The Scripps Research Institute, Department of Neuropharmacology, La Jolla, California 92037
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ABSTRACT |
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Madamba, Samuel G.,
Paul Schweitzer, and
George Robert Siggins.
Nociceptin Augments K+ Currents in Hippocampal CA1
Neurons by Both ORL-1 and Opiate Receptor Mechanisms.
J. Neurophysiol. 82: 1776-1785, 1999.
We previously
reported (see also the accompanying paper) that dynorphin A
significantly enhanced the voltage-dependent K+ M-current
(IM) in CA3 and CA1 hippocampal pyramidal
neurons (HPNs). Because the opioid-receptor-like-1 (ORL-1) receptor
shares a high sequence homology with opioid receptors and is expressed
in rat hippocampus, we examined the effects of orphanin FQ or
nociceptin, the endogenous ligand for the ORL-1 receptor, using the rat
hippocampal slice preparation and intracellular voltage-clamp
recording. Current-voltage (I-V) relationships from CA1
HPNs revealed that nociceptin superfusion induced an outward current
reversing near the equilibrium potential for K+ ions.
Ba2+ (2 mM) blocked this effect. The nociceptin-induced
current was largest at depolarized membrane potentials, where
IM is largely activated. Nociceptin
concentrations of 0.5-1 µM (but not 0.1 µM) significantly
increased IM relaxation amplitudes with
recovery on washout. Interestingly, both the general opiate antagonist naloxone and the receptor antagonist nor-binaltorphimine (nBNI) inhibited the nociceptin-induced IM
increases and outward currents in the depolarized range but not the
inward current induced at hyperpolarized potentials. The putative ORL-1
receptor antagonist, [Phe1
(CH2-NH)Gly2]NC(1-13)NH2
(hereafter ORLAn), blocked most of the nociceptin current near rest but
not the IM increase. However, ORLAn alone had direct effects similar to those of nociceptin, indicating that
ORLAn might be a partial agonist. Our results suggest that nociceptin
postsynaptically modulates the excitability of HPNs through ORL-1 and
-like opiate receptors linked to different K+ channels.
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INTRODUCTION |
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The primary structure of the recently identified
heptadecapeptide called nociceptin (Meunier et al. 1995)
or orphanin FQ (Reinscheid et al. 1996
) (hereafter
nociceptin) shares a high sequence homology with opioid peptides, and
especially dynorphin A (Meunier et al. 1995
). Indeed,
Zhang and Yu (1995)
have identified dynorphin A as a
potential ligand for the opioid-receptor-like-1 (ORL-1) receptor expressed in Xenopus oocytes. This receptor also shares a
high sequence homology with opiate receptors (Bunzow et al.
1994
) and is localized in rat hippocampus (Anton et al.
1996
; Bunzow et al. 1994
). Nociceptin has been
shown to hyperpolarize neurons by activating an inwardly rectifying
K+ conductance in neurons of locus coeruleus
(Connor et al. 1996a
), dorsal raphe (Vaughan and
Christie 1996
), hypothalamus (Lee et al. 1997
),
and CA3 hippocampus (Ikeda et al. 1997
). Nociceptin also
inhibits Ca2+ currents in cultured hippocampal
neurons (Knoflach et al. 1996
), reduces field excitatory
postsynaptic potentials (EPSPs) and prevents the induction of long-term
potentiation (LTP) in CA1 hippocampus (Yu et al. 1997
;
for review see Meunier 1997
).
Recent electrophysiological studies from our laboratory (Madamba
et al. 1997; Moore et al. 1994
; see also the
accompanying paper, Madamba et al. 1999
) showed that dynorphin A
enhanced the noninactivating voltage-dependent K+
M-current (IM) in both CA3 and CA1
hippocampal pyramidal neurons (HPNs). This effect of dynorphin was
blocked by naloxone and nor-binaltorphimine (nBNI). In most CA3 and
some CA1 HPNs, the
agonist U50,488h also increased
IM. However, U69,593, a selective
1 receptor agonist, did not increase
IM relaxation amplitudes in CA1 HPNs,
suggesting that a novel
receptor subtype or ORL-1 might be
involved. Although
receptor agonists decreased
IM in CA3, opiate agonists selective for
and µ receptors had no effect on
IM or other postsynaptic membrane
properties in CA1.
