Postsynaptic mGluR mediated excitation of neurons in midbrain periaqueductal grey.

Abstract

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 μM) produced an inward current in all PAG neurons tested which was associated with a near parallel shift in the current-voltage relationship. By contrast, the group II and III mGlu receptor agonists DCG-IV (1 μM) and l-AP4 (3 μM) produced an outward current in only 10-20% of PAG neurons tested. The DHPG induced current was concentration dependent (EC(50) = 1.4 μM), was reduced by the mGlu1 antagonist CPCCOEt (100 μM), and was further reduced by CPCCOEt in combination with the mGlu5 antagonist MPEP (10 μM). The glutamate transport blocker TBOA (30 μM) also produced an inward current, however, this was largely abolished by CNQX (10 μM) plus AP5 (25 μM). Slow EPSCs were evoked following train, but not single shock stimulation, which were enhanced by TBOA (30 μM). 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'.

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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
Article history:
Received 5 July 2011
Received in revised form
25 June 2012
Accepted 26 June 2012
Keywords:
Metabotropic glutamate receptors
Transporter
Postsynaptic
Synaptic transmission
Pain
Midbrain periaqueductal grey
a b s t r a c t
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 (EC50¼ 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 ? 2012 Published by Elsevier Ltd. All rights reserved.
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
beenclassifiedintothreemainsubtypes,namelygroupI(mGlu1and
5),groupII(mGlu2and3)andgroupIII(mGlu4,6,7and8)receptors
(Pin and Duvoisin, 1995). There is strong behavioural and electro-
physiological evidence that mGlu receptors are involved in noci-
ceptive processing, although mostof thesestudies havefocussed on
actions at the level of the peripheral nociceptor and the spinal cord
(Neugebauer, 2002). mGlu receptors are also present in a number
brainregionsinvolvedinthemodulationofnociceptiveinformation,
includingtheamygdalaandthemidbrainperiaqueductalgrey(PAG)
(Ohishi et al.,1995; Shigemoto et al.,1992; Tamaru et al., 2001).
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 pro-
nociceptive, 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
shownthat,likeopioids
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 post-
synaptic 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.
andcannabinoids,activation
* Corresponding author.
E-mail address: chris.vaughan@sydney.edu.au (C.W. Vaughan).
URL: http://www.pmri.med.usyd.edu.au
Contents lists available at SciVerse ScienceDirect
Neuropharmacology
journal homepage: www.elsevier.com/locate/neuropharm
0028-3908/$ e see front matter Crown Copyright ? 2012 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.neuropharm.2012.06.057
Neuropharmacology 66 (2013) 348e354

