Department of Psychiatry, University of Pennsylvania, Veterans Affairs Medical Center (151), Philadelphia, Pennsylvania 19104
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ABSTRACT |
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Ivanov, Alexander and Gary Aston-Jones. Local Opiate Withdrawal in Locus Coeruleus Neurons In Vitro. J. Neurophysiol. 85: 2388-2397, 2001. Noradrenergic neurons of the brain nucleus locus coeruleus (LC) become hyperactive during opiate withdrawal. It has been uncertain to what extent such hyperactivity reflects changes in intrinsic properties of these cells. The effects of withdrawal from chronic morphine on the activity of LC neurons were studied using intracellular recordings in rat brain slices. LC neurons in slices from chronically morphine-treated rats exhibited more than twice the frequency of spontaneous action potentials after naloxone compared with LC neurons from control rats. However, after naloxone treatment, the resting membrane potential (MP) of LC neurons from dependent rats was not significantly different from that in control rats. Neither resting MP nor spontaneous discharge rate (SDR) was altered by naloxone in LC neurons from control rats. Neither kynurenic acid nor a cocktail of glutamate and GABA antagonists (6-cyano-7-nitroquinoxalene-2,3-dione + 2-amino-5-phosphonopentanoic acid + bicuculline) blocked the hyperactivity of LC neurons precipitated by naloxone in slices from morphine-dependent rats. The effects of ouabain on MP and SDR were similar in LC neurons from control and morphine-dependent rats. These results indicate that an adaptive change in glutamatergic or GABAergic synaptic mechanisms or altered Na/K pump activity does not underlie the withdrawal-induced activation of LC neurons in vitro. Specific inhibitors of protein kinase A [Rp-cAMPS or N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide (H-89)] partially suppressed the withdrawal hyperactivity of LC neurons, and activators of cAMP (forskolin) or protein kinase A (Sp-cAMPS) increased the discharge rate of LC neurons from control rats. These results suggest that upregulation of cAMP-dependent protein kinase A during chronic morphine treatment is involved in the withdrawal-induced hyperactivity of LC neurons.
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INTRODUCTION |
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Recent results
clearly establish that extrinsic glutamate inputs from the
ventrolateral medulla play a prominent role in the hyperactivity of
locus coeruleus (LC) neurons after morphine withdrawal (e.g.,
Akaoka and Aston-Jones 1991; Ennis and
Aston-Jones 1988
; Rasmussen and Aghajanian
1989
). However, a possible additional intrinsic mechanism has
been controversial. Some previous studies did not observe an increase
in the spontaneous firing rate of LC neurons in brain slices from
chronically morphine-treated rats (Andrade et al. 1983
;
Bell and Grant 1998
; Christie et al.
1987a
), indicating that an intrinsic mechanism may not exist.
In another study, LC neurons recorded extracellularly in slices from
chronically morphine-treated rats were reported to have elevated
impulse rates (Kogan et al. 1992
). Another recent study
in vivo (Aston-Jones et al. 1997
) found increased
activity of LC neurons of dependent rats after local microinfusion of
methyl naloxone, consistent with a local withdrawal mechanism. However,
the possible involvement of residual synaptic inputs in these studies
was not evaluated. Thus it remains uncertain to what degree changes in
intrinsic mechanisms within LC neurons participate in their
hyperactivity during opiate withdrawal. Demonstration of intrinsic
changes with opiate withdrawal would reveal neuronal-level alterations
produced by chronic morphine and could be important for understanding
the cellular basis of opiate dependence as well as for developing pharmacotherapies for opiate addiction.
The purpose of this study was to characterize the intracellular responses of LC neurons to naloxone in brain slices from drug-naive and chronically morphine-treated rats to evaluate possible intrinsic changes in LC neurons during chronic morphine exposure that contribute to LC hyperactivity during opiate withdrawal.
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METHODS |
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Male Sprague-Dawley rats (n = 55; ~150 g) were
used in these experiments. Brain slices were prepared as described
previously (Ivanov and Aston-Jones 1996).
"Quasihorizontal" brain slices (angled ventrorostral to caudodorsal
to maximize inclusion of LC dendrites) (Shipley et al.
1996
) containing the LC were incubated in artificial
cerebrospinal fluid (ACSF) of the following composition (in mM): 126 NaCl, 5 KCl, 2.4 CaCl2, 1.3 MgSO4, 1.2 NaH2PO4, 24 NaHCO3, and 11 glucose. The ACSF was pH 7.4 and
continuously saturated with 95% O2-5%
CO2.
