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.
Dynorphin Selectively Augments the M-Current in Hippocampal CA1
Neurons by an Opiate Receptor Mechanism.
J. Neurophysiol. 82: 1768-1775, 1999.
Most electrophysiological
studies of opioids on hippocampal principal neurons have found indirect
actions, usually through interneurons. However, our laboratory recently
found reciprocal alteration of the voltage-dependent K+
current, known as the M-current (IM), by and
opioid agonists in CA3 pyramidal neurons. Recent
ultrastructural studies have revealed postsynaptic
opiate receptors
on dendrites and cell bodies of CA1 and CA3 hippocampal pyramidal
neurons (HPNs). Reasoning that previous electrophysiological studies
may have overlooked voltage-dependent postsynaptic effects of the
opioids in CA1, we reevaluated their role in CA1 HPNs using the rat
hippocampal slice preparation for intracellular current- and
voltage-clamp recording. None of the
and µ receptor-selective
opioids tested, including
{D-Pen2,5}-enkephalin (DPDPE),
{D-Ala2}-deltorphin II (deltorphin),
{D-Ala2, NMe-Phe4,
Gly-ol}-enkephalin (DAMGO), and {D-Ala2,
D-Leu5} enkephalin (DADLE), altered membrane
properties such as IM or Ca2+-dependent spikes in CA1 HPNs. The nonopioid,
Des-Tyr-dynorphin (D-T-dyn), also had no effect. By contrast, dynorphin
A (1-17) markedly increased IM at low
concentrations and caused an outward current at depolarized membrane
potentials. The opioid antagonist naloxone and the
receptor
antagonist nor-binaltorphimine (nBNI) blocked the
IM effect. However, the
-selective
agonists U69,593 and U50,488h did not significantly alter
IM amplitudes when averaged over all cells
tested, although occasional cells showed an
IM increase with U50,488h. Our results
suggest that dynorphin A postsynaptically modulates the excitability of
CA1 HPNs through opiate receptors linked to voltage-dependent
K+ channels. These findings also provide pharmacological
evidence for a functional
opiate receptor subtype in rat CA1 HPNs
but leave unanswered questions on the role of
receptors in CA1 HPNs.
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INTRODUCTION |
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Although the rodent hippocampus contains opioid
peptides and opiate receptors, the functional role of these elements is
still under investigation. Nonetheless, after two decades of research, several concepts have emerged (see Siggins et al. 1986);
these include 1) the reported lack of direct effect of the
opioids on pyramidal neurons (Nicoll et al. 1980
;
Siggins 1990
; Siggins and Zieglgänsberger
1981
; Zieglgänsberger et al. 1979
),
2) the well-known disinhibitory effect of
and µ opioids mediated by inhibitory opiate receptors on interneurons
(Lupica and Dunwiddie 1991
; Madison et al.
1987
; Zieglgänsberger et al. 1979
),
and 3) the presence of presynaptic
opiate receptors that
inhibit transmitter release (Castillo et al. 1996
;
Salin et al. 1995
; Wagner et al. 1992
; Weisskopf et al. 1993
) and may be involved in depressing
some forms of long-term potentiation (LTP) (Wagner et al.
1993
; Weisskopf et al. 1993
). However,
subsequent intracellular studies in our laboratory (Moore et al.
1994
) showed that opioids could act directly on rat CA3
hippocampal pyramidal neurons (HPNs): agonists selective for
opiate
receptors enhanced the voltage-dependent
IM, whereas
(but not µ) agonists
reduced IM. In contrast, Salin
et al. (1995)
reported that CA3 HPNs in several species did not
show such direct postsynaptic effects of opioids.
Autoradiographic studies have revealed receptor binding in the rat
hippocampal pyramidal cell layer (Tempel and Zukin 1987
; Zukin et al. 1988
). In an immunohistochemical study,
Arvidsson et al. (1995b)
showed that
receptors are
located on both post- and presynaptic elements in rat and guinea pig.
In contrast, Drake et al. (1996)
reported that
receptors are located presynaptically in the guinea pig hippocampal
formation. Electrophysiological studies have suggested that dynorphin
may activate µ but not
receptors in CA1 neurons (Chavkin
et al. 1985a
; Neumaier et al. 1988
). Similarly,
results of previous electrophysiological and binding studies suggested
that opiate receptors (mostly µ and
) were located primarily on
elements presynaptic to CA1 and CA3 HPNs (Unnerstall et al.
