurons tested (Fig. 3A, C, p < 0.0001, n ¼ 20). Another glutamate transport blocker DHK (200 mM) produced a similar inward current in 4/5 neurons tested (Fig. 3C, p ¼ 0.02, n ¼ 5). To determine whether these were mediated by ionotropic or metabotropic receptors, we examined the effect of TBOA and DHK in the presence of CNQX (10 mM) and AP5 (50 mM), at concentrations which completely abolished spontaneous and evoked fast, ionotropic glutamate receptor mediated EPSCs (see inset Fig. 4A). In the presence of CNQX, AP5, strychnine (3 mM) and SR95531 (10 mM), application of TBOA (100 mM) produced an inward current in 5 out of 9 neurons tested, which was significant when averaged across all neurons (Fig. 3B, C, p ¼ 0.006). Under these conditions, the TBOA induced current was less than that observed in the absence of CNQX, AP5, strychnine and SR95531 (Fig. 3C, p ¼ 0.006). DHK (200 mM) did not
Fig. 2. The DHPG induced inward current is mediated by group I mGlu1 and mGlu5 receptors. (A) Concentration response curve for the inward current produced by DHPG. (B) Bar chart showing the mean current produced during consecutive applications of DHPG (10 mM, 1st and 2nd), in which either no antagonist (Ctl, Control), LY341495 (LY, 100 and 0.3 mM), MPEP (10 mM), CPCCOEt (CP, 100 mM), or both MPEP and CPCCOEt were applied continuously after washout of the first application of DHPG. (C, D) Current traces of two neurons during repeated application of DHPG (10 mM) at a 12 min interval, and then baclofen (10 mM). In (D) MPEP (10 mM) and CPCCOEt (100 mM) were added after the first washout of DHPG. In (A) each data point represents the mean S.E.M. over the number of neurons shown for that concentration. In (B) **, # denote p < 0.01, 0.0001.
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Fig. 3. Glutamate transport blockade produces mainly ionotropic glutamate receptor mediated inward currents in PAG neurons. Current traces of two PAG neurons during superfusion of TBOA (30 mM), baclofen (10 mM) and CGP55845 (CGP, 1 mM) in the (A) absence, and (B) presence of CNQX (10 mM) plus AP5 (50 mM). (C) Bar chart showing the mean current produced by TBOA (100 mM) and DHK (200 mM), in the absence (open bars) and presence of CNQX and AP5 (filled bar). In (B) *, ** and # denote p < 0.05, 0.01 and 0.0001.
produce a significant inward in the presence of CNQX, AP5, strychnine and SR95531 (Fig. 3C, n ¼ 4, p ¼ 0.1). Subsequent application of baclofen (10 mM) produced an outward current in all of these neurons (Fig. 3A, B, n ¼ 38). These observations suggest that TBOA produces largely ionotropic glutamate receptor mediated currents under basal conditions. 3.4. Synaptically released glutamate activates mGlu receptors We next examined whether synaptically released glutamate evoked EPSCs and whether these were enhanced by blockade of
glutamate transporters. In the presence of a cocktail of antagonists, including strychnine (3 mM), SR95531 (10 mM), plus GABAB (1 mM CGP55845) and 5-HT (1 mM WAY100135 and 1 mM ketanserin) and mAChR (1 mM atropine) receptor antagonists, fast EPSCs were evoked by single electrical stimuli which were abolished by addition of CNQX (10 mM) and AP5 (50 mM) (Fig. 4A, B). When trains of 20 stimuli (rate ¼ 100 s1) were applied in the presence of the antagonist cocktail, an additional slower EPSC was evoked in these neurons (Fig. 4A, n ¼ 7). Addition of CNQX/AP5 abolished the fast component, but did not affect the slow component of train stimulus evoked EPSCs (Fig. 4B, n ¼ 7). In the presence of CNQX/AP5 plus the
Fig. 4. Train stimulation evokes slow EPSCs which are enhanced by glutamate transport blockade. Individual traces of EPSCs evoked by a single stimulus (T) and a train of 20 stimuli (20T, rate ¼ 100 s1) in a PAG neuron, in (A) the presence of an antagonist cocktail (Pre ¼ GABAB, 5-HT1/2, mACh antagonists, see text), (B) after addition of CNQX (10 mM) and AP5 (50 mM), and (C) then after addition of TBOA (30 mM). (D) Plots of the mean evoked EPSC charge transfer elicited by trains of 1, 2, 5, 10 and 20 stimuli in the presence of the antagonist cocktail, CNQX and AP5 (CNQX/AP5), then after addition (þTBOA). In (AeC) the stimulus artefact is blanked and the strokes indicate the time of electrical stimulation. In (D) *, # denote p < 0.05, 0.0001 (CNQX/AP5 versus þTBOA). The inset in (A) shows the single evoked EPSC expanded in time, before and after addition of CNQX and AP5 (from A and B, respectively).
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antagonist cocktail, the total charge transfer of evoked EPSCs progressively increased with the number of stimuli in the train (Fig. 4D, n ¼ 7). In these neurons, both the fast and slow components of train evoked EPSCs were abolished by addition of tetrodotoxin (1 mM, n ¼ 4). In the presence of CNQX, AP5 and the antagonist cocktail, addition of TBOA (30 mM, n ¼ 6) produced an increase in the charge transfer of EPSCs evoked by train stimuli, but had no effect on EPSCs evoked by single stimuli (Fig. 4B, D, n ¼ 7). The effect of TBOA on evoked EPSC charge transfer varied with the number of stimuli in the train (p < 0.0001, F4,48 ¼ 8.27), TBOA only producing an increase in charge transfer of EPSCs evoked by longer duration trains (Fig. 4D, p < 0.05 and 0.0001 for 10 and 20 stimuli, see also control in Fig. 5C). The TBOA enhancement was associated with an increase in the decay time constant of EPSC elicited by a train of 20 stimuli (Fig. 5D, p ¼ 0.01). In another group of neurons we examined whether the TBOA induced increase in slow EPSCs was mediated by group I, or II/III mGlu receptors, by examining the effect of mGlu receptor antagonists on EPSCs evoked by trains of 20 stimuli in the presence of CNQX, AP5 and the above antagonist cocktail. Addition of LY341495 (300 nM) had no effect on the charge transfer, or decay time constant of evoked EPSCs (Fig. 5A, C and D, p > 0.05, n ¼ 7). Subsequent addition of TBOA (30 mM) produced an increase in charge transfer and decay time constant of evoked EPSCs (Fig. 5A, C and D, p < 0.001 and 0.05). By contrast, addition of MPEP (10 mM) plus CPCCOEt (100 mM) produced a reduction in charge transfer and decay time constant of evoked EPSCs (Fig. 5B, C and D, p < 0.05, n ¼ 6). Furthermore, subsequent addition of TBOA (30 mM) did not