Experimental groups
Membrane potential and the frequency of spontaneous discharge of
LC neurons were examined in three experimental groups: control groupslices prepared from morphine-naive (untreated) rats;
acute morphine group
slices prepared from untreated rats
that were continuously maintained in morphine (5 µM) for the duration
of experiments; and morphine-dependent group
slices taken
from rats treated chronically with morphine and continuously maintained
in morphine (5 µM) for the duration of experiments. Two methods were
used for chronic morphine treatment: 1) pellets containing
75 mg of morphine base each were implanted subcutaneously on alternate
days for 5 days (1, 2, then 2 pellets). In this case, chronically
treated rats were used for electrophysiological experiments 2 days
after the final implantation. 2) Only two pellets were
implanted, and rats were used in experiments 4 days after implantation.
The results were identical with these two treatments and were pooled.
Each of these treatments has been previously shown to induce dependence as indicated by strong withdrawal behaviors following opiate antagonist administration in waking rats (Christie et al. 1987b
;
Gold et al. 1994
; Harris and Aston-Jones
2001
). Sham pellets were not used as our previous behavioral
and electrophysiological studies showed no difference from untreated
controls. Slices in the acute morphine and morphine-dependent groups
were continuously superfused with morphine (5 µM) during the
electrophysiological experiments. Previous data indicate that this
mimics the level of morphine in the brain during chronic morphine
treatment (Christie et al. 1987a
).
Electrophysiological recording and drugs
Electrodes for intracellular recording were made on a
Brown-Flaming puller from Kwik-fil glass micropipettes and were filled with 2 M KCI (resistance = 30-50 M). Intracellular potentials were amplified using an Axoclamp 2A amplifier (Axon Instruments) and
monitored on a chart recorder on-line (Gould 2200) or digitized with a
CED 1401 interface (CED, Cambridge, UK) and stored on a computer disk.
Data analysis was performed on the computer employing Chart 2.0, Spike
2, and Sigma-Plot software. All average values presented in the
following text are means ± SE.
The following drugs were applied to the slice perfusion solution: naloxone (1 µM), tetrodotoxin (TTX, 1 µM; Sigma), bicuculline (15 µM; Sigma), kynurenic acid (500 µM; Sigma), 6-cyano-7-nitroquinoxalene-2,3-dione (CNQX, 20 µM; RBI), 2-amino-5-phosphonopentanoic acid (AP5, 20-50 µM; RBI), forskolin (10-30 µM; RBI), 1,9-dideoxyforskolin (10 µM; RBI), ouabain (0.5-1 µM; RBI), Rp-cAMPS (10-50 µM; RBI), N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide (H-89, 10 µM; Calbiochem), and Sp-cAMP (10 µM; RBI). In some experiments, BaCI2 (2 mM) was added to ACSF in which NaH2PO4 was omitted.
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RESULTS |
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Control group
Twenty-five neurons in 20 slices taken from control rats were
studied. Only neurons that had a stable frequency of spontaneous discharge and a stable membrane potential close to 60 mV for 1 h
were included in the data analysis. The spontaneous discharge rate
(SDR) of LC neurons in these slices ranged from 0.5/s to 1.4/s
(mean = 1.0 ± 0.1/s; n = 25); the range of
resting membrane potential (RMP) recorded was
58 to
65 mV
(mean = 60.4 + 0.3 mV; Fig.
1B). In eight cells from this
group, the effect of naloxone was tested. No significant change in RMP
(
60.0 ± 0.4 vs.
60.3 ± 0.7 mV) or SDR (1.1 ± 0.2/s vs. 1.1 ± 0.2/s) was observed after 5-10 min.
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Acute morphine group
The RMP of 23 neurons (12 slices) from the acute morphine group
(1 h superfusion with 5 µM morphine) was significantly lower (72.9 ± 1.0 mV; n = 23) than in the control
group without morphine (
60.4 ± 0.3 mV; n = 25;
Fig. 1B). Spontaneous discharge was suppressed in all LC
neurons tested in the presence of morphine in this group. All neurons
were depolarized to control values (by ~13 mV) when naloxone was
added (average membrane potential after naloxone =
60.6 ± 0.1 mV; n = 23; Fig. 1, A and B).
RMP and SDR recorded in this group of neurons in the presence of
naloxone were not significantly different from the values observed in
LC neurons in the control group (Fig. 1C; 1.3 ± 0.2/s,
n = 23 vs. 1.0 ± 0.1/s; n = 25, P > 0.05; t-test). The preceding data
indicate that intrinsic properties reflected in RMP and SDR were not
changed by superfusion with morphine for 1-2 h.
