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The Journal of Neuroscience, September 15, 1998, 18(18):7084-7098
Opioid Inhibition of Hippocampal Interneurons via Modulation of
Potassium and Hyperpolarization-Activated Cation
(Ih) Currents
Kurt R.
Svoboda1 and
Carl R.
Lupica1, 2
1 Department of Pharmacology and 2 Program
in Neuroscience, University of Colorado Health Sciences Center,
Denver, Colorado 80262
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ABSTRACT |
The actions of mu- and delta-opioid agonists (DAMGO and DPDPE,
respectively) on GABAergic interneurons in stratum oriens of area CA1
of the hippocampus were examined by using whole-cell voltage-clamp
recordings in brain slices. Both agonists consistently generated
outward currents of similar magnitude (15-20 pA) in the majority of
cells. However, under control conditions, current-voltage (I/V) relationships revealed that only a small number of
these cells (3 of 77) demonstrated clear increases in membrane
conductance, associated with the activation of the potassium current
known as Girk. These interneurons
also exhibited a slowly activating, inwardly rectifying current known
as Ih on hyperpolarizing step commands.
Ih was blocked by the extracellular
application of cesium (3-9 mM) or ZD 7288 (10-100
µM) but was insensitive to barium (1-2 mM).
In an effort to determine whether the holding current changes were
attributable to the modulation of Girk
and/or Ih, we used known blockers of
these ion channels (barium or cesium and ZD 7288, respectively).
Extracellular application of cesium (3-9 mM) or ZD 7288 (25-100 µM) blocked Ih and
significantly reduced the opioid-induced outward currents by 58%.
Under these conditions the opioid agonists activated a potassium
current with characteristics similar to
Girk. Similarly, during barium (1-2
mM) application the opioid-induced outward currents were
reduced by 46%, and a clear reduction in Ih
and the whole-cell conductance was revealed. These data suggest that
the opioids can modulate both Ih and
Girk in the same population of stratum
oriens interneurons and that the modulation of these ion channels can
contribute to the inhibition of interneuron activity in the
hippocampus.
Key words:
delta-receptor; electrophysiology; enkephalin; GABA; hippocampus; opioid receptor; mu-receptor; nonselective cation current; oriens/alveus interneurons; queer current
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INTRODUCTION |
Opioid receptors are members of a
class of Gi/Go-coupled receptors
that inhibit both adenylyl cyclase and voltage-dependent Ca2+ channels (VDCCs) in central and peripheral
neurons (Childers, 1993
; Moises et al., 1994). In addition to these
actions, opioids activate a G-protein-coupled inwardly rectifying
potassium conductance known as Girk,
resulting in the hyperpolarization of neurons throughout the CNS
(Williams et al., 1982; Madison and Nicoll, 1988; North, 1989; Wimpey
and Chavkin, 1991). These opioid effects on ion channels generally
inhibit neuronal activity. However, in some cellular circuits, when the
opioid-sensitive neuron releases an inhibitory neurotransmitter such as
GABA, the net result can be the excitation or disinhibition
of the postsynaptic neuron. Such is the case for the principal cells of
the CA1 region of the hippocampus, where opioids produce profound
increases in pyramidal neuron activity after the inhibition of GABA
release from local circuit inhibitory interneurons (Zieglgansberger et
al., 1979; Lee et al., 1980; Nicoll et al., 1980; Pang and Rose, 1989;
Lupica and Dunwiddie, 1991). These interneurons constitute only
10-20% of the neuronal population in the hippocampus and are
heterogeneous with regard to their membrane properties,
neurotransmitter sensitivity, and neuropeptide content (for review, see
Freund and Buzsáki, 1996). However, these cells exert pandemic
control over hippocampal activity, because they each make multiple
synaptic contacts on hundreds of principal neurons.