Therefore because of the similarities between dynorphin and nociceptin,
and the possibility that ORL-1 receptors might mediate the dynorphin
IM effect, we examined the effects of
nociceptin on CA1 neurons of the hippocampal slice preparation. Here we
report that nociceptin, like dynorphin A, enhances
IM by a -like opiate receptor but
also activates another K+ conductance via a
nonopiate, putative ORL-1 receptor.
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METHODS |
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Slice preparation
We used standard intracellular voltage-clamp recording
techniques in the rat hippocampal slice, prepared as described
previously (Madamba et al. 1996; Schweitzer et
al. 1993
) and in the accompanying paper (Madamba et al. 1999
).
In brief, male Sprague-Dawley rats (100-170 g) were anesthetized with
halothane (3%), decapitated, and their brains rapidly removed and
placed in ice-cold artificial cerebrospinal fluid (ACSF) gassed with
95% O2-5% CO2. We used sharp glass micropipettes filled with 3 M KCl (tip resistance, 73 ± 3 M
; mean ± SE) to penetrate CA1 neurons. Methods of
superfusion, voltage-clamp recording, cell identification, drug
administration, and data analysis were as described previously
(Madamba et al. 1996
; Schweitzer et al.
1993
) and in the accompanying paper (Madamba et al. 1999
).
Electrophysiological methods
Current and voltage records were acquired, stored, and analyzed
as described in the accompanying paper (Madamba et al. 1999). Tetrodotoxin (TTX; 1 µM) was added to the superfusate to block Na+-dependent action potentials and inhibit
synaptic transmission.
Electrophysiological protocols
CURRENT-VOLTAGE (I-V) ANALYSIS.
We evaluated I-V relationships from a holding potential of
61 ± 1 mV with hyperpolarizing and depolarizing steps (1.5 s
duration, 7 s apart). The I-V curves were constructed
from the current values measured at the end of the voltage steps
(steady-state) just before command offset, and the values obtained in
control condition were subtracted from those in the presence of the
tested substance to obtain the net current elicited.
M-CURRENT ANALYSIS.
In CA1 HPNs, IM is commonly seen with
holding potentials around 45 mV and hyperpolarizing steps of 5-25 mV
and 1-s duration (Halliwell and Adams 1982
; Moore
et al. 1994
); it then appears as a slow inward "relaxation"
following the instantaneous (ohmic) inward current. We measured
IM amplitude with software (Clampfit, Axon Instruments) that fitted second-order exponential curves to the
IM relaxation and used the difference
between the current values just after command onset and just before
command offset to quantify the amplitude of
IM.
Q-CURRENT ANALYSIS.
We elicited the inwardly-rectifying IQ
(or Ih) from a holding potential
around 60 mV using a series of hyperpolarizing voltage steps in
increments of 15 mV and 1.5 s duration. The
IQ relaxation amplitudes were analyzed
identically to those of IM.
Drug administration
Drugs and peptides were added from a stock solution to the ACSF
in known concentrations immediately before administration to the slice
chamber. In this study the cells were depolarized to a mean of
47 ± 0.5 mV for IM analysis
but were held near resting potential between these periods (control,
drug, and wash out) to avoid the instabilities that may develop with
prolonged depolarization (see Halliwell and Adams 1982
).
For tests of the opiate antagonists, the usual protocol involved first
applying the agonist (e.g., nociceptin), followed by washout with ACSF
alone ("washout"), then adding naloxone or nBNI (to test their
possible effects on membrane properties) followed by addition of
nociceptin again but now together with the antagonist. In several
neurons, we applied nBNI first (for 6-7 min), followed by addition of
nociceptin in the presence of the
antagonist. Similarly, in
other studies we superfused the ORL-1 receptor antagonist,
[Phe1
(CH2-NH)Gly2]NC(1-13)NH2
(ORLAn) first followed by nociceptin in the presence of the
antagonist. At the end of some recordings, 2 mM
Ba2+ was added to the superfusate to verify that
IM relaxation currents were due to
K+ currents.
We obtained nociceptin and ORLAn from Tocris Cookson (Ballwin, MO), naloxone from Sigma (St. Louis, MO), nBNI from Research Biochemicals International (Natick, MA), and TTX from Calbiochem-Novabiochem (San Diego, CA).