Morphine-dependent group
The majority of LC neurons (19 of 24) from this group exhibited
spontaneous firing at an average rate of 0.3 ± 0.1/s in the presence of 5 µM morphine. The remaining neurons were silent. This is
in contrast to LC neurons in control or acute morphine groups in which
a complete suppression of impulse activity occurred with 5 µM
morphine (described in the preceding text). The membrane potential of
neurons in the morphine-dependent group (23 slices) ranged from 60 to
68 mV (mean =
61.7 ± 0.6 mV; n = 24) in
the presence of 5 µM morphine (Fig. 2,
A and B). This was significantly hyperpolarized
compared with control slices without morphine (Fig. 2B;
P = 0.04, t-test). However, morphine
hyperpolarized LC neurons significantly less in slices from the
morphine-dependent group compared with the acute morphine group
(
72.9 ± 1.0 mV). These data indicate that a prominent tolerance
to morphine developed in LC neurons during the chronic morphine
treatment. Nonetheless, morphine in the bath had a significant effect
on LC neurons because the frequency of spontaneous discharge was lower
in the morphine-dependent group than observed in the control group.
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A clear depolarization was observed in 16 of 24 neurons in the
morphine-dependent group after naloxone. The mean depolarization for
this group of 16 neurons was 3.5 ± 0.5 mV. The overall average depolarization in LC neurons after naloxone in the morphine-dependent group was ~2 mV (from 61.7 ± 0.6 to
59.5 ± 0.6 mV,
n = 24 cells, P < 0.05; Fig.
2B). The remaining eight cells from this group did not show
depolarization after naloxone (
59.6 ± 0.5 vs. 60.1 ± 0.8 mV; P > 0.05).
Overall, LC neurons from dependent animals exhibited a higher SDR after naloxone than either the control or acute morphine groups. This increase in SDR occurred similarly in cells of the morphine-dependent group that demonstrated no depolarization after naloxone (2.6 ± 0.3/s; n = 8) and cells that were depolarized by naloxone (2.5 ± 0.3/s; n = 16). Thus the higher frequency of spontaneous impulses in LC neurons from dependent rats after naloxone was not obviously dependent on a naloxone-induced depolarization.
It is important to note that the average RMP in LC neurons from dependent rats in the presence of naloxone did not differ significantly from the RMP in LC neurons from control rats (Fig. 2B). These results indicate that chronic morphine treatment did not significantly change the RMP in LC neurons from morphine-dependent rats compared with control rats. However, although their RMP postnaloxone was not different from control-naive cells, LC neurons from dependent rats displayed a markedly higher SDR after naloxone compared with that recorded from LC neurons in control rats (2.5 ± 0.3 vs. 1.0 ± 0.1/s, P < 0.01; Fig. 2C). Thus a comparison of LC neurons after naloxone from control versus dependent rats revealed a more than twofold difference in SDR without a significant difference in the average RMP.
To further examine the possible role of RMP in the elevated discharge
of LC neurons after withdrawal, we calculated the rate of spontaneous
discharge in LC neurons from control rats as a function of RMP. Cells
with an average RMP of 57.2 ± 0.4 mV (n = 4)
had an average discharge frequency of 1.4 ± 0.1/s; cells whose
RMP =
60.0 ± 0.1 mV (n = 6) had an average
discharge frequency of 1.1 ± 0.1/s; and cells with an RMP of
62.5 ± 0.3 mV (n = 6) had a corresponding
discharge of 0.8 ± 0.1/s. Thus a difference of 5 mV in membrane
potential caused a relatively small change in discharge frequency (from
0.8 to 1.4/s) compared with that observed in neurons from
morphine-dependent rats after naloxone.
We also examined the effect of repolarizing the MP to the prenaloxone
level in three cells from morphine-dependent rats (average MP = 61.5 ± 0.3 mV; rate of spontaneous spikes, 0.3 ± 0.1/s). Naloxone depolarized these cells 3.4 ± 0.1 mV on average and
increased the rate of spontaneous spikes to 2.4 ± 0.5/s.
Hyperpolarization of these neurons to the membrane potential before
naloxone only partially suppressed the spontaneous discharge rate
(1.1 ± 0.3/s). Note that this rate was still significantly higher
(P < 0.05) than the rate of spontaneous discharge in
these cells before naloxone. Together these data support the
possibility that depolarization is not the only mechanism responsible
for increasing spontaneous LC discharge rate in morphine-dependent rats
after naloxone.
TTX and Ba2+
TTX was used to block possible residual synaptic inputs to LC
neurons in the slice during naloxone-precipitated withdrawal. We found,
as in our previous report (Ivanov and Aston-Jones 1995), that TTX (1 µM) not only blocked synaptic responses [excitatory and
inhibitory postsynaptic potentials (EPSPs and IPSPs)] but also
suppressed spontaneous impulse activity in 93% of LC neurons (n = 59). Spontaneous spikes could be easily elicited
in all LC neurons tested during TTX superfusion by injection of a small inward current through the microelectrode (Fig.