Although it is likely that opioids play an important role in the
modulation of processes like learning and memory, epilepsy, and drug
reinforcement via actions on interneurons in the hippocampus (Siggins
et al., 1986; Stevens et al., 1991; Xie and Lewis, 1991), there have
been few direct studies of opioid effects on these cells. Most have
relied, instead, on indirect measures such as GABA-mediated
IPSPs/IPSCs from pyramidal cells (Lee et al., 1980; Nicoll et
al., 1980; Cohen et al., 1992; Lupica et al., 1992; Lupica,
1995). To date, studies using direct recordings from CA1 interneurons
have examined only the effects of opioids on
K+ channels and VDCCs (Madison and Nicoll, 1988;
Wimpey and Chavkin, 1991; Lambert and Wilson, 1996). These
investigators found that Girk was activated by
the µ-selective agonist DAMGO in acutely dissociated CA1/subicular
interneurons (Wimpey and Chavkin, 1991) or by a nonselective opioid
agonist
(D-Ala2-Met5-enkephalinamide)
in stratum pyramidale interneurons (Madison and Nicoll, 1988). However,
another study found that DAMGO did not activate
Girk, nor did it inhibit VDCCs in stratum
radiatum interneurons (Lambert and Wilson, 1996).
Another conductance that is prominent in hippocampal interneurons found
in stratum oriens is known as Ih (Maccaferri and
McBain, 1996). This slowly developing inward cation current is
activated by hyperpolarization, is carried by Na+
and K+ ions, and does not inactivate, even with
prolonged hyperpolarization (Halliwell and Adams, 1982; Mayer and
Westbrook, 1983; Maccaferri and McBain, 1996). Because of these
characteristics, Ih probably contributes to the
resting membrane potential and the generation of rhythmic
pacemaker-like depolarizations in central neurons and cardiac cells
(DiFrancesco, 1981; Bal and McCormick, 1996; Maccaferri and McBain,
1996; Gasparini and DiFrancesco, 1997) (for review, see Pape,
1996). Therefore, modulation of Ih by
neurotransmitters would be expected to alter the oscillatory activity
of individual neurons and, in turn, the network of cells with which
they communicate (Freund and Buzsáki, 1996) (for review, see
Pape, 1996). In addition, because of its properties,
Ih might interact with other conductances by
opposing the membrane hyperpolarization initiated by inhibitory neurotransmitters. Among the several neuromodulators that modulate Ih are the opioids, which inhibit this current
in peripheral neurons by inhibiting adenylyl cyclase (Ingram and
Williams, 1994). However, at this time no studies of the opioid
modulation of Ih in central neurons have been
described. In the present investigation we demonstrate that µ- and
-opioid receptor agonists inhibit Ih and
activate Girk in the same hippocampal
interneurons, and we postulate that these effects contribute to the
sustained inhibition of interneuron activity by the opioids.
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MATERIALS AND METHODS |
Electrophysiology. Hippocampal slices were prepared
and maintained as previously described (Miller et al., 1997). Briefly, 14- to 30-d-old male Sprague Dawley rats (Sasco, Omaha, NE) were killed
by rapid decapitation. Their brains were removed and placed in ice-cold
oxygenated artificial CSF (aCSF; see below). Brain slices
containing the hippocampus were cut transverse to the
anterior-posterior axis at 300 µm nominal thickness, using a
vibrating tissue slicer (Technical Products International, St. Louis,
MO). Then the slices were suspended on netting in a beaker containing
aCSF that was aerated continuously with 95% O2/5%
CO2 at room temperature. Control aCSF consisted of (in
mM): 126 NaCl, 3.0 KCl, 1.5 MgCl2, 2.4 CaCl2, 1.2 NaH2PO4,
11.0 glucose, and 26 NaHCO3, saturated with 95%
O2/5% CO2. Interneurons were visualized
in stratum oriens of area CA1, using a fixed stage upright microscope
equipped with differential interference contrast optics and infrared
illumination, as previously described in detail (Miller et al., 1997).
Whole-cell recordings were obtained at room temperature (20-23°C)
with an Axopatch-200A amplifier (Axon Instruments, Foster City, CA) and
electrodes pulled from thick-walled borosilicate capillary tubing
(inner diameter, 0.75 mm; outer diameter, 1.5 mm; Sutter Instrument,
Novato, CA). The electrodes had resistances of 4-7 M
when filled
with (in mM): 125.0 K-gluconate, 10.0 KCl, 10.0 HEPES, 1.0 EGTA, 0.1 CaCl2, 2.0 Mg2+-ATP,
and 0.2 Na+-GTP (adjusted to pH 7.2-7.4 with 1 M KOH, and brought to 270-280 mOsm with de-ionized water).