Quantification and statistics
All measurements are reported as means ± SE. We usually determined statistical significance by two-way ANOVA for repeated measures, followed by the Newman-Keuls post hoc test and simple main effects as warranted by significant group effects and interactions. For within-subject effects with at least 2 degrees of freedom, we used the more conservative Huynh-Feldt P value to reduce type 1 errors.
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RESULTS |
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We recorded from a total of 45 CA1 HPNs. These neurons had an
average resting membrane potential (RMP) of 68 ± 1 mV with a
mean spike amplitude of 104 ± 3 mV (n = 28).
Nociceptin alters I-V relationships
We generated I-V relationships to study the overall
effects of nociceptin on steady-state membrane properties in the
depolarized and hyperpolarized ranges (Fig.
1A). Superfusion of 0.5 and 1 µM nociceptin elicited an outward current in the depolarized range that reversed at 93 mV (mean). Figure 1B shows
control-subtracted graphs generated from the I-V
relationships, as described in METHODS, to isolate the
current elicited by 0.1, 0.5, and 1 µM nociceptin. Superfusion of 0.1 µM nociceptin did not significantly (P > 0.5) alter
steady-state currents, although a slight outward current was seen at
depolarized potentials (Fig. 1B, left panel;
n = 4). A higher concentration of nociceptin (0.5 µM;
Fig. 1, middle panel; n = 6) elicited a significant
[F(7,35) = 10.118, P < 0.01] outward current at membrane potentials of
51 to
65 mV and an inward current
at potentials of
121 to
135 mV, with a reversal at
94 ± 7 mV. Similarly, 1 µM nociceptin (Fig. 1, right panel;
n = 6) induced a significant [F(7,35) = 43.228, P < 0.001] outward current at potentials of
47 to
61 mV, an effect reversing at
93 ± 6 mV. Although 1 µM nociceptin also induced an inward current at hyperpolarized
potentials (
117 to
131 mV), this was not significantly different
compared with washout (perhaps because of persistence of effect or
incomplete washout).
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The theoretical reversal potential for K+
calculated by the Nernst equation, in ACSF containing 3.5 mM
K+ and assuming an intracellular concentration of
150 mM K+, is 98 mV. The experimental reversal
potentials we obtained (
94 and
93 mV with 0.5 and 1 µM
nociceptin, respectively), were thus near the theoretical reversal
potential for K+ ions. To further confirm the
ionic nature of the nociceptin effect, we superfused
Ba2+ at a concentration of 2 mM to
nonspecifically block K+ conductances. In the
presence of 2 mM Ba2+, nociceptin no longer
affected steady-state current recordings generated with I-V
protocols (Fig. 2, A and
B). Comparison of the net effect of 0.5 µM nociceptin in
the absence (n = 6) or presence (n = 5)
of Ba2+ (Fig. 2C) indicated that
Ba2+ prevented most of the nociceptin effect,
consistent with involvement of K+ conductances.
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Nociceptin augments the M-current
Superfusion of 0.5-1 µM nociceptin enhanced
IM relaxation amplitudes. In the HPN
recording shown in Fig. 3A,
superfusion of 1 µM nociceptin enhanced
IM amplitudes to 229% of control,
increased input conductance and induced an outward current (391 pA) at
holding potential (- - -), with recovery on washout. The average
of six such experiments with 1 µM nociceptin is shown in Fig.
3D: nociceptin significantly [F(2,10) = 15.798, P < 0.01; Newman-Keuls, P < 0.01 for control and wash] enhanced mean
IM amplitudes with recovery on wash
out. In contrast, on average 0.1 µM nociceptin did not significantly
(P > 0.5; n = 5) enhance
IM amplitudes (Fig. 3B). An
intermediate concentration (0.5 µM) significantly
[F(2,30) = 13.832, P < 0.01;
Newman-Keuls, P < 0.001 for control and wash; n = 16] increased IM
relaxation amplitudes with recovery on washout (Fig. 3C).
Because there are reports of ORL-1 desensitization (Connor et
al. 1996a; Ma et al. 1997
), in another set of
five neurons we superfused 0.5 µM nociceptin twice with washout (mean 27 min) between applications. Both nociceptin applications increased IM amplitudes similarly to 263 ± 94% of control for the first and 240 ± 59% of control for the
second application (measured at the 15-mV step), suggesting a minimal
tachyphylaxis over this time course.