3A). Spikes evoked by slight
depolarization during TTX perfusion were classified as
Ca2+ spikes because they were persistent in the
presence of 1 µM TTX, had lower amplitude (~40 mV) and longer
duration (>10 ms) than spikes in control solutions, and were blocked
by Co2+ (2 mM) or nifedipine (15 µM). On chart
records these spikes exhibited relatively large amplitude because of
their long duration (Fig. 3A). TTX did not change the
membrane potential in LC neurons from morphine-dependent rats
(n = 6).
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Although spontaneous impulses were also absent in neurons from dependent slices in the presence of TTX, naloxone superfusion evoked depolarization and tonic impulse activity (calcium spikes; see Fig. 3B). Thus TTX did not prevent the naloxone-precipitated depolarization in LC neurons from dependent rats. The frequency of impulse activity during naloxone and TTX superfusion in LC neurons from dependent rats was significantly lower (1.1 ± 0.1/s, Fig. 3C; n = 6) than without TTX (2.5 ± 0.3/s; n = 24). However, note that the increase in impulse activity due to naloxone was similar with or without TTX.
The naloxone-induced depolarization might arise as a result of
decreased outward potassium conductance (e.g., blockade of the residual
effect of morphine by naloxone) or as a result of a new inward current
caused by adaptive changes during chronic morphine exposure. On
average, the RMP of cells that did not demonstrate depolarization after
naloxone was significantly smaller (60.1 ± 0.8;
n = 8, P < 0.05) than that of cells
that with depolarization after naloxone (
62.9 ± 0.5 mV;
n = 16). After naloxone treatment, the MPs of both
groups of cells were similar (59.2 ± 0.7 and 59.5 ± 0.4 mV). These data indicate that the naloxone-induced depolarization observed here may primarily reflect blockade a residual effect of morphine.
To further examine this issue, experiments with TTX + Ba2+ were performed. 1 µM TTX + 2 mM
Ba2+ depolarized LC neurons by ~10 mV (to
52.1 ± 1.1 mV; n = 6) and increased the number
of cells without depolarization following naloxone from ~33% in
control ACSF to ~66% (4 of 6 cells). However, a clear depolarization
(~3 mV) was still observed after naloxone in the two remaining cells
when their membrane potential was artificially repolarized to the level
observed before TTX + Ba2+ (Fig.
4A). Thus neither TTX nor
Ba2+ blocked the naloxone-induced depolarization
in some dependent LC neurons.
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We also observed subthreshold membrane oscillations in LC cells when
spontaneous discharge was suppressed by hyperpolarization to 65 to
70 mV in the presence of TTX + Ba 2+, similar
to those previously reported (Williams et al. 1984
). The
frequency of subthreshold membrane oscillations in LC neurons from
morphine-dependent rats after naloxone were higher (1.3 to 2.5/s) than
that in LC neurons from control rats (0.5-1.1/s). Further studies are
necessary to determine the possible role of these oscillations in the
response of LC neurons to withdrawal.
In additional experiments, cells were hyperpolarized to 85 mV (close
to Ek with 5 mM
K+ in the ACSF) (Andrade and Aghajanian
1984a
) or external K+ was increased to 10 mM and cells were hyperpolarized to between
70 and
80 mV. Under
these conditions and in the presence of 1 µM TTX, direct
stimulation of neurons (10-ms inward current pulses) revealed no
afterhyperpolarization following evoked spikes, confirming that the
membrane potential was close to Ek
(Fig. 4B, inset). In these recordings, a
naloxone-precipitated depolarization (4.1 ± 0.5 mV;
n = 6) was observed in 6 of 11 cells (Fig.
4B). The remaining cells (n = 5) did not
yield a depolarization. These data indicate that the naloxone-induced
depolarization in some cells results from elimination of the
stimulation of potassium channels by residual morphine. However, the
fact that depolarization was observed in several cells despite setting
their membrane potential near or at Ek
indicates that another current may also be involved.
Glutamate and GABA antagonists
Previous studies have shown that naloxone given to
morphine-dependent rats causes a large release of glutamate within the LC (Aghajanian et al. 1994; Akaoka and
Aston-Jones 1991
). To test whether such synaptic release plays
a role in the elevated SDR found in LC neurons from dependent rats
after naloxone, we studied the effects of a nonselective antagonist of
glutamatergic receptors (kynurenic acid), a GABA antagonist
(bicuculline), or a cocktail of glutamate and GABA antagonists (CNQX + AP5 + bicuculline), in morphine-dependent rats. Pretreatment with
kynurenic acid (500 µM) for 5 min before naloxone administration
slightly (
4 mV) hyperpolarized membrane potential and suppressed SDR
(SDR was decreased on average from 0.3 to 0.1/s; n = 4;
the remaining 2 neurons became silent). Such kynurenic acid treatment
did not prevent a naloxone-precipitated depolarization (
60.6 ± 0.7 vs.