1983
; Zieglgänsberger et al. 1979
).
However, the recent ultrastructural studies of Commons and
Milner (1997)
, using antisera raised against the cloned
opiate receptor, have revealed postsynaptic
opiate receptors on
dendrites and cell bodies of CA1 and CA3 HPNs. Thus these anatomic
findings seemed to corroborate our previous positive findings in CA3
HPNs that
agonists postsynaptically reduced the
IM K+ conductance (Moore
et al. 1994
). Therefore, reasoning that previous studies may
have overlooked voltage-dependent postsynaptic effects of the opioids
in CA1 HPNs, we have now reevaluated the action of various opioids on
these principal neurons. We found that CA1 HPNs showed no response to
application of several
- and µ-selective agonists, yet low
concentrations of dynorphin A more selective for
receptors
augmented the voltage-sensitive IM.
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METHODS |
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Slice preparation
We used standard intracellular current- and voltage-clamp
recording techniques in the rat hippocampal slice, prepared as
described previously (Madamba et al. 1996). In brief,
male Sprague-Dawley rats (100-170 g) were anesthetized with 3%
halothane, decapitated, and their brains rapidly removed and placed in
ice-cold artificial cerebral spinal fluid (ACSF) gassed with 95%
O2-5% CO2. We cut transverse hippocampal slices 350 µm thick on a McIlwain-type brain
slicer, incubated them in an interface configuration for ~30 min, and
then completely submerged and continuously superfused the slices with
gassed ACSF of the following composition (in mM): 130 NaCl, 3.5 KCl,
1.25 NaH2PO4, 1.5 MgSO4·7H2O, 2.0 CaCl2, 24 NaHCO3, and 10 glucose. Other ions and drugs were added to the ACSF in known
concentrations. Slices were superfused with warm (31°C), gassed ACSF,
at a continuous, constant flow rate of 2-4 ml/min. We used sharp glass
micropipettes filled with 3 M KCl (tip resistance, 67 ± 2 M
;
mean ± SE) to penetrate HPNs. Methods of superfusion,
voltage-clamp recording, cell identification, drug administration, and
data analysis were as described previously (Madamba et al.
1996
; Moore et al. 1994
; Schweitzer et
al. 1993
).
Electrophysiological methods
Current and voltage were recorded with an Axoclamp-2A
preamplifier (Axon Instruments) and filtered at 0.3 kHz. Continuous current and voltage records were stored on polygraph paper. We digitized data by D/A sampling and acquisition software (pClamp; Axon
Instruments) and stored them on computer disk for data analysis via
Clampfit software (Axon Instruments). Tetrodotoxin (TTX; 1 µM) was
added to block Na+-dependent action potentials
and inhibit synaptic transmission. In discontinuous single-electrode
voltage-clamp mode, the switching frequency was 3-4 kHz; we
continuously monitored electrode settling time and input capacitance
neutralization at the headstage on a separate oscilloscope
(Finkel and Redman 1985). Current-voltage (I-V) relationships were generated from a holding potential
of
60 ± 0.3 mV with hyperpolarizing and depolarizing steps of
1.5 s duration. Problems (e.g., space clamping) associated with
voltage clamp in neurons with extended processes are discussed
elsewhere (Finkel and Redman 1985
; Halliwell and
Adams 1982
; Johnston and Brown 1983
). These
problems are less acute when studying relative changes following drug
application (see Madison et al. 1987
), especially in the
presence of agents such as TTX and Cd2+ that
block large Na+ and Ca2+ conductances.
M-current analysis
In CA1 HPNs, IM is seen with
holding potentials around 45 mV and hyperpolarizing steps of 5-25 mV
and 700-1,000 ms durations (Halliwell and Adams 1982
;
Moore et al. 1994
); under these conditions it appears as
a slow inward "relaxation" following the instantaneous (ohmic)
inward current drop (see Fig. 1). We
measured IM amplitude with software
(Clampfit, Axon Instruments) that fitted two exponential curves to the
IM relaxation and used the difference
between the instantaneous peak current at command onset and the
steady-state current just before command offset to quantify the
amplitude of IM (Fig. 1). Tail
(off-command) currents were not analyzed because of possible
contamination with other currents (e.g.,
IA or
IT). Usually, an initial current
(capacitative) artifact of 5-20 ms duration was present at command
onset (Halliwell and Adams 1982
). Therefore we fitted
the current relaxation via Clampfit (Chebyshev method) to the peak of
the initial current that usually fell within 5-20 ms of step onset
after complete settling of the capacity transient (see Fig. 1).