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Nociceptin-induced currents have an opiate receptor-mediated component
There are conflicting reports regarding opioid receptor mediation
of nociceptin effects (Connor et al. 1996a;
Knoflach et al. 1996
; Nicol et al. 1996
;
Rossi et al. 1996
; Wang et al. 1996
; Zhang and Yu 1995
). Therefore we superfused opiate
antagonists to determine whether nociceptin effects in CA1 neurons
could be mediated by an opiate mechanism (Fig.
4). We first performed I-V analysis in the presence of 4 µM naloxone, a broad spectrum opiate antagonist, to investigate a possible alteration of the
nociceptin-induced steady-state currents. Although naloxone had no
significant effect on the I-V curve analyzed over the entire
voltage range, it significantly reduced [F(1,4) = 27.14, P < 0.01] the nociceptin-induced steady-state outward current in the depolarized range (Fig. 4, B and
C), where IM is largely
activated. In contrast, at hyperpolarized potentials (
85 to
139
mV), naloxone did not alter the nociceptin-induced inward current (Fig.
4B).
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We then investigated the effect of the specific -opioid receptor
antagonist nBNI. As with naloxone, superfusion of 200-400 nM nBNI
significantly [F(1,5) = 20.79, P < 0.01] inhibited the nociceptin-induced outward current in the
depolarized range (Fig. 4, A, D, and E), while
leaving the nociceptin-induced inward component unaffected. Figure 4,
C and E, shows the nociceptin-induced components sensitive to naloxone and nBNI (currents elicited by nociceptin alone
minus currents elicited by nociceptin in the presence of opioid
antagonist). Thus isolating the naloxone- and nBNI-sensitive currents
revealed two nociceptin effects: one sensitive and one insensitive to
opiate antagonists.
To again ensure that desensitization to nociceptin was not a confound
in these experiments, another five neurons were exposed to nociceptin
only once, in the presence of nBNI. Such nBNI pretreatment significantly [ANOVA between subjects; F(1,42) = 9.075, P < 0.01; measured at 48 to
79 mV] blunted
the nociceptin-induced outward steady-state current, indicating that
the lack of effect of nociceptin was not due to desensitization caused
by repeated applications.
Opiate antagonists block nociceptin M-current effects
Using the IM protocol, 4 µM naloxone inhibited both the nociceptin-induced IM increase and the outward holding steady-state current (Fig. 5A). Mean data pooled from a different set of five neurons (Fig. 5B) showed that nociceptin (0.5 µM, n = 3; 1 µM, n = 2) significantly [F(4,16) = 24.107, P < 0.001; Newman-Keuls, P < 0.05 for all conditions] increased IM relaxation amplitudes with recovery on washout. In the same neurons, naloxone (4 µM) alone did not significantly alter IM but prevented the nociceptin enhancement of IM amplitudes (Fig. 5B). Although naloxone alone did not affect IM, it induced a slight inward current in the depolarized range (Fig. 5C), perhaps due to block of opiate receptors activated constitutively or by endogenous opioids.
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Because of the high structural homology between ORL-1 and receptors, and because nociceptin and dynorphin had similar effects on
IM, we tested nBNI, the specific
receptor antagonist. Superfusion of 200 nM nBNI alone did not alter
IM amplitudes nor change the holding
current, but blocked the nociceptin-induced
IM increase and outward holding
current (Fig. 6A). Subsequent
application of 2 mM Ba2+ induced an inward
current and totally blocked IM,
verifying the involvement of K+ channels. Figure
6B shows the average of six experiments like that shown in
Fig. 6A. As before, 0.5 µM nociceptin significantly [F(4,20) = 9.825, P < 0.05;
Newman-Keuls, P < 0.001 for all conditions] increased
mean IM amplitudes. Superfusion of 200 nM nBNI alone (n = 6) did not significantly
(P > 0.05) alter IM
but blocked the nociceptin-induced IM
increase. In another set of five neurons, nociceptin was superfused
only once to eliminate the possibility of tachyphylaxis: we pretreated
slices with 200-400 nM nBNI first, followed by 0.5 µM nociceptin
together with nBNI. In the presence of nBNI, 0.5 µM nociceptin did
not significantly (P > 0.5) alter IM relaxation amplitudes (Fig.
6C).