The reversal potential for K+ currents
(EK) using these stated internal (135.0 mM) and external (3.0 mM) K+
concentrations was predicted to be approximately 
96 mV at 20°C, according to the Nernst equation. Unless otherwise stated, all interneurons were voltage-clamped at 66 mV after correction for a
liquid junction potential. Series resistance was generally <15 M
and was monitored throughout the experiments by using the capacitative currents generated by small (5 to 10 mV, 250 msec) voltage steps. Cells were rejected from analysis if the series resistance changed by
10-15%. Voltage-clamp protocols were delivered by a pulse generator
(Master 8, A.M.P.I., Jerusalem, Israel), and signals were
acquired by a PC-based data acquisition system (Strathclyde Electrophysiology Software, courtesy of John Dempster, Strathclyde University, UK).
Histology. Biocytin (0.25%; Sigma, St. Louis, MO) was added
to the internal solution for post hoc evaluation of the
anatomical location of the neurons. A majority of the interneuron
recordings were >1 hr in duration. In these cases, at the end
of the recording period the patch pipette was withdrawn slowly from the
slice, which then was transferred to chilled (4°C) 4%
paraformaldehyde in PBS. In recordings of shorter duration the pipette
was left in place in the slice, and the preparation was left
undisturbed in the recording chamber for an additional one-half hour to
permit diffusion of biocytin into the cell. Slices were stored in the chilled fixative for 3-7 d. After fixation, they were rinsed in PBS
(three times for 5 min) and then exposed to avidin-biotin horseradish
peroxidase complex (ABC, Vector Laboratories, Burlingame, CA) for
4 d. On the fourth day, they were rinsed again in PBS and then
incubated in 0.04% diaminobenzidine (DAB) for 20-30 min. The DAB
reaction was stopped by rinsing in PBS. Then the slices were dehydrated
in incrementing concentrations of ethanol (70, 95, 100, 100%). After
dehydration, they were cleared in Hemo-D (three times for 5 min) and
then mounted on slides. Cells were reconstructed with the aid of a
camera lucida drawing tube attached to an Olympus microscope.
Chemicals. Drugs were obtained from the following sources:
DPDPE (D-Pen2,D-Pen5-enkephalin)
and DAMGO
(Tyr-D-Ala2,N-CH3-Phe4,Gly-ol-enkephalin),
National Institute on Drug Abuse Drug Supply System (Rockville, MD);
tetrodotoxin (TTX), Alomone Laboratories (Jerusalem, Israel) or Sigma;
CsCl and BaCl2 (Sigma); ZD 7288, Tocris Cookson (Ballwin,
MO). All drugs and channel blocking agents were made at 100 times their
final concentration in de-ionized water and added to the aCSF bathing
the slice (flow rate = 2 ml/min) with calibrated syringe pumps
(Razel Scientific Instruments, Stamford, CT).
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RESULTS |
Morphology
All of the recordings were obtained from neurons that had somata
located within stratum oriens of the CA1 region of the hippocampus. Previous studies have shown that a vast majority of neurons found outside of stratum pyramidale are positive for glutamic acid
decarboxylase (GAD) and, therefore, GABAergic (Somogyi et al., 1983;
Frotscher et al., 1984; Kunkel et al., 1986). The morphology of the
stratum oriens cells was diverse, and the axonal arborizations were, in many cases, elaborate. The axons of several of these cells extended through all CA1 layers, and no consistent patterns of termination were
noted. Although the axonal distribution pattern was diverse, most of
the interneurons possessed multiple dendritic processes restricted
primarily to stratum oriens. A few of these cells exhibited dendrites
extending into the alveus (Fig. 1). The
morphology of these cells with somata and dendrites confined to stratum
oriens is consistent with previous descriptions (Lacaille et al., 1987; McBain et al., 1994; Zhang and McBain, 1995). Examples of two biocytin-filled interneurons reconstructed with the use of camera lucida are shown in Figure 1.
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Figure 1.
Camera lucida reconstructions of two
biocytin-filled CA1 stratum oriens interneurons that were included in
the data set shown in Figure 2. The large majority of these neurons had
dendritic processes confined to stratum oriens and demonstrated axons
projecting to all of the major hippocampal strata. The data shown in
Figure 2B were obtained from the cell on the
left of this figure.