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Drug administration
Drugs and peptides were made from a stock solution and added to
the ACSF in known concentrations immediately before administration to
the slice chamber. The usual drug protocol followed for agonist testing
was to record currents during superfusion of ACSF alone ("control"), followed by switching to ACSF with drug and repeating these current measures after 4-10 min of drug superfusion, followed by
switching again to ACSF alone for 10-30 min with subsequent current
measures ("washout"). The cell was depolarized to 40 to
49 mV
for IM analysis at each of the three
periods but was held near resting membrane potential (RMP) between
these periods to avoid the instabilities that might develop with
prolonged depolarization (see Halliwell and Adams 1982
).
For tests of the opioid antagonists, the usual protocol involved first
applying the opioid agonist, followed by washout with ACSF alone,
followed by adding the antagonist to the superfusate (to test possible
effects on baseline properties), followed by addition of the opioid
agonist together with the antagonist. At the end of some recordings, we
added 30 µM carbachol (CCh) or 2 mM Ba2+ to the
superfusate to verify IM relaxation.
We obtained dynorphin A, DAMGO, DPDPE, deltorphin, D-T-dyn, and DADLE from Peninsula Laboratories (Belmont, CA). U69,593, U50,488h, and nBNI were obtained from Research Biochemicals International (Natick, MA). We obtained naloxone from Sigma (St. Louis, MO) and TTX from Calbiochem-Novabiochem (San Diego, CA).
Quantification and statistics
All measures are reported as means ± SE. I-V relationships were plotted by Origin 4.0 software (Microcal Software) using B-spline lines. We determined statistical significance by two-way ANOVA for repeated measures and the Newman-Keuls post hoc test when appropriate.
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RESULTS |
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We recorded from a total of 57 CA1 HPNs. These neurons had an
average RMP of 69 ± 0.3 mV (n = 51) and average
spike amplitude of 105 ± 1 mV (n = 44). Stable
recordings could be maintained for up to 4 h, suggesting a
relative lack of injury by the electrode penetration. We studied
IM using voltage clamp at a mean
holding potential of
43 ± 0.2 mV. Several lines of evidence
suggest, as reported previously (Halliwell and Adams
1982
; Moore et al. 1994
), that the current
relaxations we recorded represented
IM: 1) the relaxations were
suppressed by the muscarinic agonist carbachol (30 µM) or by 2 mM
Ba2+, and 2) the magnitude, kinetics,
and voltage dependence of the relaxations were equivalent to those of
IM previously reported (Halliwell and Adams 1982
; Moore et al.
1988a
; Schweitzer et al. 1993
).
We tested seven different opioid agonists: 1) DPDPE, a
1-selective opioid peptide; 2)
deltorphin, a
2-selective opioid; 3) DAMGO, a µ-selective opioid; 4) DADLE, a
broadly effective opioid peptide capable of activating several receptor
subtypes but especially µ and
; 5) dynorphin A (1-17),
an opioid relatively selective for
receptors at low concentrations
but less selective at higher concentrations; 6) U50,488h,
and 7) U69,593, opioids selective for the
opiate
receptor subtype. We also tested D-T-dyn to rule out possible nonopioid
effects. For any given CA1 neuron, the effects measured were usually
opioid-induced currents at rest, changes in
IM amplitudes, and changes in
I-V relationships. For the
agonists, we also examined
Ca2+-dependent spikes.
Resting and steady-state currents
On average, superfusion of DPDPE, deltorphin, DAMGO, or DADLE
(1-8 µM concentrations) induced little current measured near RMP and
had no effect on I-V curves (Table
1; Fig.