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ORL-1 antagonist blocks nociceptin-induced steady-state current near rest, but not IM
Recently, Guerrini et al. (1998) identified a
nociceptin receptor antagonist, ORLAn. In CA1 HPNs, superfusion of 1-2
µM ORLAn alone induced steady-state currents (Fig.
7A, left panel). In the
continued presence of ORLAn, 0.5 µM nociceptin had little effect near
rest. For the net nociceptin-induced steady-state currents in the
presence of ORLAn, we show the current induced by nociceptin alone
(Fig. 7A, right panel); notice that ORLAn prevented most of the nociceptin-induced current near rest. Because ORLAn had both agonist and antagonist actions in CA1 neurons, it might
be characterized as a partial agonist.
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We also asked if ORLAn could alter IM relaxation amplitudes and block the nociceptin enhancement of IM. Interestingly, 1 µM ORLAn itself increased IM and induced an outward holding current (Fig. 7B), an effect similar to that of nociceptin. However, addition of nociceptin in the continued presence of ORLAn elicited a further enhancement of IM, suggesting lack of antagonism by ORLAn. Washout of both ORLAn and nociceptin returned IM relaxation amplitudes to control levels. A second application of 0.5 µM nociceptin again increased IM amplitudes (not shown). On average ORLAn alone and nociceptin with ORLAn both significantly [F(2,12) = 16.884, P < 0.001; Newman-Keuls; P < 0.05 for all cases] increased IM amplitudes (Fig. 7C).
In HPNs, the voltage-dependent cationic conductance termed
IQ (Halliwell and Adams
1982) or Ih is seen as a slow
inward current relaxation with hyperpolarizing voltage steps to
potentials more negative than
65 mV. In the presence of 2 mM
Ba2+ to isolate
IQ, nociceptin had little effect on
this current. Subsequent superfusion of 1 mM Cs+
inhibited the inward current, verifying
IQ identity (Fig.
8A). In the same
cells where nociceptin increased IM
amplitudes (see Fig. 6B), nociceptin did not significantly
(P > 0.05) alter IQ (Fig. 8B), although there was a slight increase at
123 mV
(to 111% of control; n = 6).
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DISCUSSION |
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Our studies have shown that 1) nociceptin augmented
Ba2+-sensitive currents that reversed near the
equilibrium potential for K+; 2)
naloxone and nBNI inhibited most of the nociceptin current at
depolarized potentials but not that in the hyperpolarized range; 3) the ORL-1 antagonist ORLAn blocked the nociceptin
currents near RMP; 4) nociceptin increased
IM relaxation amplitudes;
5) naloxone and nBNI, but not ORLAn, prevented the
enhancement of IM; and 6)
nociceptin had little effect on IQ.
These data suggest that nociceptin can postsynaptically modulate the
excitability of HPNs through both ORL-1 and -like opiate receptors
linked to different K+ channels.
Nociceptin-evoked currents
The I-V relationships from CA1 HPNs showed that
nociceptin currents reversed near 98 mV in 3.5 mM
K+, suggesting that they are carried by
K+ ions. Furthermore, Ba2+
inhibited the nociceptin effect, further indicating involvement of
K+ conductances. Other studies have found that
nociceptin hyperpolarized neurons by activating an inwardly rectifying
K+ conductance in locus coeruleus (Connor
et al. 1996a
) and dorsal raphe (Vaughan and Christie
1996
). Similarly, nociceptin hyperpolarized mouse CA3 HPNs by
activating an inwardly rectifying K+ channel
current coupled to a G-protein, and this effect was blocked by
Ba2+ (Ikeda et al. 1997
). In our
hands, a low (100 nM) concentration of nociceptin had little effect on
steady-state currents or IM amplitudes, whereas a slightly higher concentration (0.5 µM) elicited a marked effect throughout the voltage range tested. Superfusion of 1 µM nociceptin did not elicit an effect beyond that with 0.5 µM,
indicating that the maximum effect was reached with 0.5 µM nociceptin. Therefore nociceptin has a fairly steep dose-response relationship with an apparent EC50 probably
falling between 0.1 and 0.5 µM.
