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The Journal of Neuroscience, December 15, 1998, 18(24):10269-10276
Enhanced Opioid Efficacy in Opioid Dependence Is Caused by an
Altered Signal Transduction Pathway
Susan L.
Ingram,
Christopher W.
Vaughan,
Elena E.
Bagley,
Mark
Connor, and
MacDonald J.
Christie
Department of Pharmacology and The Medical Foundation, The
University of Sydney, Sydney, New South Wales 2006, Australia
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ABSTRACT |
Chronic morphine administration induces adaptations in neurons
resulting in opioid tolerance and dependence. Functional studies have
implicated a role for the periaqueductal gray area (PAG) in the
expression of many signs of opioid withdrawal, but the cellular
mechanisms are not fully understood. This study describes an increased
efficacy, rather than tolerance, of opioid agonists at µ-receptors on
GABAergic (but not glutamatergic) nerve terminals in PAG after chronic
morphine treatment. Opioid withdrawal enhanced the amplitudes of
electrically evoked inhibitory synaptic currents mediated by
GABAA receptors and increased the frequency of spontaneous miniature GABAergic synaptic currents. These effects were not blocked
by 4-aminopyridine or dendrotoxin, although both Kv channel blockers
abolish acute opioid presynaptic inhibition of GABA release in PAG.
Instead, the withdrawal-induced increases were blocked by protein
kinase A inhibitors and occluded by metabolically stable cAMP analogs,
which do not prevent acute opioid actions. These findings indicate that
opioid dependence induces efficacious coupling of µ-receptors to
presynaptic inhibition in GABAergic nerve terminals via adenylyl
cyclase- and protein kinase A-dependent processes in PAG. The potential
role of these adaptations in expression of withdrawal behavior was
supported by inhibition of enhanced GABAergic synaptic transmission by
the  2 adrenoceptor agonist clonidine. These findings
provide a cellular mechanism that is consistent with other studies
demonstrating attenuated opioid withdrawal behavior after injections of
protein kinase A inhibitors into PAG and suggest a general mechanism
whereby opioid withdrawal may enhance synaptic neurotransmission.
Key words:
opioid efficacy; opioid dependence; opioid withdrawal; sensitization; periaqueductal gray; adenylyl cyclase; protein kinase A; synaptic plasticity
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INTRODUCTION |
Opioid addiction is a complex
phenomenon that consists of components including tolerance,
drug-seeking or craving, and physical dependence characterized by
withdrawal avoidance behaviors (Koob and LeMoal, 1997 ). Opioid
tolerance and withdrawal are believed to result from adaptations that
develop in multiple neural systems after chronic exposure to opioids.
Tolerance, or a diminished responsiveness to the inhibitory actions of
opioids, has been widely demonstrated to occur in opioid-sensitive
cells and is thought to involve functional uncoupling between opioid
receptors and their effectors (Law et al., 1982; Chavkin and Goldstein, 1984; Christie et al., 1987; Puttfarcken et al., 1988). However, opioid
receptor-effector uncoupling cannot fully account for physical dependence that is characterized by withdrawal signs or abnormal rebound responses in single neurons after administration of an opiate antagonist.
Biochemical indices of withdrawal rebound, such as hypertrophy of
adenylyl cyclase activity, have been widely reported during opioid
withdrawal (Avidor-Reiss et al., 1997). Enhanced excitability of CNS
neurons has also been reported during opioid withdrawal in cerebral
cortex and striatum (Fry et al., 1980), hypothalamus (Russell et al.,
1995), and dorsal horn of the spinal cord (Johnson and Duggan, 1981).
However, clear evidence of withdrawal rebound in the membrane
properties of single neurons and the link to biochemical adaptations
has usually been elusive, despite the demonstration of tolerance in the
same cells (Andrade et al., 1983; Christie et al., 1987; Wimpey et al.,
1989; Kennedy and Henderson, 1992).
Functional and biochemical studies have suggested a role for the
periaqueductal gray area (PAG) in the expression of many withdrawal
signs (Bozarth and Wise, 1984; Maldonado et al., 1992; Bozarth, 1994;
Chieng and Christie, 1996; Christie et al., 1997). The PAG is rich in
opioid receptors and endogenous opioids, and is a major site mediating
opioid analgesia (Reichling et al., 1988; Fields et al., 1991; Bandler
and Shipley, 1994; Mansour et al., 1995). Acutely, µ-opioids directly
inhibit a subpopulation of PAG neurons via activation of a postsynaptic
membrane K conductance (Chieng and Christie, 1994; Osborne et al.,
1996). During opioid withdrawal, opioid-sensitive neurons in PAG
display excessively enhanced action potential activity caused by
induction of a novel opioid-sensitive current distinct from the K
conductance acutely modulated by opioids (Chieng and Christie, 1996).