2C). At more depolarized
membrane potentials (near 45 mV), these opioid peptides only induced
small, statistically insignificant inward currents (mean
32 ± 49,
15 ± 22, and
18 ± 57 pA, respectively; DADLE effects were not determined; see Fig. 3
and Table 1). In contrast, 0.5 µM dynorphin had a marked effect near
45 mV: it induced a mean outward current of 178 ± 13 pA, but
only 3 ± 11 pA near RMP. Similarly 1 µM dynorphin induced 214 pA near
45 mV and only 12 pA near RMP (Table 1). Figure
4A shows the effect of
dynorphin on the steady-state current values of a CA1 neuron held at
59 mV and subjected to hyperpolarizing and depolarizing voltage
steps. Superfusion of 0.5 µM dynorphin induced an outward current at depolarized membrane potentials that reversed around
90 mV (Fig. 4A) but induced little current near RMP (
69 mV for this
cell). A lower concentration of dynorphin (0.1 µM) had little effect (4 pA) near
45 mV in two cells. Although on average 4-8 µM
U50,488h had no significant effect on mean
IM amplitudes (see
M-currents), it evoked a mean outward current of 98 ± 22 pA near
45 mV in three cells. In contrast, 1 µM U69,593 induced
little current across the voltage range tested. Similarly, 1 µM
D-T-dyn had no effect on steady state currents (Table 1).
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Ca2+-dependent spikes
Because of the relative lack of effect of agonists on resting
currents, and because of the clear presence of
receptors on CA1
HPNs (Commons and Milner 1997
), we also explored a
possible effect of
agonists on Ca2+-dependent
spikes. Because of difficulties in adequately recording Ca2+ currents in slice preparations, these
experiments were performed in current-clamp mode in the presence of 1 µM TTX to block Na+-dependent spikes.
Superfusion of the selective
1-receptor
agonist DPDPE (1 µM) had little effect on the
Cd2+-sensitive
Ca2+-dependent spikes elicited by depolarizing
voltage steps (Fig. 2A; n = 2). Similarly,
the selective
2 receptor agonist deltorphin (1 µM) also had little effect on Ca2+-dependent
spikes, RMP, or slope resistance in CA1 HPNs (Fig. 2, B and
C; n = 2).
M-currents
None of the µ- or -selective opiate agonists altered
IM relaxation amplitudes in CA1
neurons (Fig. 3). Thus, averaging data from seven neurons, superfusion
of 1-5 µM DPDPE did not significantly (P > 0.1)
alter IM amplitudes when compared with
control values (Fig. 3C, left panel). Likewise, the
selective
2-receptor agonist deltorphin (1 µM) did not significantly change IM
amplitudes in five cells (Fig. 3, B and C, middle
panel; P > 0.9). The µ-selective agonist DAMGO
(1-2 µM; n = 5) also did not significantly
(P > 0.5) change IM
amplitudes (Fig. 3C, right panel). In two cells, 4-8 µM
DADLE also had little effect on IM
amplitudes (data not shown).
Interestingly, in the same cells where 1 µM deltorphin had no
effect on IM amplitude, subsequent
superfusion of 0.5 µM dynorphin clearly augmented
IM (data not shown). Dynorphin A (0.5 µM) markedly induced an outward holding current concomitant with an
increased IM amplitude, with recovery
on washout (Fig. 4B). In the same cell after 23 min washout
of the first exposure, a second application of 0.5 µM dynorphin again
increased IM amplitudes, suggesting a
lack of tachyphylaxis over this period. In data pooled from eight
cells, superfusion of 0.5 µM dynorphin significantly
[F(2,14) = 79.11, P < 0.001;
Newman-Keuls, P < 0.01; for all conditions] increased
mean IM relaxation amplitudes to 160%
of control with recovery on washout (Fig.
5B1). Similarly, in three
cells 1 µM dynorphin increased IM
amplitudes to 169% of control (averaged values taken from 10 to 20 mV
hyperpolarizing steps). In contrast, a lower concentration of dynorphin
(0.1 µM) had no effect on IM (n = 2; data not shown). Thus the maximally effective
concentration for dynorphin was 0.5 µM, and the apparent
EC50 for this relatively steep dose-response
relationship must fall between 0.1 and 0.5 µM. Figure 5A
shows a representative recording: superfusion of 0.5 µM dynorphin A
enhanced IM amplitudes, elicited a
steady-state outward current (cf. dashed line) and increased input
conductance in a CA1 HPN; this response was prevented by the selective
antagonist nBNI (1 µM). Subsequent superfusion of 30 µM
carbachol (CCh; n = 4) elicited an inward steady-state
current and totally blocked the inward current relaxations, verifying
the identity of the relaxations as due to
IM. On average, 1 µM nBNI
(n = 3) or 4 µM naloxone (n = 1)
totally prevented the augmenting effect of 0.5 µM dynorphin on
IM (Fig. 5B2).