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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, NaH2PO41.4,
MgCl21.2, CaCl22.4, glucose 11, NaHCO325, as described previously (Drew et al.,
2005). The slices were maintained at 34?C in a submerged chamber containing
ACSF equilibrated with 95% O2and 5% CO2. Individual slices were then transferred to
a chamber and superfused continuously (1.8 ml min?1) with ACSF at 34?C.
2.2. Drug solutions
DL-2-Amino-5-phosphonovaleric acid (AP5), (?)-baclofen, 7-(Hydroxyimino)-
cyclopropa[b]chromen-1a-carboxylateethyl
(Carboxymethyl)-4-isopropylpyrrolidine-2-carboxylic
DHK),(RS)-3,5-dihydroxyphenylglycine
hydrochloride were obtained from Sigma (Sydney, Australia);
phosphonobutyric acid (L-AP4), (2S)-3-[[(1S)-1-(3,4-Dichlorophenyl)ethyl]amino-2-
hydroxypropyl](phenylmethyl)phosphinic
3-[2-[4-(4-Fluorobenzoyl)-1-piperidinyl]ethyl]-2,4[1H,3H]-quinazolinedione tartrate
(ketanserin), (2S,20R,30R)-2-(20,30-dicarboxycyclopropyl)glycine (DCG-IV), and DL-
threo-b-benzyloxyaspartic acid (TBOA) from Tocris Cookson (Bristol, UK); 6-Cyano-7-
nitroquinoxaline-2,3-dione disodium (CNQX), 2-Methyl-6-(phenylethynyl)pyridine
hydrochloride (MPEP), SR95531, (S)-N-tert-Butyl-3-(4-(2-methoxyphenyl)-piperazin-
1-yl)-2-phenylpropanamide dihydrochloride (WAY100135) and tetrodotoxin (TTX)
fromAbcamBiochemicals(Cambridge,U.K.). Stock solutions ofall drugswere madein
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) imme-
diately before use and applied by superfusion.
ester(CPCCOEt),
acid
picrotoxin
(2S,3S,4R)-3-
(dihydrokainic
and
L-(þ)-2-amino-4-
acid,
(DHPG),strychnine
acidhydrochloride(CGP55845),
2.3. Electrophysiology and analysis
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, MgCl22, HEPES 10, EGTA 11,
MgATP 2, NaGTP 0.3 and QX-314 3; with pH of 7.3 and osmolality of 280e285
mosmol l?1. 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 s?1) 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 Scien-
tific 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
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 dorsolat-
eral 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 aver-
aged across all neurons, DCG-IV and
a significant change in membrane current (Fig. 1B, mean cur-
rents ¼ 3 ? 2 pA and 1 ? 1 pA, p ¼ 0.2 and 0.4). Subsequent
application of the GABABagonist baclofen (10 mM) produced an
outward current in all of these neurons which was reversed by the
GABABantagonist 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 relation-
ship (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 (10mM) 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).
L-AP4 did not produce
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
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 EC50of 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 GABAAantagonist 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, F4,26¼ 4.2). When noantagonist
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 concentrationwhich 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 ¼14 ? 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 (100mM) reduced the inward current produced
by DHPG (Fig. 2B, p < 0.01, n ¼ 6, DHPG 2:1 ¼61 ?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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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 medi-
ated 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 addi-
tion of CNQX (10 mM) and AP5 (50 mM) (Fig. 4A, B). When trains of
20 stimuli (rate ¼ 100 s?1) 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 notaffect the slowcomponentof train stimulus
evoked EPSCs (Fig. 4B, n ¼ 7). In the presence of CNQX/AP5 plus the
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.
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 s?1) 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 compo-
nents of train evoked EPSCs were abolished by addition of tetro-
dotoxin (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 bytrain 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, F4,48¼ 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 antago-
nists 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 (100mM) produced a reduction in charge transferand
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 byendogenous 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 EC50similar to that
forbothgroupImGlu1andmGlu5receptorsubtypes(ConnandPin,
1997). The DHPG induced current was reduced by broad spectrum
mGlu receptor antagonism (LY341495,100 mM), but not bygroup 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 toa 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 s?1) 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 neu-
rokinin, 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 L-
AP4 had no effecton 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 acti-
vation of an inwardly rectifying Kþconductance, as observed for
other Gi/o-coupled GPCRs within PAG, including m-opioid, somato-
statin 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 addi-
tion, 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
predominantlymediated by ionotropic
(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 bygroup I (MPEP plus CPCCOET), but notgroup
II/III mGlu receptor antagonists. This suggests that the slow EPSCs
were at least partly mGlu1/5 receptor mediated, with the incom-
plete 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 indi-
catethat groupI mGlu receptor mediated synapticcurrents 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 iono-
tropic 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
transmissionwithin the PAG. We have previouslyshown that group
I, II and III mGlu receptoractivationproduces presynaptic inhibition
of neurotransmitter release from GABAergic terminals throughout
the PAG (Drew and Vaughan, 2004). The group II and III induced
glutamatereceptors
inhibition is mediated via presynaptic Gi/o-coupled mGlu receptors,
while group I induced inhibition is mediated by postsynaptic Gq-
coupled mGlu5- induced endocannabinoid signalling which acti-
vates presynaptic cannabinoid CB1receptors (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 (Drewet al., 2009, 2008; Mitchell et al., inpress; 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.
Analgesicssuch asopioids
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 receptorsin 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 anti-
and 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 under-
lying these differences because both group II and III mGlu receptor
agonists inhibited small subpopulations of PAG neurons. In addi-
tion, 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.
andcannabinoidsactivate
Acknowledgements
This work was supported by Australian National Health and
Medical Research Council grant 1003097 to CWV.
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