Acutely, µ-opioids also inhibit presynaptic GABAergic
neurotransmission in the PAG through production of metabolites of
arachidonic acid that activate 4-aminopyridine (4-AP) and
dendrotoxin-sensitive K channels (Kv channels, Vaughan and Christie,
1997; Vaughan et al., 1997). Opioid inhibition of GABAergic synaptic
transmission causes disinhibition of PAG projection neurons, which
leads to the activation of descending antinociceptive pathways as well
as other autonomic and somatic sequelae (Basbaum and Fields, 1984;
Reichling et al., 1988; Fields et al., 1991; Bandler and Shipley,
1994). Because the PAG is involved in the expression of withdrawal
behaviors, and withdrawal excites opioid-sensitive neurons in PAG, we
were interested in examining the effects of chronic morphine
administration on GABAergic synaptic transmission in the PAG.
A preliminary account of these findings has been presented elsewhere
(Ingram et al., 1997).
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MATERIALS AND METHODS |
Chronic treatment with morphine. Physical dependence
on morphine was induced by a series of injections of a sustained
release preparation of morphine base on alternate days over 5 d
(Chieng and Christie, 1996) and animals were used for recordings 1 or 2 d later. Morphine base was suspended with 0.1 ml mannide
mono-oleate (Arlacel A) and 0.4 ml of light liquid paraffin and made up
to 1 ml with 0.9% w/v NaCl in water. The subcutaneous injections (2 ml/kg) were made while the rats were under light halothane anesthesia.
Control rats were injected with vehicle solution. Animals were treated
with 100 mg/kg morphine in most experiments. In preliminary
experiments, injections of 50 mg/kg and 100 mg/kg morphine resulted in
greater naloxone-precipitated enhancement of GABAergic
neurotransmission than 25 mg/kg (235 ± 42%, n = 5; 201 ± 28%, n = 15; and 141 ± 9%,
n = 5, respectively).
Preparation of tissue and recording. Midbrain PAG slices
(280 µm) were cut from 4-6-week-old Sprague Dawley rats and were maintained at 34°C in a submerged chamber containing physiological saline equilibrated with 95% O2 and 5% CO2
and were later transferred to a superfusing chamber (32°C) for
recording. The extracellular solution contained (in mM):
NaCl, 126; KCl, 2.5; NaH2PO4, 1.4; MgCl2, 1.2; CaCl2, 2.4; glucose,
11; and NaHCO3, 25. Unless otherwise stated, brain
slices were maintained in vitro in 5 µM
morphine immediately after slicing until termination of experiments.
PAG neurons were visualized using infrared Nomarski optics, and
whole-cell voltage-clamp recordings were made using patch electrodes
(2-5 M ) containing (in mM): CsCl, 140; EGTA, 10; HEPES,
5; CaCl2, 2; and MgATP, 2, pH 7.3, osmolarity
270-290 mosmol/l. Series resistance (<12M) was compensated by 80%
and continuously monitored during experiments. Liquid junction
potentials of  4 mV were corrected.
GABAergic evoked IPSCs (eIPSCs) were elicited in neurons voltage
clamped at 75 mV via bipolar tungsten stimulating electrodes placed
near the recording electrode (rate: 0.03 Hz; stimuli: 5-50 V, 20-400
msec) in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 5 µM), and four to six consecutive responses were averaged (pClamp; Axon Instruments, Foster City, CA). Glutamatergic evoked EPSCs (eEPSCs) were evoked in the presence of bicuculline (30 µM). Spontaneous miniature IPSCs (mIPSCs) were obtained
in the presence of TTX (0.3 µM) and CNQX (3 µM), filtered at 2 kHz, and recorded on video tape (via a
Sony PCM501). mIPSCs were sampled at 5 kHz (Fetchex) for later off-line
analysis (Axograph; Axon Instruments). Events were detected by
selecting events in which nonconsecutive 1 msec segments exceeded a
preset threshold (set to 6-15 pA for a rejection rate of at least
10%) and were ranked by amplitude and interevent interval to construct
cumulative probability distributions. Time-mIPSC frequency plots were
constructed by counting the number of events above a preset threshold
(6-15 pA) in 30-60 sec epochs (Superscope; GW Instruments). Frequency
versus time plots were generated by normalizing the mIPSC frequency to the average of 5 min in control solution. All data are expressed as
mean ± SEM.
Drugs, reagents, and solutions. -Dendrotoxin
and staurosporin were obtained from Alamone Laboratories
(Jerusalem, Israel). Rp-8(4-chlorophenylthio)-adenosine-3',5'-cyclic
monophosphorothioate, 8-para-chlorophenylthioadenosine-3',5'-cyclic
monophosphorothioate (RP-8-CPT-cAMP-S) was from Biolog (Bremen,
Germany). N-[2(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89) was from Biomol (Plymouth, PA).
D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) was donated by the National Institute on Drug Abuse
(Bethesda, MD). Clonidine, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX),
and naloxone HCl were obtained from Research Biochemicals (Natick, MA).