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The blockade by nBNI of the dynorphin-induced
IM augmentation indicated that this
effect was mediated by receptors. We therefore tested the selective
agonist U50,488h. Superfusion of 4 µM U50,488h clearly can
increase IM amplitude in some
individual CA1 HPNs (Fig. 6, A
and B), with recovery on washout. In addition, in the same
cell a 0.5 µM dynorphin-induced IM
increase was blocked by 4 µM naloxone. However, when averaged across
all six cells studied, superfusion of 4-8 µM U50,488h only slightly
increased IM amplitudes. This effect
did not reach statistical significance (n = 6;
P > 0.1; Fig. 6C). Similarly, in five cells
1 µM U69,593 had no significant (P > 0.5) effect on
IM amplitude.
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DISCUSSION |
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In this study we found that, in the presence of TTX,
superfusion of opiates selective for 1,
2 and µ receptors did not alter IM or other postsynaptic properties in
rat CA1 HPNs. By contrast, dynorphin A significantly enhanced
IM amplitudes, and this effect was
blocked by naloxone and the selective
receptor antagonist, nBNI.
However, U69,593 and U50,488h did not significantly alter IM amplitudes, although some
individual cells showed a clear IM augmentation with U50,488h. Both U50,488h and dynorphin A induced an
outward steady-state current near
45 mV where
IM persists. These findings suggest
that a pharmacologically functional
receptor subtype exists in rat
CA1 neurons and that opiates may have more complex actions than the
simple disinhibitory or presynaptic effects that have received the most
attention in hippocampus.
Absence of voltage-dependent effects of and µ agonists on
HPNs
The selective 1 and
2 receptor agonists (DPDPE and deltorphin) did
not alter Ca2+-dependent spikes in CA1 HPNs,
suggesting a lack of effect on voltage-dependent
Ca2+ currents. However, there is the caveat that
such current-clamp studies offer a relatively insensitive index of drug
effects on Ca2+ currents, and that reciprocal
opioid effects on the different subtypes of Ca2+
currents could result in false-negative results. Unfortunately, voltage-clamping Ca2+ currents in CA1 slice
neurons with extended processes is difficult due to space-clamp
confounds combined with the positive step potentials needed (around 0 mV) and the typically large Ca2+ current
amplitudes. However, M-currents have smaller current amplitudes and are
easier to record at more negative potentials (near
45 mV).
In voltage-clamp recordings, opioid peptides selective for
1,
2, and µ receptors had little effect on the voltage-dependent IM. On average, these peptides had no
effect near RMP or near
45 mV (see Table 1). In view of the recent
observation of
receptors on CA1 HPNs (Commons and Milner
1997
) and our finding of IM
depression by
agonists in CA3 (Moore et al. 1994
),
the lack of postsynaptic effect of
receptor agonists on
IM in CA1 HPNs was unexpected.
Similarly, the µ agonist DAMGO had no clear effect on
IM or Ca2+ spikes, despite the
reported postsynaptic localization of µ receptors in CA1 neurons
(Arvidsson et al. 1995b
). It may be argued that we have
not examined several other voltage-sensitive currents, such as
IA, ID,
IT, and delayed rectifier currents. These
are inactivating currents that may not influence resting properties. Modulatory effects of the opioids on other postsynaptic transmitter receptors, such as those for glutamate (see Deisz et al.
1988
; Martin et al. 1997
), could also be a site
of action. Thus postsynaptic
receptors could play a role in
phosphorylation and desensitization of receptors, leading to
neuroadaptive processes as demonstrated for the µ receptor
(Zhang et al. 1996
).
Nevertheless, our results indicating a lack of clear postsynaptic
effect of - or µ-selective agonists in CA1 HPNs are in agreement
with previous studies supporting the disinhibition hypotheses (Nicoll et al. 1980
; Siggins and Gruol
1986
; Siggins and Zieglgänsberger 1981
;
Zieglgänsberger et al. 1979
). These opiate
receptor agonists have been shown to reduce GABAergic transmission in
hippocampus (Lupica and Dunwiddie 1991
; Lupica et
al. 1992
), and an intracellular study of hippocampal
interneurons showed that they were directly hyperpolarized by
enkephalins (Madison and Nicoll 1988
).