produce an increase in charge transfer and decay time constant of evoked EPSCs (Fig. 5B, C, and D p > 0.05). 4. Discussion In the present study it has been demonstrated that group I mGlu receptor activation produces an inward current in all PAG neurons, while group II and III mGlu receptor activation produces an outward current in only a minority of PAG neurons. The group I mGlu receptor induced current was likely to be mediated by both mGlu1 and mGlu5 receptors. Group I mGlu receptors were also activated by endogenous glutamate, but mainly under conditions of enhanced activity and when glutamate uptake was reduced. A number of observations indicated that the inward current produced by the group I agonist DHPG in the present study was mediated via postsynaptic mGlu1 and mGlu5 subtypes. First, the inward excitatory current produced by DHPG was observed in the presence of TTX, CNQX, AP5, strychnine and picrotoxin, indicating that it was a direct postsynaptic action. Second, the DHPG induced current was concentration-dependent, with an EC50 similar to that for both group I mGlu1 and mGlu5 receptor subtypes (Conn and Pin, 1997). The DHPG induced current was reduced by broad spectrum mGlu receptor antagonism (LY341495, 100 mM), but not by group II/ III mGlu receptor antagonism (LY341495, 0.3 mM). In addition, the DHPG induced current was reduced by the mGlu1 antagonist CPCCOEt (100 mM), but not by the mGlu5 antagonist MPEP. The DHPG induced current, however, was reduced to a greater extent by a combination of both CPCCOEt and MPEP. This suggests that while mGlu1 receptors predominate, both mGlu1 and mGlu5 receptors
Fig. 5. The enhancement of slow EPSCs by TBOA is largely group I mGlu receptor mediated. (AeB) Individual traces of evoked EPSCs elicited in two PAG neurons by a train of 20 stimuli (20T, rate ¼ 100 s1) in the presence of an antagonist cocktail (GABAB, 5-HT1/2, mACh antagonists, see text), CNQX (10 mM) and AP5 (50 mM) (Pre); after addition of mGlu receptor antagonists; then after addition TBOA (30 mM). The antagonists used in these two neurons were (A) LY341495 (LY, 0.3 mM), and (B) MPEP (10 mM) plus CPCCOEt (100 mM) (MPEP/CP). Scatter plots of the (C) charge transfer and (D) decay time constant (tau) of slow evoked EPSCs elicited by a train of 20 stimuli before (Pre), then after addition of mGlu receptor antagonists (LY 0.3 mM, or MPEP 10mM þ CPCCOET 100 mM), then TBOA (30 mM). In (CeD), control data is also shown in which no antagonists were added (Control, same data as in Fig. 4D); horizontal bars are the mean values; *, ** and *** denote p < 0.05, 0.01 and 0.001.
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contribute to the DHPG induced current. The DHPG induced inward current was likely to be mediated by activation of a non-selective cation conductance and inhibition of an inwardly rectifying Kþ conductance because it was associated with a near parallel shift in the currentevoltage relationship, as observed previously for neurokinin, neurotensin and cholecystokinin Gq-coupled GPCRs within the PAG (Drew et al., 2005; Mitchell et al., in press, 2009). In contrast to DHPG, the group II and III agonists DCG-IV and LAP4 had no effect on most PAG neurons, and produced an inhibitory outward current in only a small subpopulation of PAG neurons. This difference between the incidence of postsynaptic group I and group II/III mediated actions is consistent with anatomical evidence for a relatively greater expression of postsynaptic group I mGlu receptors within the PAG (Azkue et al., 1997; de Novellis et al., 2003; Marabese et al., 2007b). The receptor subtypes and conductances underlying the group II and III induced outward currents were not examined because of the relatively small proportion of responders, but was likely to be mediated by activation of an inwardly rectifying Kþ conductance, as observed for other Gi/o-coupled GPCRs within PAG, including m-opioid, somatostatin and nociception/orphaninFQ receptors (Chieng and Christie, 1994; Connor et al., 2004; Vaughan et al., 1997). The above experiments indicate that synthetic agonists activate postsynaptic group I mGlu receptors within the PAG. We therefore examined whether enhancing endogenous glutamate levels with glutamate transport blockers could induce mGlu receptor mediated postsynaptic currents. While both TBOA and DHK produced inward currents, these were largely abolished by CNQX and AP5. In addition, single and paired electrical stimulation evoked fast EPSCs which were completely abolished by CNQX and AP5, and were subsequently unaffected by TBOA. This suggests that, under basal conditions and during low rates of stimulation, endogenously released glutamate induces postsynaptic currents which are predominantly mediated by ionotropic glutamate receptors (Vaughan and Christie, 1997). In contrast to single and paired stimulation, slow EPSCs were evoked during prolonged repetitive stimulation in the presence of ionotropic glutamate receptor blockers, plus antagonists for metabotropic GABAB, 5-HT1/2 and mACh receptors. These slow EPSCs were reduced by group I (MPEP plus CPCCOET), but not group II/III mGlu receptor antagonists. This suggests that the slow EPSCs were at least partly mGlu1/5 receptor mediated, with the incomplete blockade of slow EPSCs by MPEP/CPCCOEt being due to surmountable group I mGlu receptor antagonism. Indeed, this was the case for the DHPG induced current. While these experiments were carried out in the presence of a cocktail of GPCR antagonists, a role for other unidentified metabotropic receptors cannot be excluded. Finally, TBOA increased the charge transfer and duration of these slow EPSCs, and this enhancement was abolished by group I, but not group II/III mGlu receptor blockade. These findings indicate that group I mGlu receptor mediated synaptic currents are only observed under conditions in which spill-over occurs, such as that during enhanced neuronal activity, and that this is enhanced by blockade of glutamate uptake. It is therefore likely that group I mGlu receptors are distal to the synaptic zone, compared to ionotropic receptors, and that their activation is tightly regulated by transporters, as observed in the spinal cord and other regions of the nervous system (e.g. Batchelor et al., 1994; Bengtson et al., 2004; Galik et al., 2008; Kim et al., 2003). The mGlu receptor induced postsynaptic currents observed in the present study differ to their effects on GABAergic synaptic transmission within the PAG. We have previously shown that group I, II and III mGlu receptor activation produces presynaptic inhibition of neurotransmitter release from GABAergic terminals throughout the PAG (Drew and Vaughan, 2004). The group II and III induced