Morphine base was from Glaxo. 4-AP, Arlacel A, bicuculline, 8-bromo-cAMP (8-Br-cAMP), D-Ala-Met-enkephalin-Gly-ol
(DAMGO), forskolin, met-enkephalin, and tetrodotoxin (TTX; Alamone
Laboratories) were from Sigma (St Louis, MO). CNQX was from Tocris
(Bristol, UK). Stock solutions of all drugs were diluted to working
concentrations in the extracellular solution immediately before use and
applied by superfusion. -Dendrotoxin was dissolved in extracellular
solution containing 0.1% bovine serum albumin. 8-Br-cAMP and
RP-8-CPT-cAMP-S, were dissolved directly in extracellular solution.
Forskolin, staurosporin, H-89, and CNQX were superfused with
0.01-0.1% DMSO. Stock solutions of all other drugs were made in
distilled water. The composition of physiological saline was altered in
experiments using CdCl2
(NaH2PO4-free).
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RESULTS |
Opioid efficacy is increased during dependence
Sprague Dawley rats were treated chronically with morphine to
induce dependence (Chieng and Christie, 1996). Whole-cell voltage-clamp recordings were made from brain slices containing the PAG that were
maintained in either morphine (5 µM) to prevent
spontaneous opioid withdrawal or normal external solution to study
spontaneous withdrawal. In studies in which slices were maintained in
morphine (5 µM), GABAA-mediated synaptic
currents were evoked in the absence and presence of naloxone (1 µM). Superfusion of naloxone potentiated GABAergic
synaptic currents in neurons from dependent animals compared with
vehicle controls (Fig.
1A; see Fig.
7A). The selective µ-receptor antagonist CTAP (1 µM, Kramer et al., 1989), also increased the amplitude of
eIPSCs in neurons from dependent animals (246 ± 43% of
prenaloxone baseline, n = 5 vs 129 ± 11%,
n = 4 in vehicle controls). This increase was specific
for GABAergic eIPSCs because naloxone produced only a 17 ± 6%
(n = 4) increase in glutamatergic eEPSCs in
neurons from dependent animals (see Fig. 7A). These results
suggest that the sustained presence of morphine present in tissue
in vitro (5 µM) produces greater inhibition of
GABAergic synaptic transmission in slices from dependent than control
animals.
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Figure 1.
Opioid withdrawal enhances GABAergic eIPSCs, and
morphine is more efficacious in neurons from dependent animals.
A, Averaged eIPSC traces elicited from single neurons
from vehicle-treated (Vehicle) and dependent animals
(Dependent) maintained in morphine (5 µM)
before and during superfusion of naloxone (1 µM).
B, eIPSC traces from single neurons from vehicle-treated
(Vehicle) or dependent animals
(Dependent) during spontaneous withdrawal (no morphine
for >1 hr) before (control), in the presence of
superfused morphine (10 µM; morphine), and
subsequently during cosuperfusion of naloxone (1 µM
naloxone; trace overlaid on
control trace).
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To directly test whether morphine was more efficacious after chronic
morphine treatment, brain slices were incubated in the absence of
morphine for 1-6 hr (spontaneously withdrawn). DAMGO is a full agonist
and morphine is a partial agonist in the PAG slice preparation, i.e.,
maximum inhibition of eIPSCs by morphine is consistently less than that
produced by the more efficacious µ-receptor agonists met-enkephalin
and DAMGO (Vaughan et al., 1997). In untreated tissue, morphine
produced 21 ± 9% (n = 6), 20 ± 8%
(n = 9), and 19 ± 2% (n = 6)
inhibition of eIPSCs at concentrations of 5, 10, and 30 µM, respectively. Moreover, the inhibition produced by
morphine (10 µM) was also less than that produced by
met-enkephalin (100 µM) when tested in the same cells
(28 ± 8% vs 71 ± 5%; n = 5;
p < 0.05). A supramaximal concentration of morphine
(10 µM) produced a greater inhibition of eIPSCs in
neurons from dependent animals (Fig. 1B; 43 ± 4% inhibition; n = 6) compared with vehicle controls
(21 ± 6%; n = 10; p < 0.025).
There were no residual effects of morphine in the spontaneously
withdrawn slices because naloxone reversed the effects of superfused
morphine only to control levels (Fig. 1B). Therefore,
the increased maximal effect of this partial agonist in slices from
dependent animals suggests enhanced efficacy of coupling between
µ-receptors and eIPSC inhibition. In addition, the potency of the
full agonist DAMGO was increased. Concentration-response curves showed
that DAMGO was fourfold more potent in spontaneously withdrawn neurons
(EC50 = 16 nM) than in vehicle controls
(EC50 = 65 nM; Fig.
2).
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Figure 2.
DAMGO is more efficacious in neurons from
dependent animals. Concentration-response relationship for percentage
inhibition of eIPSC amplitudes by the selective µ-opioid agonist
DAMGO in neurons from morphine-dependent (closed
circles, no morphine for >1 hr) and vehicle-treated
(open circles) animals. Each point shows the mean (± SEM) of responses of three to eight neurons. A logistic function was
fitted to the concentration-response curves to determine the
EC50 (dependent, 16 nM;
vehicle, 65 nM).