Postsynaptic effects of dynorphin
In contrast to the lack of effects of µ and agonists
on CA1 HPNs, we found that dynorphin A significantly enhanced
IM amplitudes, with recovery on
washout. Dynorphin A also induced an outward steady-state current in
the depolarized range, probably a consequence of its effects on
IM. Although dynorphin is an agonist
at the
receptor (Chavkin et al. 1982
), previous
extracellular studies have suggested that higher concentrations of
dynorphin A increased CA1 population spike amplitude by acting on
presynaptic µ receptors (Chavkin et al. 1985a
;
Neumaier et al. 1988
) to reduce release of an inhibitory
neurotransmitter. However, in the present studies DAMGO did not affect
CA1 HPNs, suggesting that the µ receptor subtype is not involved in
dynorphin enhancement of IM. By
contrast, the nBNI block of dynorphin A-induced augmentation of
IM suggests the presence of a
pharmacologically functional postsynaptic
receptor in CA1 neurons.
U50,488h slightly (but not significantly) enhanced
IM amplitudes (see Fig. 6); this
apparent negative effect might be due to
receptor-subtype variation
(see next paragraph). Similarly, U69,593 had little effect on
IM amplitudes or on steady-state currents,
further implying that a
1 receptor was not involved. This finding was unexpected because the dynophin A enhancement of
IM amplitudes was blocked by nBNI,
suggesting
receptor mediation. In keeping with its slight
enhancement of IM, U50,488h also induced a
clear outward current (mean: 98 ± 22 pA) near
45 mV but not near RMP (see Table 1). The dynorphin A enhancement of
IM amplitude is not likely to be a
nonspecific peptide effect because D-T-dyn had no effect on membrane
properties in HPNs.
There are reports of dynorphin-like immunoreactive fibers located in
the hippocampal CA1 region (Chavkin et al. 1985b;
McGinty et al. 1983
), and several
receptor subtypes
(e.g.,
1,
2, or epsilon) are located in
the rat brain (Nock et al. 1988
, 1990
, 1993
;
Zukin et al. 1988
), where the
2 receptor
subtype predominates. Dynorphin has a higher potency than U50,488h for
the
2 subtype (Zukin et al. 1988
). The
lower affinity of the
2 receptor for U50,488h may
explain the lack of significant IM
enhancement by U50,488h. Furthermore, U69,593 is specific for
1 receptor and is inactive at the
2
receptor, suggesting that a
1 receptor is not involved.
However, our finding of nBNI blockade of dynophin enhancement of
IM might suggest
1 receptor
mediation. Although it has been suggested that nBNI is a selective
1 antagonist (Nock et al. 1990
), it may
also interact with the
2 receptor subtype (see
Wollemann et al. 1993
for review). Previously, we
reported that U50,488h significantly enhanced
IM amplitudes in CA3 HPNs (Moore et
al. 1994
), with nBNI blockade of this effect. It is possible
that some new, uncharacterized
receptor subtype or splice variant
is responsible for the dynorphin effect on
IM in CA1 HPNs (see Clark et al.
1989
; and our accompanying paper Madamba et al.
1999
). Furthermore, the exact localization of
receptors in
the rat CA1 region is presently unknown.
Our finding of large IM increases induced by
dynorphin in rat CA1 HPNs is highly consistent with the significant
dynorphin-induced IM increases we reported
previously for rat CA3 HPNs (Moore et al. 1994). In
contrast to our CA3 findings of decreased IM
by higher dynorphin concentrations (>0.5 µM), CA1 neurons showed only augmentation of IM at all dynorphin
concentrations tested above 0.1 µM. However, Salin et al.
(1995)
found no postsynaptic dynorphin effects in CA3 HPNs of
several species, including rat. The inability to replicate our
IM data may arise from several differences
in our experimental conditions, such as 1) possible contamination of the apparent IM relaxation
with the Q-current relaxation that is not altered by opioids in CA3
neurons (Moore et al. 1994
); 2) the
method for quantifying the IM relaxations; 3) bath temperature; 4) dynorphin
concentrations; and 5) the presence or absence of
Ca2+ channel antagonists.