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inhibition is mediated via presynaptic Gi/o-coupled mGlu receptors, while group I induced inhibition is mediated by postsynaptic Gqcoupled mGlu5- induced endocannabinoid signalling which activates presynaptic cannabinoid CB1 receptors (Drew et al., 2008; Drew and Vaughan, 2004). These findings indicate that group I mGlu receptor actions predominate at the postsynaptic level, while group II/III actions predominate at the presynaptic level. The postsynaptic receptors and the transporters involved in group I mGlu receptor mediated postsynaptic currents and retrograde endocannabinoid signalling also differed. In the present study the DHPG induced inward current was mediated by both mGlu1 and mGlu5 receptors, and was induced by both broad spectrum (TBOA) and glial cell selective EAAT2 (DHK) transport blockers. This differs to retrograde endocannabinoid signalling within the PAG which is induced exclusively by the mGlu5 subtype and by TBOA, but not by DHK (Drew et al., 2009, 2008; Mitchell et al., in press; Mitchell et al., 2009). These findings suggest that postsynaptic coupling to ion channels and endocannabinoid production cascades are regulated by spatially distinct group I mGlu receptor subtypes and glutamate transporters in the postsynaptic membrane. Analgesics such as opioids and cannabinoids activate a descending pathway which projects via the midbrain PAG and rostroventral medial medulla to produce analgesia at the level of the spinal cord dorsal horn (Fields et al., 2006). Microinjection of group I mGlu receptor agonists into the PAG produces analgesia (Maione et al., 1998, 2000). This group I mGlu receptor induced analgesia is likely to be produced by direct excitation and indirectly via presynaptic inhibition of GABAergic inputs impinging upon PAG neurons. In the present study, both mGlu1 and mGlu5 activation produced postsynaptic excitation. In addition, we have previously shown that postsynaptic mGlu5 activation indirectly inhibits GABAergic synaptic transmission via retrograde endocannabinoid signalling (Drew et al., 2009, 2008; Drew and Vaughan, 2004). These divergent cellular findings parallel the differing roles of mGlu1 and mGlu5 receptors in cannabinoid induced analgesia from within the PAG (de Novellis et al., 2005). The influence of group II and III mGlu receptors within the PAG on nociception is even more complex. Intra-PAG microinjection of group II agonists have antiand pro-nociceptive actions in different pain assays, while agonists at the mGlu7 and mGlu8 group III mGlu receptor subtypes have pro- and anti-nociceptive effects, respectively (Maione et al., 1998, 2000; Marabese et al., 2007a; Marabese et al., 2007b). The present study, however, could not resolve the cellular mechanisms underlying these differences because both group II and III mGlu receptor agonists inhibited small subpopulations of PAG neurons. In addition, we have previously reported that group II and III mGlu receptor activation presynaptically inhibits GABAergic synaptic transmission to a similar extent in all PAG neurons (Drew and Vaughan, 2004). The latter is consistent with an analgesic action, although the roles of specific group II and III mGlu receptor subtypes have not been explored. Overall, these findings indicate that group I, II and II mGlu receptors modulate analgesia from within the PAG in a complex manner which is related to the actions of specific mGlu receptor subtypes on pre- and postsynaptic elements within this brain structure. Acknowledgements This work was supported by Australian National Health and Medical Research Council grant 1003097 to CWV. References Anwyl, R., 1999. Metabotropic glutamate receptors: electrophysiological properties and role in plasticity. Brain Res. Rev. 29, 83e120.
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Azkue, J.J., Knöpfel, T., Kuhn, R., Mateos, J.M., Grandes, P., 1997. Distribution of the metabotropic glutamate receptor subtype mGluR5 in rat periaqueductal grey and relationship with ascending spinofugal afferents. Neurosci. Lett. 228, 1e4. Batchelor, A.M., Madge, D.J., Garthwaite, J., 1994. Synaptic activation of metabotropic glutamate receptors in the parallel Fibre-Purkinje cell pathway in rat cerebellar slices. Neuroscience 63 (4), 911e915. Bengtson, C.P., Tozzi, A., Bernardi, G., Mercuri, N.B., 2004. Transient receptor potential-like channels mediate metabotropic glutamate receptor EPSCs in rat dopamine neurones. J. Physiol. 555 (Pt 2), 323e330. Chieng, B., Christie, M.J., 1994. Hyperpolarization by opioids acting on m-receptors of a sub-population of rat periaqueductal gray neurones in vitro. Brit. J. Pharmacol. 113 (1), 121e128. Conn, P.J., Pin, J.P., 1997. Pharmacology and functions of metabotropic glutamate receptors. Annu. Rev. Pharmacol. Toxicol. 37, 205e237. Connor, M., Bagley, E.E., Mitchell, V.A., Ingram, S.L., Christie, M.J., Humphrey, P.P.A., et al., 2004. Cellular actions of somatostatin on rat periaqueductal grey neurons in vitro. Brit. J. Pharmacol. 142 (8), 1273e1280. de Novellis, V., Marabese, I., Palazzo, E., Rossi, F., Berrino, L., Rodella, L., et al., 2003. Group I metabotropic glutamate receptors modulate glutamate and gammaaminobutyric acid release in the periaqueductal grey of rats. Eur. J. Pharmacol. 462 (1e3), 73e81. de Novellis, V., Mariani, L., Palazzo, E., Vita, D., Marabese, I., Scafuro, M., et al., 2005. Periaqueductal grey CB1 cannabinoid and metabotropic glutamate subtype 5 receptors modulate changes in rostral ventromedial medulla neuronal activities induced by subcutaneous formalin in the rat. Neuroscience 134 (1), 269e281. Drew, G.M., Lau, B.K., Vaughan, C.W., 2009. Substance P Drives endocannabinoidmediated disinhibition in a midbrain descending analgesic pathway. J. Neurosci. 29 (22), 7220e7229. Drew, G.M., Mitchell, V.A., Vaughan, C.W., 2008. Glutamate spillover modulates GABAergic synaptic transmission in the rat midbrain periaqueductal grey via metabotropic glutamate receptors and endocannabinoid signaling. J. Neurosci. 28 (4), 808e815. Drew, G.M., Mitchell, V.A., Vaughan, C.W., 2005. Postsynaptic actions of substance P on rat periaqueductal grey neurons in vitro. Neuropharmacology 49 (5), 587e595. Drew, G.M., Vaughan, C.W., 2004. Multiple metabotropic glutamate receptor subtypes modulate GABAergic neurotransmission in rat periaqueductal grey neurons in vitro. Neuropharmacology 46 (7), 927e934. Fields, H.L., Basbaum, A.I., Heinricher, M.M., 2006. Central nervous systems mechanisms of pain modulation. In: McMahon, S.B., Koltzenburg, M. (Eds.), Textbook of Pain, fifth ed. Elsevier, Churchill Livingston, Philadelphia, pp. 125e142. Galik, J., Youn, D.H., Kolaj, M., Randic, M., 2008. Involvement of group I metabotropic glutamate receptors and glutamate transporters in the slow excitatory synaptic transmission in the spinal cord dorsal horn. Neuroscience 154 (4), 1372e1387. Keay, K.A., Bandler, R., 2001. Parallel circuits mediating distinct emotional coping reactions to different types of stress. Neurosci. Biobehav. Rev. 25 (7e8), 669e678.