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Increased opioid efficacy is mediated via changes in
presynaptic terminals
The effects of naloxone on TTX-insensitive spontaneous
mIPSCs in neurons from dependent animals were examined to
determine the site of increased sensitivity to morphine. GABAergic
mIPSCs were isolated in the presence of CNQX (3 µM) and
TTX (0.3 µM). Raw current traces are shown in Figure
3A. Comparisons of basal rate
between cells were not possible because of the large variation in basal
mIPSC frequency (range in dependent 0.3-3.5 Hz, in vehicle controls
0.5-3.3 Hz). All comparisons were made within each cell and normalized
to control amplitudes or frequencies. Naloxone increased the frequency
of mIPSCs only in neurons from dependent animals maintained in morphine
(Fig. 3A-C; see Fig. 7B), and had no
effect on the amplitudes of the mIPSCs in either group (98 ± 8%
of baseline in slices from dependent animals, n = 14 and 85 ± 6%, n = 7 in vehicle controls) (Fig.
3B). CTAP (1 µM) also increased the frequency
of mIPSCs in neurons from dependent animals (188 ± 34% of
baseline in slices from treated animals, n = 4 vs 123 ± 9%, n = 3 in vehicle controls) without
affecting the amplitude of mIPSCs (pooled dependent and vehicle
amplitudes were 89 ± 8%, n = 7). These findings
demonstrate that enhanced efficacy of µ-opioids to inhibit IPSCs in
PAG neurons results from adaptations that develop within GABAergic
nerve terminals after chronic treatment with morphine.
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Figure 3.
Opioid withdrawal enhances mIPSCs in neurons from
dependent animals. A, Consecutive raw current traces of
mIPSCs recorded from neurons maintained in morphine (5 µM) in the absence and presence of naloxone (1 µM). B, Cumulative distribution plots of
mIPSC amplitude and frequency from the dependent neuron shown above in
A for the presence of morphine (5 µM,
solid line) and after superfusion of naloxone (1 µM, dashed line). C, Time
course of normalized mIPSC frequency in neurons maintained in morphine
(5 µM) during superfusion of naloxone (1 µM) in dependent (closed circles) and
vehicle-treated (open circles) animals. Each point
represents the mean (± SEM) of four to six neurons.
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Enhanced GABAergic evoked currents are blocked by clonidine
If enhanced GABAergic transmission in PAG is involved in
expression of withdrawal behavior, then other manipulations that suppress withdrawal might also be expected to overcome the
naloxone-precipitated enhancement of GABAergic neurotransmission. The
2 adrenoceptor agonist clonidine is known to suppress
many of the characteristic signs of the opioid withdrawal syndrome
(Redmond and Krystal, 1984; Christie et al., 1997). Superfusion of
clonidine (1 µM) produced significantly greater
inhibition of eIPSC amplitudes during withdrawal (in the presence of
naloxone, 1 µM) than in slices from vehicle controls
(Fig. 4). The effects of clonidine were
reversed by idazoxan (1 µM, data not shown). Moreover,
the inhibition produced by clonidine during withdrawal was sufficient to overcome the enhancement of eIPSC amplitude produced by naloxone in
slices from dependent animals (302 ± 95% of baseline in
naloxone, 109 ± 35% of baseline after addition of clonidine; 1 µM; n = 3; p > 0.6 for
clonidine plus naloxone vs baseline).
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Figure 4.
Clonidine (1 µM) inhibits eIPSCs to
a greater extent during withdrawal (in the presence of naloxone, 1 µM, or no morphine for >1 hr) than in neurons from
vehicle-treated animals (n = 6 per group). The
asterisk signifies statistical significance for
comparison between dependent and vehicle groups (unpaired
t test, p < 0.005).
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Enhanced opioid efficacy is not mediated by a 4-AP and
dendrotoxin-sensitive K channel
Acute µ-receptor-mediated inhibition of GABAergic synaptic
transmission in the PAG occurs through activation of 4-AP and
dendrotoxin-sensitive, voltage-dependent K channels (Vaughan et al.,
1997). However, 4-AP (100 µM) did not significantly
affect the naloxone-precipitated potentiation of eIPSCs in neurons from
dependent animals (Fig. 5A,
7A). Neither 4-AP nor dendrotoxin (100 nM)
affected inhibition of eIPSCs by DAMGO during spontaneous withdrawal
from dependent slices, but abolished inhibition of eIPSCs by DAMGO (100 nM) in vehicle controls (Fig. 5B; see Fig.
8).
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Figure 5.