62.8 ± 1.5 mV; n = 6). The frequency of
spikes during naloxone in LC neurons with kynurenic acid treatment from
dependent rats (1.8 ± 0.1/s; n = 6) was
significantly higher then that in control rats (1.0 ± 0.1/s;
P < 0.01), indicating that kynurenic acid did not
block the withdrawal-induced hyperactivity in LC neurons in vitro. The
average increase in SDR after naloxone was slightly but not
significantly lower in LC neurons from dependent rats with kynurenic
acid then without kynurenate (Fig.
5B), and impulse frequency
increased when kynurenic acid was washed from these slices. Note that
kynurenate similarly decreased LC impulse activity in slices from
dependent rats (see preceding text) and from control rats (from
1.1 ± 0.2 to 0.9 ± 0.2/s; n = 9; data not
shown). This indicates that this agent slightly decreased activity in
LC neurons from control and morphine-treated rats in a
similar manner.
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The effect of more selective and potent antagonists of glutamate receptors (20 µM CNQX +50 µM AP5), in combination with bicuculline (15 µM), was also examined. This cocktail suppressed synaptic responses evoked by stimulation of the slice through a bipolar electrode during 5 min of superfusion in all three cells tested (Fig. 5A). This cocktail did not prevent the naloxone-precipitated elevation in impulse frequency in any of the eight cells tested (activity increased by 1.8 ± 0.4/s after naloxone in the presence of the cocktail; P < 0.05). The average elevations in the frequencies of discharge for naloxone, naloxone + kynurenate and naloxone + cocktail are illustrated on Fig. 5B.
In addition, the hyperactivity of LC neurons after naloxone in dependent rats was not prevented by 15 µM bicuculline (activity increased by 1.9 ± 0.4/s after naloxone in the presence of bicuculline, P < 0.05, n = 7; not illustrated). Bicuculline had an inconsistent effect on membrane potential in LC neurons from control and dependent rats (most cells were slightly hyperpolarized in the presence of bicuculline).
Thus although kynurenic acid or the antagonist cocktail slightly altered the baseline frequency in control and morphine-dependent rats, these treatments did not prevent the naloxone-precipitated hyperactivity of LC neurons from dependent rats. These results indicate that release of glutamate or GABA from residual synaptic inputs during naloxone-precipitated withdrawal does not produce the hyperactivity observed in LC neurons in slices from dependent rats.
Ouabain
A recent study indicated that S neurons of the myenteric plexus in
chronically morphine-treated animals were depolarized by ~10 mV
compared with S neurons from naive animals and that this difference was
associated with a reduction in electrogenic
Na+/K+ pumping (Kong
et al. 1992, 1997
). As noted in the preceding text, we did not
find a significant difference in RMP of LC neurons from control versus
morphine-dependent rats (see Fig. 2). However, it seemed possible that
chronic morphine could reduce
Na+/K+ pump activity in LC
neurons, leading to hyperexcitability, which would become apparent
after opiate withdrawal. To test this possibility, we examined the
effect of ouabain on the SDR and membrane potential of LC neurons from
control and dependent slices. SDR in LC neurons from control and
morphine-dependent rats began to increase 1-1.5 min following the
onset of superfusion with ouabain (0.5-1 µM; Fig.
6A2). We noted that the
increase in firing rate of LC neurons from dependent rats evoked by
ouabain always developed with a slowly progressing depolarization that
was never observed following naloxone. After 4 min of superfusion with
ouabain, LC neurons were depolarized by ~10 mV on average (53.0 ± 1.2 vs. 62.0 ± 1.0 mV, Fig. 6B; n = 5). The frequency of SDR in LC neurons from both control and dependent
rats strongly increased (up to 6 spikes/s, mean = 5.0 ± 0.6/s in control and 6.0 ± 1.2/s in dependent rats) after 4 min
of superfusion with ouabain (Fig. 6C). Notably, there was no
significant difference between LC neurons from control versus
morphine-dependent rats in the depolarization or increased impulse
activity following ouabain. Naloxone added after washing ouabain from
slices of dependent rats for 10 min increased the impulse activity of
LC neurons without any depolarization (Fig. 6A2). These
results indicate that the increased excitability of LC neurons in
dependent rats after naloxone is unlikely to be due to an inhibition of
an electrogenic Na+/K+ pump as a consequence of
chronic morphine treatment.
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Protein kinase inhibitors
The effect of naloxone before and 10 min after superfusion with
protein kinase inhibitors (Rp-cAMPS or H-89) was tested on LC neurons
from control and morphine-dependent rats. In slices from control rats,
Rp-cAMPS (10 µM) did not significantly change LC membrane potential
(64.4 ± 1.1 vs.