Kim, S.J., Calejesan, A.A., Zhuo, M., 2002. Activation of brainstem metabotropic glutamate receptors inhibits spinal nociception in adult rats. Pharmacol. Biochem. Behav. 73, 429e437. Kim, S.J., Kim, Y.S., Yuan, J.P., Petralia, R.S., Worley, P.F., Linden, D.J., 2003. Activation of the TRPC1 cation channel by metabotropic glutamate receptor mGluR1. Nature 426 (6964), 285e291. Maione, S., Marabese, I., Leyva, J., Palazzo, E., de Novellis, V., Rossi, F., 1998. Characterisation of mGluRs which modulate nociception in the PAG of the mouse. Neuropharmacology 37 (12), 1475e1483. Maione, S., Oliva, P., Marabese, I., Palazzo, E., Rossi, F., Berrino, L., et al., 2000. Periaqueductal gray matter metabotropic glutamate receptors modulate formalininduced nociception. Pain 85 (1e2), 183e189. Marabese, I., de Novellis, V., Palazzo, E., Scafuro, M.A., Vita, D., Rossi, F., et al., 2007a. Effects of (S)-3,4-DCPG, an mGlu8 receptor agonist, on inflammatory and neuropathic pain in mice. Neuropharmacology 52 (2), 253e262. Marabese, I., Rossi, F., Palazzo, E., de Novellis, V., Starowicz, K., Cristino, L., et al., 2007b. Periaqueductal gray metabotropic glutamate receptor subtype 7 and 8 mediate opposite effects on amino acid release, rostral ventromedial medulla cell activities, and thermal nociception. J. Neurophysiol. 98 (1), 43e53. Mitchell, V.A., Jeong, H.J., Drew, G.M., Vaughan, C.W. Cholecystokinin exerts an effect via the endocannabinoid system to inhibit GABAergic transmission in midbrain periaqueductal gray. Neuropsychopharmacology, in press. Mitchell, V.A., Kawahara, H., Vaughan, C.W., 2009. Neurotensin inhibition of GABAergic transmission via mGluR-induced endocannabinoid signalling in rat periaqueductal grey. J. Physiol. 587 (11), 2511e2520. Neugebauer, V., 2002. Metabotropic glutamate receptors e important modulators of nociception and pain behavior. Pain 98, 1e8. Ohishi, H., Akazawa, C., Shigemoto, R., Nakanishi, S., Mizuno, N., 1995. Distributions of the mRNAs for L-2-amino-4-phosphonobutyrate-sensitive metabotropic glutamate receptors, mGluR4 and mGluR7, in the rat brain. J. Comp. Neurol. 360, 555e570. Pin, J.-P., Duvoisin, R., 1995. The metabotropic glutamate receptors: structure and functions. Neuropharmacology 34, 1e26. Shigemoto, R., Nakanishi, S., Mizuno, N., 1992. Distribution of the mRNA for a metabotropic glutamate receptor (mGluR1) in the central nervous system: an in situ hybridization study in adult and developing rat. J. Comp. Neurol. 322, 121e135. Tamaru, Y., Nomura, S., Mizuno, N., Shigemoto, R., 2001. Distribution of metabotropic glutamate receptor mGluR3 in the mouse CNS: differential location relative to pre- and postsynaptic sites. Neuroscience 106, 481e503. Vaughan, C.W., Christie, M.J., 1997. Presynaptic inhibitory action of opioids on synaptic transmission in the rat periaqueductal grey in vitro. J. Phsyiol. (Lond.) 498, 463e472. Vaughan, C.W., Ingram, S.L., Christie, M.J., 1997. Actions of the ORL(1) receptor ligand nociceptin on membrane properties of rat periaqueductal gray neurons in vitro. J. Neurosci. 17 (3), 996e1003.
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Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm
Postsynaptic mGluR mediated excitation of neurons in midbrain periaqueductal grey Adrianne R. Wilson-Poe, Vanessa A. Mitchell, Christopher W. Vaughan* Pain Management Research Institute, Level 13, Kolling Building, Kolling Institute of Medical Research, Northern Clinical School, The University of Sydney at Royal North Shore Hospital, St Leonards, NSW, Australia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 July 2011 Received in revised form 25 June 2012 Accepted 26 June 2012
Metabotropic glutamate (mGlu) receptors modulate pain from within the midbrain periaqueductal grey (PAG). In the present study, the postsynaptic mGlu receptor mediated effects on rat PAG neurons were examined using whole-cell patch-clamp recordings in brain slices. The selective group I agonist DHPG (10 mM) produced an inward current in all PAG neurons tested which was associated with a near parallel shift in the currentevoltage relationship. By contrast, the group II and III mGlu receptor agonists DCG-IV (1 mM) and L-AP4 (3 mM) produced an outward current in only 10e20% of PAG neurons tested. The DHPG induced current was concentration dependent (EC50 ¼ 1.4 mM), was reduced by the mGlu1 antagonist CPCCOEt (100 mM), and was further reduced by CPCCOEt in combination with the mGlu5 antagonist MPEP (10 mM). The glutamate transport blocker TBOA (30 mM) also produced an inward current, however, this was largely abolished by CNQX (10 mM) plus AP5 (25 mM). Slow EPSCs were evoked following train, but not single shock stimulation, which were enhanced by TBOA (30 mM). The TBOA enhancement of slow EPSCs was abolished by MPEP plus CPCCOEt. These findings indicate that endogenously released glutamate, under conditions in which neurotransmitter spill-over is enhanced, activates group I mGlu receptors to produce excitatory currents within PAG. Thus, postsynaptic group I mGlu receptors have the potential to directly modulate the analgesic, behavioural and autonomic functions of the PAG. This article is part of a Special Issue entitled ‘Metabotropic Glutamate Receptors’. Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
Keywords: Metabotropic glutamate receptors Transporter Postsynaptic Synaptic transmission Pain Midbrain periaqueductal grey
1. Introduction Metabotropic glutamate G-protein-coupled (mGlu) receptors exert a wide range of cellular actions within the central nervous system (Anwyl, 1999). Based on their sequence homology and biochemical and pharmacological profiles, mGlu receptors have been classified into three main subtypes, namely group I (mGlu1 and 5), group II (mGlu2 and 3) and group III (mGlu4, 6, 7 and 8) receptors (Pin and Duvoisin, 1995). There is strong behavioural and electrophysiological evidence that mGlu receptors are involved in nociceptive processing, although most of these studies have focussed on actions at the level of the peripheral nociceptor and the spinal cord (Neugebauer, 2002). mGlu receptors are also present in a number brain regions involved in the modulation of nociceptive information, including the amygdala and the midbrain periaqueductal grey (PAG) (Ohishi et al., 1995; Shigemoto et al., 1992; Tamaru et al., 2001).
* Corresponding author. E-mail address: [email protected] (C.W. Vaughan). URL: http://www.pmri.med.usyd.edu.au
The PAG plays a pivotal role in integrating an animal’s somatomotor, autonomic and behavioural responses to threat, stress and pain, and is a major site of the analgesic actions of opioids and cannabinoids (Fields et al., 2006; Keay and Bandler, 2001). Microinjection of mGlu receptor agonists into brain regions such as the PAG can be either antinociceptive, or pronociceptive, depending upon the mGlu receptor subtype and pain model used (Kim et al., 2002; Maione et al., 1998, 2000; Marabese et al., 2007b). This analgesia could be mediated via distinct pre- and postsynaptic mechanisms. We have previously shown that, like opioids and cannabinoids, activation of presynaptic Gi/o-coupled group II and III mGlu receptors directly inhibits GABAergic synaptic transmission within the PAG (Drew and Vaughan, 2004). In addition, activation of postsynaptic Gq-coupled group I mGlu receptors inhibits GABAergic synaptic transmission indirectly through a process of retrograde endocannabinoid signalling and presynaptic cannabinoid CB1 receptors (Drew et al., 2008; Drew and Vaughan, 2004). The postsynaptic effects of group I, II and III mGlu receptor activation on rat PAG neurons in vitro are unknown and were the subject of the present study.