Withdrawal-induced enhancement of eIPSC amplitude
and efficacy of morphine are not affected by 4-AP. A,
Averaged eIPSC traces elicited from single neurons from vehicle-treated
(Vehicle) and dependent animals
(Dependent) maintained in morphine (5 µM)
before and during superfusion of naloxone (1 µM) in the
presence of 4-AP (100 µM) throughout. B,
eIPSC traces from spontaneously withdrawing slices (no morphine for >1
hr) before (control), in the presence of
superfused DAMGO (100 nM; DAMGO), and then
after superfusion of naloxone (1 µM
naloxone; trace overlaid on
control trace) in the presence of 4-AP (100 µM) throughout.
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Naloxone also increased the frequency of mIPSCs in neurons from
dependent animals in the presence of 4-AP or dendrotoxin (Figs. 6,
7B) without affecting
amplitude distributions (pooled dependent and vehicle amplitudes in
4-AP, 98 ± 5%, n = 19; pooled amplitudes in
dendrotoxin, 102 ± 5%, n = 11). µ-Opioid
inhibition of synaptic transmission in spinal dorsal horn neurons has
been shown to be mediated by inhibition of presynaptic Ca entry (Hori
et al., 1992). However, the naloxone-induced increase in the frequency
of mIPSCs in PAG neurons from dependent animals persisted in the
presence of Cd (100 µM), high Mg (10 mM) and
absence of extracellular Ca (167 ± 19% of prenaloxone baseline,
n = 5 vs 95 ± 15%, n = 4 in vehicle control neurons). Naloxone had no effect on the amplitude of
mIPSCs in slices from dependent and vehicle control animals (pooled
amplitudes 95 ± 5%, n = 9). These results
demonstrate that a mechanism distinct from Ca entry or the K channels
that are normally modulated by µ-opioids in GABAergic nerve terminals underlies sensitization to µ-opioids.
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Figure 6.
Withdrawal-induced enhancement of mIPSC frequency
is not affected by 4-AP. Time course of normalized mIPSC frequency in
neurons maintained in morphine (5 µM) and 4-AP (100 µM) during superfusion of naloxone (1 µM)
from dependent (closed circles) and vehicle-treated
(open circles) animals. Each point represents the mean
(± SEM) of four to six (vehicle) and eight to twelve
(dependent) neurons.
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Figure 7.
Withdrawal-induced enhancement of GABAergic
transmission is blocked by inhibitors of protein kinase A and occluded
by a metabolically stable analog of cAMP. A, Naloxone
induced increase in eIPSC amplitude in neurons from dependent animals
(closed bars) and vehicle controls (open
bars). Naloxone (1 µM) does not increase the
amplitude of glutamatergic eEPSCs (GLUTS; hatched
bar). 4AP represents 4-aminopyridine (100 µM), Stauro represents staurosporine (1 µM), RP represents RP-8-CPT-cAMP-S (100 µM), and 8Br represents 8-Br-cAMP (1 mM). B, Naloxone-induced increase in mIPSC
frequency in neurons from dependent animals (closed
bars) and vehicle controls (open bars).
Asterisks signify statistical significance for
comparisons between dependent and vehicle groups (unpaired
t tests, p < 0.05).
DTX represents dendrotoxin (100 nM).
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Adenylyl cyclase and protein kinase A become the predominant signal
transduction pathway involved in opioid inhibition during
dependence
The nonspecific serine-threonine kinase inhibitor staurosporine
(primarily protein kinases A and C, 1 µM) abolished the
naloxone-induced increase in eIPSCs in slices from dependent animals
(Fig. 7A) but had no effect on the increase in vehicle
controls (128 ± 10%, n = 8). The more selective
protein kinase A inhibitors, H-89 (10 µM) and
RP-8-CPT-cAMP-S (100 µM) also attenuated the
naloxone-induced increase in the eIPSCs (Fig. 7A).
Similarly, H89 and RP-8-CPT-cAMP-S blocked the naloxone-induced
increase in frequency of mIPSCs in neurons from dependent animals (Fig.
7B). RP-8-CPT-cAMP-S had no effect in matched vehicle
controls (frequency = 136 ± 15%, n = 5 of
control in presence of naloxone). These results suggest that the
naloxone-induced enhancement of GABAergic neurotransmission in neurons
from dependent animals requires the activation of protein kinase A.
If excessive cAMP formation and protein kinase A activity is indeed
responsible for enhanced GABAergic neurotransmission during opioid
withdrawal, then high concentrations of metabolically stable cAMP
analogs should stimulate GABAergic neurotransmission and occlude the
stimulatory effects of naloxone. In brain slices maintained in morphine
(5 µM), the metabolically stable cAMP analog, 8-Br-cAMP (1 mM), increased eIPSC amplitudes in neurons from
dependent animals to 161 ± 34% (n = 5) and in
vehicle controls to 186 ± 40% (n = 5). The
8-Br-cAMP-induced increase in GABAergic transmission occluded the
increase in eIPSC amplitude by naloxone in neurons from dependent animals (Fig. 7A).