The nociceptin effect at depolarized membrane potentials, where
IM is activated, was mostly inhibited
by naloxone and nBNI. Several studies have found that naloxone did not
inhibit nociceptin effects, in dorsal raphe nucleus (Vaughan and
Christie 1996), Xenopus laevis oocytes
coexpressing ORL-1 with potassium channel subunits (Matthes et
al. 1996
), and periaqueductal gray neurons (Vaughan et
al. 1997
). Similarly, in our study naloxone and nBNI did not
alter the nociceptin current seen at hyperpolarized potentials, suggesting that a nonopioid mechanism may be involved in this voltage
range (see Fig. 4).
We used a recently identified nociceptin antagonist to investigate
specific ORL-1 mediated effects in CA1 HPNs. Guerrini et al.
(1998) reported that ORLAn prevented the nociceptin-induced contractions of guinea pig ileum. In the presence of ORLAn, most of the
nociceptin-induced steady-state current near RMP was blocked in CA1
HPNs. However, superfusion of ORLAn alone elicited an effect similar to
that with nociceptin, suggesting that it may act as a partial agonist.
Interestingly, Butour et al. (1998)
reported that ORLAn
acted as an agonist in transformed CHO cells expressing the human ORL-1
receptor. These studies indicate that ORLAn may need further
characterization and also suggest possible multiple ORL-1 receptor
types (see Nociceptin augments IM).
Our findings indicate that nociceptin inhibits CA1 HPNs by
concomitantly activating at least two conductances: an opiate
receptor-mediated outward current (most probably
IM) at depolarized potentials and an
ORL-1-mediated current near rest. A possible candidate for this latter
ORLAn-sensitive current might be either the inward rectifier
(Ikeda et al. 1997) or a voltage-independent
K+ current (so-called "resting" current), that are both
sensitive to Ba2+, although a more complete
characterization of this current is needed. Interestingly, we reported
that somatostatin also inhibits HPNs by activating both
IM and a voltage-insensitive K+
current (Schweitzer et al. 1998
). In rat hippocampus,
IQ (or Ih) is a
mixed Na+/K+ inwardly rectifying conductance
activated at hyperpolarized membrane potentials. We found that
nociceptin (like somatostatin) did not significantly alter
IQ, indicating that this conductance is
unlikely to account for the nociceptin current at hyperpolarized potentials.
Nociceptin augments IM
The nociceptin increase in IM
relaxation amplitudes was blocked by naloxone and nBNI, suggesting that
nociceptin may interact with a opiate receptor. Still, naloxone
inhibition of nociceptin effects is controversial. The original studies
that identified nociceptin/orphanin FQ (Meunier et al.
1995
; Reinscheid et al. 1995
) showed that
nociceptin shares a high homologous sequence with dynorphin A but did
not interact with the opiate receptors. However, there are some reports
suggesting that the nociceptin system may interact with the opioid
system. Thus Zhang and Yu (1995)
reported that dynorphin
inhibited forskolin-stimulated cyclic AMP through ORL-1 receptors
expressed in Xenopus oocytes. Nociceptin-induced analgesia
was blocked by naloxone in mice and rats (Rossi et al.
1996
, 1998
). In a microdialysis study of rat striatum, nociceptin-induced dopamine release was attenuated by naloxone (Konya et al. 1998
). Furthermore, dynorphin A
displaced the binding of nociceptin to ORL-1 receptor in the range of
10-100 nM (Meng et al. 1996
), and naloxone and nBNI in
the micromolar range inhibited nociceptin binding to ORL-1 in a CHO
cell line expressing the human ORL-1 receptor (Fawzi et al.
1997
).
We (Madamba et al. 1999, accompanying paper) have found
that dynorphin A also increased IM
amplitudes in CA1 HPNs, an effect also prevented by nBNI. However, the
selective
agonists U69,593 and U50,488h did not significantly
increase IM amplitudes, suggesting that classic
receptors (e.g.,
1) might not
be involved. The receptors activated in this study could represent a
different
receptor subtype (e.g.,
2) or a
splice variant not yet characterized (see Rossi et al.
1996
). Another possibility is that the nociceptin (and perhaps
dynorphin) enhancement of IM is
mediated via a novel nociceptin receptor sensitive to opiate
antagonists. In accord with this idea, ORLAn alone increased
IM amplitudes but did not prevent the
nociceptin enhancement of IM. Earlier
studies characterizing the ORL-1 receptor have suggested splice
variants that differ in conformation (Halford et al.