0028-3908/$ e see front matter Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuropharm.2012.06.057
A.R. Wilson-Poe et al. / Neuropharmacology 66 (2013) 348e354 2. Materials and methods 2.1. Animals and slice preparation Experiments were carried out on male and female Sprague-Dawley rats (15e24 days old), following the guidelines of the National Health and Medical Research Council ‘Australian code of practice for the care and use of animals for scientific purposes’ and with the approval of the Royal North Shore Hospital Animal Care and Ethics Committee. Animals were deeply anaesthetized with isoflurane, decapitated and coronal midbrain slices (300 mm) containing PAG were cut using a vibratome (VT1000S, Leica Microsystems, Nussloch, Germany) in ice-cold artificial cerebrospinal fluid (ACSF), of the following composition: (in mM): NaCl 126, KCl 2.5, NaH2PO4 1.4, MgCl2 1.2, CaCl2 2.4, glucose 11, NaHCO3 25, as described previously (Drew et al., 2005). The slices were maintained at 34 C in a submerged chamber containing ACSF equilibrated with 95% O2 and 5% CO2. Individual slices were then transferred to a chamber and superfused continuously (1.8 ml min1) with ACSF at 34 C. 2.2. Drug solutions DL-2-Amino-5-phosphonovaleric acid (AP5), ()-baclofen, 7-(Hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt), (2S,3S,4R)-3(Carboxymethyl)-4-isopropylpyrrolidine-2-carboxylic acid (dihydrokainic acid, DHK), (RS)-3,5-dihydroxyphenylglycine (DHPG), picrotoxin and strychnine hydrochloride were obtained from Sigma (Sydney, Australia); L-(þ)-2-amino-4phosphonobutyric acid (L-AP4), (2S)-3-[[(1S)-1-(3,4-Dichlorophenyl)ethyl]amino-2hydroxypropyl](phenylmethyl)phosphinic acid hydrochloride (CGP55845), 3-[2-[4-(4-Fluorobenzoyl)-1-piperidinyl]ethyl]-2,4[1H,3H]-quinazolinedione tartrate 0 0 0 0 (ketanserin), (2S,2 R,3 R)-2-(2 ,3 -dicarboxycyclopropyl)glycine (DCG-IV), and DLthreo-b-benzyloxyaspartic acid (TBOA) from Tocris Cookson (Bristol, UK); 6-Cyano-7nitroquinoxaline-2,3-dione disodium (CNQX), 2-Methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP), SR95531, (S)-N-tert-Butyl-3-(4-(2-methoxyphenyl)-piperazin1-yl)-2-phenylpropanamide dihydrochloride (WAY100135) and tetrodotoxin (TTX) from Abcam Biochemicals (Cambridge, U.K.). Stock solutions of all drugs were made in distilled water, except MPEP and TBOA which were made in dimethylsulfoxide. All agents were diluted to working concentrations in ACSF (solvent 0.03% v/v) immediately before use and applied by superfusion. 2.3. Electrophysiology and analysis
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neurons, using concentrations which we have previously shown to produce maximal presynaptic inhibition within PAG (Drew and Vaughan, 2004). DHPG (10 mM) produced an inward current in all neurons tested throughout the ventrolateral, lateral and dorsolateral PAG (Fig. 1A, B, mean current ¼ 27 3 pA, p < 0.0001, n ¼ 25). By contrast, DCG-IV (1 mM) and L-AP4 (3 mM) had no effect in most neurons, producing an outward current in 10% and 18% of PAG neurons, respectively (Fig. 1A, B, n ¼ 2/20 and 4/22). When averaged across all neurons, DCG-IV and L-AP4 did not produce a significant change in membrane current (Fig. 1B, mean currents ¼ 3 2 pA and 1 1 pA, p ¼ 0.2 and 0.4). Subsequent application of the GABAB agonist baclofen (10 mM) produced an outward current in all of these neurons which was reversed by the GABAB antagonist CGP55845 (1 mM) (Fig. 1A, 33 4 pA, n ¼ 25). The inward current produced by DHPG (10 mM) was associated with a near parallel inward shift in the currentevoltage relationship (Fig. 1C). The mean slope conductance in these neurons was 1.4 0.3 nS and 2.5 0.7 nS in the absence and 1.6 0.4 nS and 2.3 0.6 nS in the presence of DHPG, when measured between 60/90 mV and 110/130 mV (n ¼ 6). By contrast, subsequent application of baclofen (10 mM) produced an outward current which reversed polarity at 110 4 mV (Fig. 1C, n ¼ 6). In these neurons, baclofen (10 mM) increased the slope conductance to 1.9 0.4 nS and 3.3 0.7 nS, when measured between 60/ 90 mV and 110/130 mV (n ¼ 6). 3.2. DHPG acts via postsynaptic mGlu1 and mGlu5 receptors DHPG produced an inward current over concentrations ranging from 0.3 to 30 mM (n ¼ 4e14). The inward current produced by
PAG neurons were visualized using infrared Dodt contrast gradient optics on an upright microscope (BX51; Olympus, Tokyo, Japan). Whole-cell voltage-clamp recordings at 60 mV (liquid junction potential corrected) were made using an Axopatch 200B (Molecular Devices, Sunnyvale, USA) with an internal solution comprising (mM): K-gluconate 95, KCl 30, NaCl 15, MgCl2 2, HEPES 10, EGTA 11, MgATP 2, NaGTP 0.3 and QX-314 3; with pH of 7.3 and osmolality of 280e285 mosmol l1. Series resistance (<25 MU) was compensated by 80% and continuously monitored during experiments. In some experiments EPSCs (excitatory postsynaptic currents) were electrically evoked via a unipolar glass electrode (tip diameter 5e20 mm) placed 50e200 mm from the recording electrode (10e55 V, 100e200 ms). Single and train stimuli (2e20 per train, rate ¼ 100 s1) were delivered every 12 s. Recordings of postsynaptic currents and evoked EPSCs were filtered (0.5, 2 kHz low-pass filter) and sampled (1, 10 kHz) for analysis (Axograph X, Axograph Scientific Software, Sydney, Australia). Neurons were considered to respond to an agonist if it produced a current of greater than 5 pA (approximately 1 standard deviation of the noise level) and reversed upon washout. Agonist induced currents were measured as the difference between the peak current during agonist application compared to that immediately prior to application. The total charge transfer of evoked EPSCs was measured as the area of the EPSC relative to the baseline over a 10 ms period prior to stimulation. The time constant of the evoked EPSC decay phase was fit to an exponential using the least square method based. The EPSCs had mixed decay phase kinetics, with some being best fit by one and others by two exponentials; this was decided by the fit with the lowest sums of squared errors. The tau values (decay time constant) presented are the weighted average. All numerical data are expressed as mean SEM. Statistical comparisons of mean drug effects were made using paired Student’s t-test, and comparisons between multiple treatment groups with a one-way ANOVA (using Dunnett’s, or the NewmaneKeuls corrections for post-hoc comparisons), or two-way ANOVA (using a Bonferroni correction for post-hoc comparisons). Comparisons of proportions were made using Chi-squared, or Fisher’s exact tests. Differences were considered significant if p < 0.05.