64.5 ± 0.7 mV, n = 3) or spontaneous discharge rate after 10-15 min of superfusion. However,
with a higher concentration (50 µM), Rp-cAMPS significantly increased
the impulse activity of LC neurons from control rats (1.9 ± 0.2 vs. 1.2 ± 0.2/s; P < 0.05) without a significant
change of membrane potential. It was not clear why Rp-cAMPS increased spontaneous discharge rate in LC neurons from control rats. One possibility is that the excessive concentration of Rp-cAMPS may activate (rather than suppress) protein kinase A (Pedarzani and Storm 1993
).
Rp-cAMPS (10 µM) was tested on the LC cells from dependent slices. First, naloxone was tested and then after ~1 h wash the same cell was perfused for 10-15 min with Rp-cAMPS (10 µM), after which naloxone was again added. Pretreatment with Rp-cAMPS (10 µM) did not change the membrane potential or spontaneous discharge rate (62.9 ± 1.3 vs. 63.1 ± 0.7 mV; n = 3) but significantly decreased the impulse activity induced by naloxone (Fig. 7, A and B; 1.6 ± 0.2 vs. 3.2 ± 0.3/s; P < 0.05).
|
Another specific inhibitor of protein kinase A (PKA), H-89 (10 µM),
did not significantly change the RMP in neurons from dependent rats
(61.2 ± 1.4 vs.
60.7 ± 1.1 mV; n = 12, Fig. 7C) but significantly suppressed the postnaloxone SDR
(1.5 ± 0.2 vs. 2.7 ± 0.3/s; P < 0.01, n = 12; Fig. 7D). Thus although inhibitors
of protein kinase A did not change the membrane potential in LC neurons
from dependent rats, they significantly suppressed the
naloxone-precipitated impulse hyperactivity.
Activators of cAMP and PKA
The PKA activator Sp-cAMPS (10 µM) slightly but significantly
hyperpolarized LC neurons from control rats (Fig.
8A; from 61.3 ± 0.9 to
63.6 ± 0.7 mV; n = 9; P < 0.05) and strongly (1.9 ± 0.3 vs. 0.9 ± 0.1/s;
P < 0.01) increased the frequency of spontaneous action potentials and subthreshold membrane oscillations (Fig. 8,
A and B). Forskolin (10-15 µM) also increased
the frequency of spontaneous discharge in four of seven LC neurons
(from 0.8 ± 0.2 to 1.6 ± 0.2/s; P < 0.05, n = 7, Fig. 8C) without obviously changing
membrane potential, in agreement with previous reports (Harris
and Williams 1991
; Wang and Aghajanian 1987
).
The inactive forskolin analogue, 1,9-dideoxyforskolin, at the same
concentration had no effect on spontaneous discharge (3 cells tested).
In contrast, forskolin at a higher concentration (30 µM) completely
suppressed spontaneous firing and hyperpolarized LC neurons (not
illustrated; 3 cells tested), in agreement with previous reports
(Osborne and Williams 1996
). Thus these results indicate
that PKA or adenylate cyclase activators significantly increased the
frequency of spontaneous spikes in LC neurons from naïve rats
without membrane depolarization.
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DISCUSSION |
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The main findings of this study are that LC neurons in slices from
chronically morphine-treated rats have a higher tonic discharge rate
after naloxone compared with control rats but do not exhibit a
significant difference in RMP. This indicates that LC neurons in vitro
exhibit a local withdrawal response as we previously reported with in
vivo experiments (Aston-Jones et al. 1997). Our experiments with neurotransmitter antagonists, and with TTX, indicate that this withdrawal-induced hyperactivity in vitro is not produced by
neurotransmitter release and presumably results from adaptations produced by chronic morphine treatment in these neurons. Our studies also indicate that this adaptation likely involves changes in the
cAMP-PKA cascade.
Previous data have demonstrated that the majority of the
withdrawal-induced activation of LC neurons in vivo is mediated
indirectly, via excitatory amino acid inputs from the rostral
ventrolateral medulla (Akaoka and Aston-Jones 1991;
Ennis and Aston-Jones 1988
; Rasmussen and
Aghajanian 1989
). However, these data did not exclude the
possible additional involvement of an intrinsic mechanism in the
hyperactivity of LC neurons during withdrawal. In fact, in these prior
studies withdrawal-induced hyperactivity was not completely eliminated
by antagonizing amino acid inputs or by lesions of LC inputs. Our
present findings are in agreement with a previous extracellular study
in vitro (Kogan et al. 1992
) in showing that LC neurons
in brain slices from morphine-dependent rats exhibit elevated impulse
activity in response to opiate withdrawal. However, that study did not
establish that the withdrawal-induced activation was due to an
intrinsic change in LC neurons and not to other factors, e.g.,
increased synaptic activation of LC cells. In addition, that prior
study did not examine the membrane potential of LC neurons during
withdrawal as extracellular recording methods were used, and possible
mechanisms involved were not examined. The present study extends that
report by showing that although withdrawal activates LC cells in vitro
above discharge rates in naïve rats, the resting membrane
potential of LC neurons from morphine-dependent rats after naloxone did
not significantly differ from that in neurons from naïve
control animals. In addition, hyperactivity of LC neurons in vitro
during withdrawal occurs without significant depolarization, is
independent of presynaptic transmitter release, and may involve a
cAMP/PKA mechanism.