1995
; Wang et al. 1994
). Similarly, Rossi
et al. (1996)
have suggested that
3
and/or KOR-3 receptors might be splice variants. Indeed, the
3 agonist naloxone benzoylhydrazone inhibited
nociceptin reduction of Ca2+ currents in rat
dorsal root ganglia (Abdulla and Smith 1997
). Interestingly, sigma receptors also have been implicated in some nociceptin effects (Kobayashi et al. 1997
).
Several reports have suggested that ORL-1 receptors may undergo acute
and homologous desensitization (Connor et al. 1996a; Ma et al. 1997
). However, we did not detect
desensitization of nociceptin enhancement of
IM amplitudes. Furthermore, the
averaged nociceptin-induced steady-state current was not significantly different between two successive applications. Similarly, the nociceptin-induced decrease of Ca2+ currents in
dissociated hippocampal neurons did not desensitize after repeated
applications of nociceptin (Knoflach et al. 1996
).
Possible functional role for nociceptin in CA1
As noted in the preceding paper, several groups have reported that
dynorphin inhibits LTP, the cellular model of learning and memory, in
guinea pig hippocampus (Terman et al. 1994;
Wagner et al. 1993
; Weisskopf et al.
1993
). Similarly, nociceptin inhibits LTP in rat CA1
hippocampus (Yu et al. 1997
) and reduces
voltage-sensitive calcium currents (required for CA1 LTP) in
dissociated rat pyramidal neurons (Knoflach et al.
1996
). In behavioral studies, both nociceptin and dynorphin
(via
receptors) diminish spatial learning in rats (Sandin et
al. 1997
, 1998
). Thus endogenous nociceptin, by
activating an outward current carried by two different
K+ conductances, could play a role in preventing
or regulating LTP induction. Conversely, dynorphin and/or nociceptin
might enhance or mediate long-term depression (LTD). This possibility
is supported by our recent data showing a novel form of LTD in CA1 that
is blocked by naloxone (Francesconi et al. 1997
).
Studies in progress should help determine whether the opiate receptors
and ion channels involved in this LTD are the same as those mediating
dynorphin and nociceptin effects.
In an immunohistochemical study, Anton et al. (1996)
found evidence for postsynaptic ORL-1 localization in the hilus of the dentate. The hilar mossy cells and somatostatin containing neurons are
believed to play a role in epilepsy and seizures (for review see
McNamara 1994
). A recent study also showed nociceptin
immunoreactivity in the hippocampus (Mitsuma et al.
1998
). It is possible that nociceptin is co-localized with
somatostatin or is located in hilar interneurons, where it could serve
as a brake to inhibit seizures, as postulated for somatostatin
(Tallent and Siggins 1997
). Indeed, nociceptin inhibits
epileptiform activity in CA3 neurons in slices treated with
Mg2+-free ACSF (M. K. Tallent, unpublished
observations). Nociceptin and somatostatin have similar actions in CA1
pyramidal neurons: augmentation of IM
amplitude and induction of an outward current at rest. Somatostatin and
nociceptin also both have inhibitory presynaptic actions in the
hippocampus (Tallent and Siggins 1997
; Yu et al.
1997
).
These and other studies show that nociceptin markedly inhibits neurons
by a variety of mechanisms, including activation of IM and an inwardly rectifying K+
conductance (Ikeda et al. 1997), reduction of various
Ca2+ currents (Abdulla and Smith 1997
;
Connor et al. 1996b
), and reduction of synaptic
transmission (Yu et al. 1997
). These effects are
consistent with an anticonvulsive function. Our findings that
nociceptin can augment at least two K+ conductances, one
active at depolarized potentials (IM) and
the other near rest and at more hyperpolarized potentials, suggest that
endogenous nociceptin could provide a powerful inhibitory influence
over a wide range of membrane potentials. This influence may serve to
clamp the membrane potential at rest and provide an intrinsic braking
mechanism against neuronal hyperexcitabilty.
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ACKNOWLEDGMENTS |
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We thank Drs. S. J. Henriksen and M. K. Tallent for valuable criticism.
This research was supported by National Institutes of Health Grants DA-03665, MH-44346, and K01-DA-00291.
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FOOTNOTES |
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Address for reprint requests: G. R. Siggins, CVN12, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 21 September 1998; accepted in final form 3 June 1999.
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REFERENCES |
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