3. Results 3.1. Group I, but not group II and III mGlu receptor activation produces an inward current in most PAG neurons We first examined the postsynaptic effects of the group I, II and III mGlu receptor agonists DHPG, DCG-IV and L-AP4 on PAG
Fig. 1. The group I mGlu receptor agonist DHGP produces an inward current in all PAG neurons. (A) Current trace of a PAG neuron during superfusion of DCG-IV (1 mM), L-AP4 (3 mM), DHPG (10 mM), baclofen (10 mM) and CGP55845 (CGP, 1 mM). (B) Scatter plot of the inward currents produced by DHPG, DCG-IV and L-AP4, with the bars indicating the mean current. (C) Currentevoltage relationship for a PAG neuron before (Pre), during DHPG, then during baclofen and following washout of baclofen (Wash). Membrane currents were evoked by voltage steps in 10 mV increments from 60 mV to 130 mV (250 ms duration). In (B) responders are shown as filled circles and non-responders as open circles; *** denotes p < 0.0001. (A) and (C) are from different neurons.
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DHPG was concentration dependent, with an EC50 of 1.4 mM (90% confidence interval 0.8e2.4 mM) and a Hill slope of 1.2 0.2 (Fig. 2A). The DHPG (10 mM) induced current was observed in the combined presence of TTX (500 nM), the non-NMDA and NMDA antagonists CNQX (5 mM) and AP5 (50 mM), the GlyR antagonist strychnine (3 mM), and the GABAA antagonist SR95531 (10 mM) (mean current ¼ 24 6 pA, n ¼ 5). We next examined whether the inward current produced by DHPG was mediated by group I, II, or III mGlu receptors. To do this, DHPG (10 mM) was applied twice at a 10e12 min interval with, or without addition of mGlu receptor antagonists after the first application of DHPG (Fig. 2C, D). The current produced by the first and second applications of DHPG differed significantly across treatment groups (Fig. 2B, p ¼ 0.03, F4,26 ¼ 4.2). When no antagonist was added, DHPG produced inward currents which were similar during the first and second applications (Fig. 2B, C, n ¼ 6, p > 0.05, DHPG 2:1 ¼ 81 10%). Addition of LY341495 (100 mM), at a concentration which antagonises group I, II and III mGlu receptors (Drew et al., 2008), reduced the DHPG induced inward current (Fig. 2B, p < 0.01, n ¼ 4, DHPG 2:1 ¼ 14 9%). Addition of LY341495 (0.3 mM), at a concentration which antagonises only group II and III mGlu receptors (Drew et al., 2008), had no effect on the DHPG induced inward current (Fig. 2B, p > 0.05, n ¼ 4, DHPG 2:1 ¼ 98 9%). While addition of the mGlu5 antagonist MPEP (10 mM) had no effect on the DHPG induced inward current (Fig. 2B, p > 0.05, n ¼ 7, DHPG 2:1 ¼ 88 15%), addition of the mGlu1 antagonist CPCCOEt (100 mM) reduced the inward current produced by DHPG (Fig. 2B, p < 0.01, n ¼ 6, DHPG 2:1 ¼ 61 11%). Addition of
both MPEP and CPCCOEt reduced the inward current produced by DHPG (p < 0.0001, n ¼ 8, DHPG 2:1 ¼ 26 7%) to a greater extent than by MPEP, or CPCCOEt alone (p < 0.05) (Fig. 2B, D). Subsequent application of baclofen (10 mM) produced an outward current in these neurons which was similar in the absence and presence of LY341495, MPEP and CPCCOEt (Fig. 2C, D, p > 0.05). 3.3. Basal endogenously released glutamate acts mainly via ionotropic glutamate receptors We next examined whether endogenously released glutamate activates mGlu receptors. Application of the glutamate transport blocker TBOA (30e100 mM) produced a slowly developing inward current which gradually reversed following washout in all neurons tested (Fig. 3A, C, p < 0.0001, n ¼ 20). Another glutamate transport blocker DHK (200 mM) produced a similar inward current in 4/5 neurons tested (Fig. 3C, p ¼ 0.02, n ¼ 5). To determine whether these were mediated by ionotropic or metabotropic receptors, we examined the effect of TBOA and DHK in the presence of CNQX (10 mM) and AP5 (50 mM), at concentrations which completely abolished spontaneous and evoked fast, ionotropic glutamate receptor mediated EPSCs (see inset Fig. 4A). In the presence of CNQX, AP5, strychnine (3 mM) and SR95531 (10 mM), application of TBOA (100 mM) produced an inward current in 5 out of 9 neurons tested, which was significant when averaged across all neurons (Fig. 3B, C, p ¼ 0.006). Under these conditions, the TBOA induced current was less than that observed in the absence of CNQX, AP5, strychnine and SR95531 (Fig. 3C, p ¼ 0.006). DHK (200 mM) did not
Fig. 2. The DHPG induced inward current is mediated by group I mGlu1 and mGlu5 receptors. (A) Concentration response curve for the inward current produced by DHPG. (B) Bar chart showing the mean current produced during consecutive applications of DHPG (10 mM, 1st and 2nd), in which either no antagonist (Ctl, Control), LY341495 (LY, 100 and 0.3 mM), MPEP (10 mM), CPCCOEt (CP, 100 mM), or both MPEP and CPCCOEt were applied continuously after washout of the first application of DHPG. (C, D) Current traces of two neurons during repeated application of DHPG (10 mM) at a 12 min interval, and then baclofen (10 mM). In (D) MPEP (10 mM) and CPCCOEt (100 mM) were added after the first washout of DHPG. In (A) each data point represents the mean S.E.M. over the number of neurons shown for that concentration. In (B) **, # denote p < 0.01, 0.0001.