Previous studies have suggested that enhanced cAMP formation during
withdrawal leads to extracellular accumulation of adenosine (adenosine
tone) that acts on presynaptic adenosine receptors to modulate
GABAergic neurotransmission in guinea pig ventral tegmental area
neurons (Bonci and Williams, 1996) and rat nucleus accumbens (Chieng
and Williams, 1998). The A1 receptor antagonist DPCPX (1 µM) did not affect eIPSCs from spontaneously withdrawn dependent or vehicle control slices (96 ± 6%, n = 11 and 101 ± 21%, n = 6 of pre-DPCPX baseline,
respectively), suggesting that adenosine tone plays no role in
modulating GABAergic neurotransmission in PAG neurons during opioid withdrawal.
The observed opioid supersensitivity could be primarily caused by
enhanced cAMP/protein kinase A regulation of GABA release in PAG
neurons in dependent animals or, alternatively, summed modulation of a
4-AP-sensitive K channel and an induced protein kinase A-dependent
mechanism in GABAergic terminals. These possibilities were examined in
the following concentration-response experiments in spontaneously
withdrawn slices (Fig. 8). DAMGO produced
inhibition of eIPSC amplitudes presence of 4-AP with at least the same
potency as in its absence in neurons from dependent animals (Fig. 8), suggesting that enhanced sensitivity to µ-opioid agonists is largely accounted for by a protein kinase A-dependent mechanism. In vehicle controls, 4-AP and dendrotoxin completely abolished the inhibitory effects of DAMGO; however, in the presence of 4-AP (100 µM) and forskolin (10 µM), DAMGO produced
robust inhibition of eIPSC amplitude. The sensitivity to DAMGO in
vehicle controls in the presence of 4-AP and forskolin suggests that
increased formation of cAMP in GABAergic nerve terminals accounts for
part of the altered µ-opioid receptor transduction in dependent
animals. However, this forskolin-dependent inhibition of eIPSCs in the
presence of 4-AP in control neurons was substantially less sensitive to
DAMGO than was observed in neurons from dependent animals in the
presence or absence of 4-AP (Fig. 8).
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Figure 8.
Opioid supersensitivity during withdrawal is
neither blocked by 4-AP, nor fully mimicked by forskolin in untreated
tissue. Concentration-response relationship for percentage inhibition
of eIPSC amplitudes by the selective µ-opioid agonist DAMGO. The
fitted logistic functions from Figure 2 for dependent (dashed
line) and vehicle controls (dotted line) are
shown. Superimposed points are for neurons from dependent animals in
the presence of 4-AP (100 µM) or dendrotoxin (100 nM) (closed circles), vehicle controls in
the presence of 4-AP (closed squares), and vehicle
controls in the presence of forskolin (10 µM) plus 4-AP
(open circles). Each point shows the mean (± SEM) of
responses of four to nine neurons.
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DISCUSSION |
Chronic morphine treatment enhances opioid efficacy in PAG
Chronic morphine treatment induces enhanced µ-opioid efficacy in
PAG neurons. A supramaximal concentration of the partial agonist,
morphine, produced greater inhibition of GABAergic IPSCs during
withdrawal than in control tissue suggesting increased efficacy of
morphine for activation of µ-receptor coupling. Moreover, the potency
of the full µ-receptor agonist DAMGO was simultaneously enhanced, as
would be expected if efficacy of coupling were increased. This
contrasts with the generally reported reduced efficacy, or tolerance,
mediated by uncoupling of µ-opioid receptors from effector systems
(Law et al., 1982; Chavkin and Goldstein, 1984; Christie et al., 1987;
Puttfarcken et al., 1988; Wimpey et al., 1989; Noble and Cox, 1996). It
is possible that coupling of µ-receptors to G-protein activation is
also diminished in GABAergic terminals of PAG by chronic morphine
treatment (Sim et al., 1996), but induction of coupling to additional
systems that modulate transmitter release (see below) more than
compensates for the deficit.
Enhanced efficacy of morphine in GABAergic presynaptic terminals
in PAG
The enhanced efficacy of morphine appears to be caused by
a presynaptic mechanism. Naloxone-precipitated withdrawal increased the
frequency without affecting the amplitude of GABAergic mIPSCs, suggesting a presynaptic mechanism of mIPSC modulation. However, high
variability of basal mIPSC frequencies precluded direct comparison of
long-term adaptations in basal release properties from GABAergic terminals.
Naloxone-precipitated withdrawal enhanced both GABAergic eIPSCs and
mIPSCs. The functional relationship between mIPSCs and eIPSCs is
unclear. Release after electrical stimulation is the result of
activation of voltage-dependent Ca channels, and the frequency of
mIPSCs is largely independent of Ca channel activation under the
present conditions (Vaughan and Christie, 1997; Vaughan et al., 1997).
One way to address whether increased frequency of mIPSCs may underlie
evoked IPSC amplitudes is to study spontaneous (absence of TTX) IPSCs.
However, the density of spontaneous IPSCs was too high, particularly in
the presence of naloxone, to resolve individual events. Nonetheless, it
is intriguing that under all circumstances the modulation of both
eIPSCs and mIPSCs mirrored one another (see also Vaughan et al., 1997).