Our findings differ in some regards from a previous intracellular study
that reported no obvious difference in the mean frequency of LC firing
between control tissue and morphine-treated rats (Christie et
al. 1987a). The reason for this discrepancy in results is not
clear but may result from differences in methods (e.g., lines of rats
used or the presence or absence of morphine in the bath of slices from
dependent rats). Nonetheless, our study does agree with this previous
report in finding no difference in RMP of LC neurons in slices from
nonmorphine-treated animals versus dependent slices after naloxone.
That previous study also found that membrane resistance
(Rin) was not different for LC neurons
from morphine-dependent versus naive rats (Christie et al.
1987a). This result is consistent with our previous
observations of Rin when LC neurons
were depolarized by other agents (glutamate and hypocretin), which
indicated that reliably detectable changes in
Rin (~10% or more) were observed only if the change of membrane potential exceeded 5 mV (Ivanov and Aston-Jones 1996
, 2000
). In the present studies,
the average depolarization of LC neurons in morphine-dependent rats
after naloxone was ~2.2 mV, and the difference between RMP in LC
neurons from control and morphine-dependent rats was not significant. Thus no difference in Rin would be expected.
Our data support earlier results that LC neurons in vitro demonstrate a
marked tolerance to morphine after chronic morphine treatment
(Andrade et al. 1983; Christie et al.
1987a
). The signs of tolerance included a diminished effect of
morphine on the membrane potential and spontaneous discharge in many LC
neurons from dependent rats in spite of morphine in the bath. However,
as also previously reported, tolerance appeared to be incomplete as LC
neurons from dependent slices exhibited spontaneous firing in the
presence of morphine at a rate that was lower than LC neurons from
control rats without morphine in the bath. In addition, morphine
hyperpolarized dependent cells, albeit to a smaller extent than in
slices from control rats. Such apparent incomplete tolerance may
reflect tolerance in multiple cellular processes that exhibit different
time courses.
It is important to note that the naloxone-precipitated depolarization
in individual LC neurons from dependent rats could not be observed in
~50% of cells whose MP was shifted close to
Ek (Fig. 4C3). These
results indicate that the depolarization observed after naloxone in at
least some LC neurons from morphine-dependent rats might be caused by
elimination of a residual effect of morphine. However, a possible
additional source of depolarization also appears likely in some cells
as depolarization was occasionally observed despite a MP near
Ek. It is notable that the appearance
of persistent depolarization after chronic morphine treatment was found
in neurons from the periaqueductal gray (Chieng and Christie
1996).
It was reported that chronic morphine treatment could modulate the
release of GABA and glutamate from synaptic terminals
(Bonci and Williams 1997; Martin et al.
1999
; Pinnock 1992
). Those results indicated that residual synaptic inputs might influence the
excitability of LC neurons in slices from morphine-dependent rats.
However, we found that withdrawal-induced hyperactivity of LC neurons
was only slightly suppressed by antagonists of glutamate and
GABAA receptors. Moreover, these antagonists had
a similar effect on LC neurons from dependent and control slices, and
there was no tendency of these antagonists to selectively suppress
withdrawal-induced activity. Therefore little if any of the withdrawal
hyperactivity in LC neurons in vitro appears to be involve amino acid
inputs. This conclusion is consistent with the results of our
experiments using TTX to block synaptic transmission. TTX completely
blocked spontaneous (pacemaker) discharge in LC neurons from control
rats (as we have previously reported) (Horvath et al.
1999
; Ivanov and Aston-Jones 1995
) but only
partially suppressed it in LC neurons from dependent rats after
naloxone. We also demonstrated in these previous studies that
depolarization of TTX-treated LC neurons restored spontaneous impulse
activity in the form of Ca2+ spikes. These
results indicate that the lower rate of discharge of LC neurons from
dependent slices treated with TTX compared with control cells probably
is not due to blockade of a withdrawal-specific current. Rather this
lower rate is due to an overall suppressive effect of TTX on LC impulse
activity, and impulses are restored by naloxone-induced withdrawal
despite TTX. Indeed a similar increase in activity occurs after
naloxone in dependent slices regardless of whether TTX is present.