Past publications (e.g., North et al. 1987;
Piguet and North 1993
; Williams and North
1984
; Williams et al. 1988
) on neuronal opiate
effects had postulated that, in addition to the well-known presynaptic
inhibitory effects of opiates on transmitter release, agonists specific
for µ and
receptors exerted their inhibitory effects by opening
K+ channels, whereas those specific for
receptors did
not, but rather inhibited Ca2+ channel function
(Gross and MacDonald 1987
). However, several studies
(Deisz et al. 1988
; Martin et al. 1997
;
Moore et al. 1988b
; Siggins and
Zieglgänsberger 1981
; Sutor and
Zieglgänsberger 1984
; Yuan et al.
1992
), including the present investigation, found that many
central neurons showed no direct inhibitory effects of µ or
agonists attributable to K+ channel activation. By
contrast, the Moore et al. (1994)
studies and those of
others (Grudt and Williams 1993
; Henry et al.
1995
; Ikeda et al. 1995
; Ma et al.
1995
; Simmons and Chavkin 1996
), have shown that
agonists clearly can activate K+ channels in several
neuron types and in Xenopus oocytes coexpressing
-opioid receptors and K+ channels. As the M-current is
carried through K+ channels, the present study, and the
accompanying paper on nociceptin, adds further evidence that
agonists open K+ channels.
Physiological role of hippocampal dynorphin
As for the function of endogenous opioids in CA1
hippocampus, regulation of the M-current could play an important role
in events that involve prolonged depolarizations, such as those
triggered during theta burst activity. Dynorphin- or nociceptin-induced augmentation of IM would be predicted
to counter prolonged depolarizations and reduce bursting activity
(Halliwell and Adams 1982). Considerable evidence
suggests that opiates play some as yet undefined role in epileptiform
activity. The epileptigenic action of µ agonists in CA1 and CA3
neurons may arise from inactivation of inhibitory interneurons in
hippocampus (Nicoll et al. 1980
; Siggins and
Gruol 1986
; Siggins and Zieglgänsberger
1981
; Zieglgänsberger et al. 1979
). By
contrast, several reports suggest that dynorphins dampen epileptiform
activity (Jones 1991
; Tortella and Holaday
1986
), consistent with an inhibitory action via
K+ channel/IM
augmentation. Changes of dynorphin levels and metabolism in hippocampus
following evoked seizure activity (Gall 1988
;
Hong et al. 1988
) also may indicate a role for dynorphin
in seizures.
Dynorphin also could be involved in some form of synaptic plasticity.
Several studies have shown that dynorphin depresses LTP, a cellular
model for learning and memory, in guinea pig hippocampus (Terman
et al. 1994; Wagner et al. 1993
;
Weisskopf et al. 1993
). However, the locus of this
effect is thought to be presynaptic, in part because dynorphin was
assumed to have no postsynaptic action. By contrast,
- and/or
µ-selective agonists enhanced LTP in rat hippocampus (Derrick
et al. 1992
; Gramham et al. 1991
; Xie and
Lewis 1995
). DAMGO facilitation of mossy fiber LTP may be
partially due to inhibition of GABAB receptor
activation (Jin and Chavkin 1999
). Although the role of
IM in LTP processing is still unknown,
it seems logical that augmenting IM
should prevent the postsynaptic depolarization needed for the
expression of many forms of LTP. Interestingly, a novel form of
long-term depression (LTD) found in the rat CA1 hippocampus is blocked
by naloxone (Francesconi et al. 1997
), suggesting that
endogenous opioids could also play a role in this form of synaptic
plasticity. Considering the
IM-augmenting (i.e., inhibitory)
effect of dynorphin in this region, dynorphin could be implicated in
mediating this form of LTD. Dynorphin could also be involved in other
phenomena such as paired-pulse inhibition or short-term potentiation.
Based on its potent hippocampal actions, it is reasonable to suggest
that endogenous dynorphin could play a role in memory function, an idea
supported by data showing naloxone-sensitive impairment of spatial
memory by dynorphin microinjection into the hippocampus (McDaniel et al. 1990
). Furthermore, dynorphin B impairs
spatial learning in rats via
receptors (Sandin et al.
1998
). Additional studies will be required to determine whether
the M-current is involved in these seizure-, plasticity-, or
memory-related phenomena and whether endogenous dynorphin in
hippocampus might have similar effects. As implied in the data of our
accompanying paper (Madamba et al. 1999
), similar
considerations apply to the possible role of nociceptin in hippocampus.
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ACKNOWLEDGMENTS |
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We thank Drs. S. J. Henriksen and M. K. Tallent for valuable criticisms.
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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