After chronic morphine, the magnitude of naloxone-induced enhancement,
insensitivity to 4-AP, and blockade by protein kinase A inhibitors were
similar for both eIPSCs and mIPSCs. This may indicate that protein
kinase A modulation of GABAergic release is subsequent to Ca
stimulation of release, as has been demonstrated at other central
synapses (Trudeau et al., 1996).
Induction of protein kinase signaling underlies enhanced
opioid efficacy
The results discussed above demonstrate that chronic
morphine treatment induces coupling of µ-opioid receptors via a
mechanism that is not prominent in GABAergic nerve terminals in
untreated animals (Fig. 9). µ-Receptors
normally produce GABAergic presynaptic inhibition in PAG by coupling to
a dendrotoxin- and 4-AP-sensitive K channel via 12-lipoxygenase
metabolites of arachidonic acid (Vaughan et al., 1997). However, these
Kv channel blockers did not prevent presynaptic inhibition by
µ-opioids after chronic morphine treatment. Instead, inhibition by
µ-opioid agonists was blocked by protein kinase A inhibitors and
occluded by metabolically stable cAMP analogs in slices from
morphine-treated animals. Chronic treatment with morphine, therefore,
induces a shift in the coupling of µ-opioid receptor activation to
GABAergic presynaptic inhibition from one predominantly regulated by
4-AP-sensitive K channels to one involving adenylyl cyclase and protein
kinase A. The target effector of protein kinase A is unknown but may be
another ion channel that regulates release through depolarization of
GABAergic terminals in the PAG or, possibly, a protein or proteins more directly involved in vesicular release mechanisms.
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|
Figure 9.
Scheme of altered signal transduction and enhanced
opioid efficacy in GABAergic nerve terminals of PAG after chronic
treatment with morphine. 1, In untreated tissue the
dominant mechanism of presynaptic inhibition by µ-opioids is
enhancement of the activity of a 4-AP and dendrotoxin-sensitive Kv
channel via formation of phospholipase A2 (PLA2)
metabolites. Inhibitors of adenylyl cyclase (AC) or
protein kinase A (PKA) do not impair acute opioid
actions (this is, therefore, a minor pathway under basal conditions).
However, opioids can acutely inhibit GABAergic neurotransmission after
stimulation of adenylyl cyclase by forskolin. 2, 4-AP
and dendrotoxin largely abolish presynaptic inhibition during acute
application of opioids but do not prevent presynaptic inhibition after
chronic morphine treatment. 3, In contrast, adenylyl
cyclase and protein kinase A inhibitors prevent presynaptic inhibition
by opioids after chronic treatment with morphine. Basal and stimulated
activity of adenylyl cyclase and perhaps other elements of the protein
kinase A signaling cascade are hypertrophied after chronic morphine
treatment, and µ-opioids can then efficaciously inhibit synaptic
transmission via this mechanism. 4, Enhanced formation
of cAMP and activity of protein kinase stimulate GABA release,
thereby enhancing GABAergic synaptic transmission during
naloxone-precipitated withdrawal.
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|
The induction of coupling of µ-opioid receptors to GABA release in
the PAG via a protein kinase A-dependent mechanism could be caused by
hypertrophy of adenylyl cyclase. Both basal and forskolin-stimulated adenylyl cyclase activity are hypertrophied during opioid withdrawal in
brain tissue and a variety of cultured cell types (Sharma et al., 1975;
Nestler and Tallman, 1988; Matsuoka et al., 1994; Avidor-Reiss et al.,
1997). Stimulation of adenylyl cyclase and protein kinase A activity
are widely known to enhance synaptic neurotransmission (Capogna et al.,
1995; Vaughan et al., 1997). Similar to our findings in the PAG, Bonci
and Williams (1997) reported enhanced forskolin stimulation of
GABAergic IPSCs in guinea pig ventral tegmental area after chronic
morphine treatment.
Enhanced µ-opioid receptor efficacy may result from hypertrophied
adenylyl cyclase if opioids are normally capable of coupling to this
transduction mechanism. Indeed, DAMGO inhibited GABAergic eIPSCs in
control tissue in the presence of both forskolin and 4-AP. The potency
of DAMGO in the presence of 4-AP was substantially greater in tissue
from dependent than control (in forskolin) animals but it remains
possible that sensitivity to DAMGO in control would have differed under
different conditions of stimulation of adenylyl cyclase (Avidor-Reiss
et al., 1997). Enhanced efficacy could not be accounted for by
summation of 4-AP-sensitive and protein kinase A-dependent mechanisms
because the potency of DAMGO after chronic morphine treatment was at
least as great in the presence of 4-AP as in its absence. These results
suggest either that chronic morphine upregulates adenylyl cyclase, or
additional adaptations also occur within GABAergic terminals to enhance
protein kinase A activity. Some studies have shown that opioid
inhibition of adenylyl cyclase activity is desensitized in PAG
membranes (Noble and Cox, 1996) and µ-receptor coupling to G-protein
activation may be diminished in PAG (Sim et al., 1996) after chronic
morphine. However, hypertrophy of specific isoforms of adenylyl cyclase
(Avidor-Reiss et al., 1997) within GABAergic terminals or other
elements of the signaling cascade (Nestler and Tallman, 1988) could
explain the enhancement in opioid efficacy.