Previous results indicated that the inhibition of a
Na+/K+ pump after chronic morphine treatment
caused depolarization and increased the excitability of S neurons from
myenteric plexus (Kong et al. 1997). The absence of a
significant difference in the RMP between LC neurons from control and
morphine-dependent rats, and a similar effect of ouabain on the RMP and
SDR in LC neurons from both groups of animals, make such a mechanism
unlikely in the withdrawal-induced hyperactivity of LC neurons.
Chronic morphine administration has previously been shown to upregulate
the cAMP second-messenger and protein phosphorylation pathways in LC
neurons (Nestler 1992; Nestler and Tallman
1988
). It was hypothesized that the elevated cAMP activates
cAMP-dependent TTX-insensitive sodium channels, which depolarize LC
neurons and increases their excitability (Alreja and Aghajanian
1991
; Wang and Aghajanian 1987
). However, this
suggestion was not consistent with other results (Travalgi et
al. 1995
). The present findings also do not support this
hypothesis. First, LC neurons from dependent rats were not
significantly depolarized compared with LC neurons from control rats,
as would be expected for cells with a persistently increased
Na+ conductance. Moreover, in ~30% of tested
cells, the withdrawal-induced increase in impulse activity of LC
neurons developed without a detectable depolarization, and a few cells
even exhibited a small hyperpolarization after naloxone. Second, the
selective activator of PKA, Sp-cAMPS, increased impulse activity
without depolarizing LC neurons from control rats, and, selective
inhibitors of PKA (Rp-cAMPS and H-89) suppressed the withdrawal-induced
hyperactivity of LC neurons without significantly hyperpolarizing their
membrane potential. Moreover, the cAMP stimulator forskolin had no
consistent effect on the membrane potential of LC neurons as was also
reported in a previous study (Harris and Williams 1991
).
Increased impulse activity following forskolin could reflect
attenuation of the AHP in LC neurons (Shiekhattar and
Aston-Jones 1994
).
Together, the present results indicate that elevated impulse activity
in LC neurons during opiate withdrawal and activation of the cAMP
cascade was not caused simply by depolarization of these cells
subsequent to an increased Na conductance. However, it is possible that
distal dendrites become depolarized during withdrawal without being
detectable with recordings in the soma. Such an effect could lead to
elevated spike rates, particularly in neurons whose axons originate
from proximal dendrites, as in the LC (Groves and Wilson
1980). This possibility requires further analysis.
In addition, the participation of an unidentified inward current that
slightly depolarizes LC neurons in dependent rats cannot be excluded by
the present results. It is logical to suggest that potassium channels
activated by morphine during chronic treatment are the primary targets
for adaptive changes. The present findings (no changes in membrane
potential and increase in spontaneous discharge rate after chronic
morphine treatment) lead us to propose that a downregulation of
potassium channels that control spontaneous discharge rate resulting
from activation of PKA causes the naloxone-precipitated hyperexcitation
in LC neurons. Indeed, the most prominent effect after naloxone
observed in this study was increased frequency of spontaneous action
potentials. Previous work indicated that two types of potassium
currents are primarily involved in the regulation of spike frequency in
central neurons: the A current and the SK current (Rudy
1988). These currents exist in LC neurons (Osmanovic and
Shefner 1993
; Williams et al. 1984
) and
participate in the regulation of SDR (Andrade and Aghajanian
1984b
). Furthermore Rp-cAMPS selectively prevented the
inhibitory effect of cAMP on the SK current in hippocampal neurons
(Pedarzani and Storm 1993
), and activation of either PKA
or PKC downregulated the transient A-type K+
channels in dendrites of hippocampal CA1 pyramidal neurons
(Hoffman and Johnston 1998
). These findings are
consistent with our hypothesis that chronic morphine administration
(and the associated increase in cAMP) lead to decreased
K+ channel activity in LC neurons, and that this
change underlies the locally induced withdrawal-precipitated
hyperactivity in LC cells.
Thus our results demonstrate that the withdrawal-induced hyperactivity in LC neurons in vitro does not involve release of neurotransmitters but presumably results from adaptations produced by chronic morphine treatment. Our studies also indicate that this adaptation may involve downregulation of conductances in the family of potassium channels. Further studies using voltage-clamp methods and a preparation with truncated LC dendrites are needed to test this hypothesis, and to elucidate the specific channel(s) that are altered during chronic morphine exposure.
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ACKNOWLEDGMENTS |
---|
We thank Dr. Glenda Harris for comments on the manuscript.
This work was supported by National Institute on Drug Abuse Grant DA-06214.
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FOOTNOTES |
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Address for reprint requests: G. Aston-Jones, Dept. of Psychiatry, University of Pennsylvania, VAMC (151), University and Woodland Aves., Philadelphia, PA 19104 (E-mail: gaj{at}mail.med.upenn.edu).
Received 23 February 2000; accepted in final form 5 February 2001.
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REFERENCES |
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