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Fig. 3. Glutamate transport blockade produces mainly ionotropic glutamate receptor mediated inward currents in PAG neurons. Current traces of two PAG neurons during superfusion of TBOA (30 mM), baclofen (10 mM) and CGP55845 (CGP, 1 mM) in the (A) absence, and (B) presence of CNQX (10 mM) plus AP5 (50 mM). (C) Bar chart showing the mean current produced by TBOA (100 mM) and DHK (200 mM), in the absence (open bars) and presence of CNQX and AP5 (filled bar). In (B) *, ** and # denote p < 0.05, 0.01 and 0.0001.
produce a significant inward in the presence of CNQX, AP5, strychnine and SR95531 (Fig. 3C, n ¼ 4, p ¼ 0.1). Subsequent application of baclofen (10 mM) produced an outward current in all of these neurons (Fig. 3A, B, n ¼ 38). These observations suggest that TBOA produces largely ionotropic glutamate receptor mediated currents under basal conditions. 3.4. Synaptically released glutamate activates mGlu receptors We next examined whether synaptically released glutamate evoked EPSCs and whether these were enhanced by blockade of
glutamate transporters. In the presence of a cocktail of antagonists, including strychnine (3 mM), SR95531 (10 mM), plus GABAB (1 mM CGP55845) and 5-HT (1 mM WAY100135 and 1 mM ketanserin) and mAChR (1 mM atropine) receptor antagonists, fast EPSCs were evoked by single electrical stimuli which were abolished by addition of CNQX (10 mM) and AP5 (50 mM) (Fig. 4A, B). When trains of 20 stimuli (rate ¼ 100 s1) were applied in the presence of the antagonist cocktail, an additional slower EPSC was evoked in these neurons (Fig. 4A, n ¼ 7). Addition of CNQX/AP5 abolished the fast component, but did not affect the slow component of train stimulus evoked EPSCs (Fig. 4B, n ¼ 7). In the presence of CNQX/AP5 plus the
Fig. 4. Train stimulation evokes slow EPSCs which are enhanced by glutamate transport blockade. Individual traces of EPSCs evoked by a single stimulus (T) and a train of 20 stimuli (20T, rate ¼ 100 s1) in a PAG neuron, in (A) the presence of an antagonist cocktail (Pre ¼ GABAB, 5-HT1/2, mACh antagonists, see text), (B) after addition of CNQX (10 mM) and AP5 (50 mM), and (C) then after addition of TBOA (30 mM). (D) Plots of the mean evoked EPSC charge transfer elicited by trains of 1, 2, 5, 10 and 20 stimuli in the presence of the antagonist cocktail, CNQX and AP5 (CNQX/AP5), then after addition (þTBOA). In (AeC) the stimulus artefact is blanked and the strokes indicate the time of electrical stimulation. In (D) *, # denote p < 0.05, 0.0001 (CNQX/AP5 versus þTBOA). The inset in (A) shows the single evoked EPSC expanded in time, before and after addition of CNQX and AP5 (from A and B, respectively).
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antagonist cocktail, the total charge transfer of evoked EPSCs progressively increased with the number of stimuli in the train (Fig. 4D, n ¼ 7). In these neurons, both the fast and slow components of train evoked EPSCs were abolished by addition of tetrodotoxin (1 mM, n ¼ 4). In the presence of CNQX, AP5 and the antagonist cocktail, addition of TBOA (30 mM, n ¼ 6) produced an increase in the charge transfer of EPSCs evoked by train stimuli, but had no effect on EPSCs evoked by single stimuli (Fig. 4B, D, n ¼ 7). The effect of TBOA on evoked EPSC charge transfer varied with the number of stimuli in the train (p < 0.0001, F4,48 ¼ 8.27), TBOA only producing an increase in charge transfer of EPSCs evoked by longer duration trains (Fig. 4D, p < 0.05 and 0.0001 for 10 and 20 stimuli, see also control in Fig. 5C). The TBOA enhancement was associated with an increase in the decay time constant of EPSC elicited by a train of 20 stimuli (Fig. 5D, p ¼ 0.01). In another group of neurons we examined whether the TBOA induced increase in slow EPSCs was mediated by group I, or II/III mGlu receptors, by examining the effect of mGlu receptor antagonists on EPSCs evoked by trains of 20 stimuli in the presence of CNQX, AP5 and the above antagonist cocktail. Addition of LY341495 (300 nM) had no effect on the charge transfer, or decay time constant of evoked EPSCs (Fig. 5A, C and D, p > 0.05, n ¼ 7). Subsequent addition of TBOA (30 mM) produced an increase in charge transfer and decay time constant of evoked EPSCs (Fig. 5A, C and D, p < 0.001 and 0.05). By contrast, addition of MPEP (10 mM) plus CPCCOEt (100 mM) produced a reduction in charge transfer and decay time constant of evoked EPSCs (Fig. 5B, C and D, p < 0.05, n ¼ 6). Furthermore, subsequent addition of TBOA (30 mM) did not
produce an increase in charge transfer and decay time constant of evoked EPSCs (Fig. 5B, C, and D p > 0.05). 4. Discussion In the present study it has been demonstrated that group I mGlu receptor activation produces an inward current in all PAG neurons, while group II and III mGlu receptor activation produces an outward current in only a minority of PAG neurons. The group I mGlu receptor induced current was likely to be mediated by both mGlu1 and mGlu5 receptors. Group I mGlu receptors were also activated by endogenous glutamate, but mainly under conditions of enhanced activity and when glutamate uptake was reduced. A number of observations indicated that the inward current produced by the group I agonist DHPG in the present study was mediated via postsynaptic mGlu1 and mGlu5 subtypes. First, the inward excitatory current produced by DHPG was observed in the presence of TTX, CNQX, AP5, strychnine and picrotoxin, indicating that it was a direct postsynaptic action. Second, the DHPG induced current was concentration-dependent, with an EC50 similar to that for both group I mGlu1 and mGlu5 receptor subtypes (Conn and Pin, 1997). The DHPG induced current was reduced by broad spectrum mGlu receptor antagonism (LY341495, 100 mM), but not by group II/ III mGlu receptor antagonism (LY341495, 0.3 mM). In addition, the DHPG induced current was reduced by the mGlu1 antagonist CPCCOEt (100 mM), but not by the mGlu5 antagonist MPEP. The DHPG induced current, however, was reduced to a greater extent by a combination of both CPCCOEt and MPEP. This suggests that while mGlu1 receptors predominate, both mGlu1 and mGlu5 receptors
Fig. 5. The enhancement of slow EPSCs by TBOA is largely group I mGlu receptor mediated. (AeB) Individual traces of evoked EPSCs elicited in two PAG neurons by a train of 20 stimuli (20T, rate ¼ 100 s1) in the presence of an antagonist cocktail (GABAB, 5-HT1/2, mACh antagonists, see text), CNQX (10 mM) and AP5 (50 mM) (Pre); after addition of mGlu receptor antagonists; then after addition TBOA (30 mM). The antagonists used in these two neurons were (A) LY341495 (LY, 0.3 mM), and (B) MPEP (10 mM) plus CPCCOEt (100 mM) (MPEP/CP). Scatter plots of the (C) charge transfer and (D) decay time constant (tau) of slow evoked EPSCs elicited by a train of 20 stimuli before (Pre), then after addition of mGlu receptor antagonists (LY 0.3 mM, or MPEP 10mM þ CPCCOET 100 mM), then TBOA (30 mM). In (CeD), control data is also shown in which no antagonists were added (Control, same data as in Fig. 4D); horizontal bars are the mean values; *, ** and *** denote p < 0.05, 0.01 and 0.001.