Enhanced "adenosine tone" does not modulate GABAergic
transmission during opioid withdrawal in PAG
GABAergic synaptic transmission has also been shown to be
modulated by extracellular adenosine acting on A1 adenosine receptors ("adenosine tone") after chronic morphine treatment in ventral tegmental area (Bonci and Williams, 1996) and nucleus accumbens (Chieng
and Williams, 1998). However, the absence of effects of the A1
antagonist DPCPX in control or treated tissue in the present study
suggests that adenosine tone plays no role in modulating GABAergic
neurotransmission in PAG neurons during opioid withdrawal.
Clonidine overcomes opioid withdrawal in PAG
The observation that the 2-adrenoceptor agonist
clonidine suppressed GABAergic synaptic transmission more effectively
during withdrawal than in control tissue is consistent with its ability to suppress many of the signs of the opioid withdrawal syndrome (Redmond and Krystal, 1984; Christie et al., 1997). 2
adrenoceptors couple to the same postsynaptic K channels as µ-opioid
receptors, as well as to adenylyl cyclase. Therefore, these results
suggest that the efficacy of clonidine may also be enhanced after
chronic morphine treatment and that characterization of drugs that
suppress the withdrawal-induced increase in GABAergic release in the
PAG (in addition to clonidine) could prove useful for management of opioid withdrawal.
Significance of enhanced opioid efficacy for
withdrawal behavior
Adaptations in adenylyl cyclase signaling systems associated with
opioid dependence have been known for a long time (Sharma et al., 1975;
Nestler and Tallman, 1988; Matsuoka et al., 1994; Avidor-Reiss et al.,
1997), but the physiological significance of this signaling system has
remained elusive (Christie et al., 1997). The present results suggest
how hypertrophy of adenylyl cyclase could mediate opioid withdrawal
behaviors elicited from PAG (Wei et al., 1973; Bozarth and Wise, 1984;
Maldonado et al., 1992; Christie et al., 1997). Opioids are thought to
elicit acute responses from PAG by disinhibiting PAG output neurons
through direct inhibition of GABAergic neurotransmission (Reichling et al., 1988; Fields et al., 1991; Bandler and Shipley, 1994; Osborne et
al., 1996; Vaughan and Christie, 1997). Descending PAG output neurons
are thought to produce antinociception and modulate somatic and
autonomic components of defensive behavior (Reichling et al., 1988;
Fields et al., 1991; Bandler and Shipley, 1994). During chronic opioid
treatment, opioid receptor supersensitivity presumably maintains opioid
disinhibition at GABAergic synapses in PAG despite the development of
tolerance elsewhere (Chieng and Christie, 1996). However, enhanced
GABAergic inhibition during withdrawal would be expected to excessively
suppress activity of descending output neurons from PAG and produce
withdrawal behaviors. The presynaptic actions described here were
observed in all neurons tested, suggesting that the conclusions also
apply to descending output neurons (see also Vaughan et al., 1997).
Consistent with this interpretation, injections of protein kinase A
inhibitors into PAG attenuate many different behaviors associated with
opioid withdrawal (Maldonado et al., 1995; Punch et al., 1997).
These mechanisms could also be responsible for withdrawal rebound and
sensitization to opioids in opioid-sensitive nerve terminals elsewhere
in the nervous system. For example, withdrawal rebound of GABAergic
synaptic transmission mediated by adenylyl cyclase-dependent mechanisms
has been reported in ventral tegmental area (Bonci and Williams, 1996,
1997) and nucleus accumbens (Chieng and Williams, 1998) and could play
a role in withdrawal craving. Enhanced efficacy of morphine was also
reported in GABAergic terminals in the latter region (Chieng and
Williams, 1998). Nonetheless, withdrawal rebound of transmitter release
does not occur at all opioid-sensitive synapses because no enhancement
of glutamatergic synaptic transmission was observed in PAG in the
present study.
| |
FOOTNOTES |
Received June 17, 1998; revised Aug. 27, 1998; accepted Sept. 25, 1998.
This work was supported by the NH & Medical Research Council of
Australia, The Human Frontier Science Program (S.L.I.), and The Medical
Foundation of The University of Sydney (M.J.C.). We gratefully
acknowledge donation of CTAP by the National Institute on Drug Abuse.
We thank K. Earle for help with the analysis.
Correspondence should be addressed to Dr. M. J. Christie,
Department of Pharmacology D06, The University of Sydney, New South Wales 2006, Australia.
| |
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Top
Abstract
Introduction