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contribute to the DHPG induced current. The DHPG induced inward current was likely to be mediated by activation of a non-selective cation conductance and inhibition of an inwardly rectifying Kþ conductance because it was associated with a near parallel shift in the currentevoltage relationship, as observed previously for neurokinin, neurotensin and cholecystokinin Gq-coupled GPCRs within the PAG (Drew et al., 2005; Mitchell et al., in press, 2009). In contrast to DHPG, the group II and III agonists DCG-IV and LAP4 had no effect on most PAG neurons, and produced an inhibitory outward current in only a small subpopulation of PAG neurons. This difference between the incidence of postsynaptic group I and group II/III mediated actions is consistent with anatomical evidence for a relatively greater expression of postsynaptic group I mGlu receptors within the PAG (Azkue et al., 1997; de Novellis et al., 2003; Marabese et al., 2007b). The receptor subtypes and conductances underlying the group II and III induced outward currents were not examined because of the relatively small proportion of responders, but was likely to be mediated by activation of an inwardly rectifying Kþ conductance, as observed for other Gi/o-coupled GPCRs within PAG, including m-opioid, somatostatin and nociception/orphaninFQ receptors (Chieng and Christie, 1994; Connor et al., 2004; Vaughan et al., 1997). The above experiments indicate that synthetic agonists activate postsynaptic group I mGlu receptors within the PAG. We therefore examined whether enhancing endogenous glutamate levels with glutamate transport blockers could induce mGlu receptor mediated postsynaptic currents. While both TBOA and DHK produced inward currents, these were largely abolished by CNQX and AP5. In addition, single and paired electrical stimulation evoked fast EPSCs which were completely abolished by CNQX and AP5, and were subsequently unaffected by TBOA. This suggests that, under basal conditions and during low rates of stimulation, endogenously released glutamate induces postsynaptic currents which are predominantly mediated by ionotropic glutamate receptors (Vaughan and Christie, 1997). In contrast to single and paired stimulation, slow EPSCs were evoked during prolonged repetitive stimulation in the presence of ionotropic glutamate receptor blockers, plus antagonists for metabotropic GABAB, 5-HT1/2 and mACh receptors. These slow EPSCs were reduced by group I (MPEP plus CPCCOET), but not group II/III mGlu receptor antagonists. This suggests that the slow EPSCs were at least partly mGlu1/5 receptor mediated, with the incomplete blockade of slow EPSCs by MPEP/CPCCOEt being due to surmountable group I mGlu receptor antagonism. Indeed, this was the case for the DHPG induced current. While these experiments were carried out in the presence of a cocktail of GPCR antagonists, a role for other unidentified metabotropic receptors cannot be excluded. Finally, TBOA increased the charge transfer and duration of these slow EPSCs, and this enhancement was abolished by group I, but not group II/III mGlu receptor blockade. These findings indicate that group I mGlu receptor mediated synaptic currents are only observed under conditions in which spill-over occurs, such as that during enhanced neuronal activity, and that this is enhanced by blockade of glutamate uptake. It is therefore likely that group I mGlu receptors are distal to the synaptic zone, compared to ionotropic receptors, and that their activation is tightly regulated by transporters, as observed in the spinal cord and other regions of the nervous system (e.g. Batchelor et al., 1994; Bengtson et al., 2004; Galik et al., 2008; Kim et al., 2003). The mGlu receptor induced postsynaptic currents observed in the present study differ to their effects on GABAergic synaptic transmission within the PAG. We have previously shown that group I, II and III mGlu receptor activation produces presynaptic inhibition of neurotransmitter release from GABAergic terminals throughout the PAG (Drew and Vaughan, 2004). The group II and III induced
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inhibition is mediated via presynaptic Gi/o-coupled mGlu receptors, while group I induced inhibition is mediated by postsynaptic Gqcoupled mGlu5- induced endocannabinoid signalling which activates presynaptic cannabinoid CB1 receptors (Drew et al., 2008; Drew and Vaughan, 2004). These findings indicate that group I mGlu receptor actions predominate at the postsynaptic level, while group II/III actions predominate at the presynaptic level. The postsynaptic receptors and the transporters involved in group I mGlu receptor mediated postsynaptic currents and retrograde endocannabinoid signalling also differed. In the present study the DHPG induced inward current was mediated by both mGlu1 and mGlu5 receptors, and was induced by both broad spectrum (TBOA) and glial cell selective EAAT2 (DHK) transport blockers. This differs to retrograde endocannabinoid signalling within the PAG which is induced exclusively by the mGlu5 subtype and by TBOA, but not by DHK (Drew et al., 2009, 2008; Mitchell et al., in press; Mitchell et al., 2009). These findings suggest that postsynaptic coupling to ion channels and endocannabinoid production cascades are regulated by spatially distinct group I mGlu receptor subtypes and glutamate transporters in the postsynaptic membrane. Analgesics such as opioids and cannabinoids activate a descending pathway which projects via the midbrain PAG and rostroventral medial medulla to produce analgesia at the level of the spinal cord dorsal horn (Fields et al., 2006). Microinjection of group I mGlu receptor agonists into the PAG produces analgesia (Maione et al., 1998, 2000). This group I mGlu receptor induced analgesia is likely to be produced by direct excitation and indirectly via presynaptic inhibition of GABAergic inputs impinging upon PAG neurons. In the present study, both mGlu1 and mGlu5 activation produced postsynaptic excitation. In addition, we have previously shown that postsynaptic mGlu5 activation indirectly inhibits GABAergic synaptic transmission via retrograde endocannabinoid signalling (Drew et al., 2009, 2008; Drew and Vaughan, 2004). These divergent cellular findings parallel the differing roles of mGlu1 and mGlu5 receptors in cannabinoid induced analgesia from within the PAG (de Novellis et al., 2005). The influence of group II and III mGlu receptors within the PAG on nociception is even more complex. Intra-PAG microinjection of group II agonists have antiand pro-nociceptive actions in different pain assays, while agonists at the mGlu7 and mGlu8 group III mGlu receptor subtypes have pro- and anti-nociceptive effects, respectively (Maione et al., 1998, 2000; Marabese et al., 2007a; Marabese et al., 2007b). The present study, however, could not resolve the cellular mechanisms underlying these differences because both group II and III mGlu receptor agonists inhibited small subpopulations of PAG neurons. In addition, we have previously reported that group II and III mGlu receptor activation presynaptically inhibits GABAergic synaptic transmission to a similar extent in all PAG neurons (Drew and Vaughan, 2004). The latter is consistent with an analgesic action, although the roles of specific group II and III mGlu receptor subtypes have not been explored. Overall, these findings indicate that group I, II and II mGlu receptors modulate analgesia from within the PAG in a complex manner which is related to the actions of specific mGlu receptor subtypes on pre- and postsynaptic elements within this brain structure. Acknowledgements This work was supported by Australian National Health and Medical Research Council grant 1003097 to CWV. References Anwyl, R., 1999. Metabotropic glutamate receptors: electrophysiological properties and role in plasticity. Brain Res. Rev. 29, 83e120.
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