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Opioids generate outward currents in stratum
oriens interneurons
Bath application of the selective µ-opioid receptor agonist
DAMGO (1 µM) or the selective -agonist DPDPE (1 µM) generated reversible outward currents in the majority
of stratum oriens interneurons. Of the 66 interneurons in which DPDPE
was tested, 40 (60.6%) responded with outward currents, whereas 24 of
55 (43.6%) neurons that were tested were sensitive to DAMGO. The
magnitudes of the outward currents were similar with either agonist
(Fig. 2A). Only 12 of
44 neurons (27%) that were tested responded to both agonists.
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Figure 2.
Effects of selective µ-opioid
(DAMGO, 1 µM; n = 12)
and -opioid (DPDPE, 1 µM;
n = 12) agonists on interneurons with somata
located in stratum oriens of area CA1 in hippocampal slices.
A, Time course of mean ± SEM opioid-induced
outward currents. All neurons were voltage-clamped at 66 mV. The
horizontal bar indicates the duration of the opioid
agonist bath application. B, Current responses obtained
from a single interneuron before (Control) and
during DAMGO application that was used in the construction of the
curves shown in C (right panel).
Voltage steps (250 msec in duration, 10 mV increments) from 66 to
136 mV were used to produce these responses. Note the prominent
voltage- and time-dependent inward sag in the current responses at
progressively hyperpolarized voltage steps. C, Mean ± SEM effect of - and µ-agonists on current-voltage
(I/V) relationships obtained during the peaks of
the holding current changes shown in A. All data were
normalized to the largest current response that was obtained by using a
250 msec voltage step to 136 mV
(I/Imax). In this and
subsequent figures the points labeled
Subtracted ( ) indicate the drug-induced change in
current obtained by subtracting the control points from those measured
during drug application.
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In neurons throughout the CNS, opioid-generated outward currents
reverse near the K+ equilibrium potential
(EK) and usually are associated with an increase in membrane conductance because of the activation
of a G-protein-coupled K+ channel known as
Girk (Williams et al., 1982; Madison and Nicoll, 1988; North, 1989; Wimpey and Chavkin, 1991). However, in the present
study, when the effects of DAMGO and DPDPE were examined on
current-voltage (I/V) relationships (using 250 msec
voltage steps from 66 to 136 mV) (Fig. 2B), we
found that there was either very little change in the whole-cell
conductance or a small net decrease, despite the clear change in
holding current (control = 7.1 ± 0.6 nS, pooled DPDPE and
DAMGO = 6.8 ± 0.7 nS; n = 17; measured at
steady state from the linear portion of the I/V curves). This is illustrated in Figure 2C, in which both DPDPE and
DAMGO produced changes in the I/V curves during the peak
outward currents that were inconsistent with an increase in whole-cell
conductance expected to result from the activation of a
K+ channel like Girk. To
evaluate whether these neurons were capable of generating outward
currents associated with the activation of
Girk, we compared the actions of the
GABAB receptor agonist baclofen, which is known to activate
this conductance (Gahwiler and Brown, 1985; Inoue et al., 1985;
Newberry and Nicoll, 1985; Christie and North, 1988), with the opioid
agonists in the same interneurons. At a holding potential of 66 mV,
baclofen (60 µM) generated outward currents (25.0 ± 4.7 pA, n = 7) that were associated with an increase in
membrane conductance of 1.7 ± 0.5 nS (Fig. 3). In addition, the
Erev for the baclofen-induced current was 91.1 ± 4.1 mV, which is close to the calculated
EK of 96 mV. These data indicated that,
whereas both the opioid and GABAB receptor agonists
produced outward currents, only baclofen activated an apparent
K+ channel associated with an increase in membrane
conductance and characteristics similar to Girk
(compare Fig. 3B,C). A small number of the stratum oriens
interneurons (8 of 94, 8.5%) exhibited current responses to voltage
steps hyperpolarized to approximately 76 mV that lacked the prominent
inward sag that is indicative of the hyperpolarization-activated cation
current known as Ih (visible in Fig.
2B). Of those eight neurons lacking
Ih, three demonstrated clear outward
currents on opioid application. In each of these cells the
I/V curves revealed that the opioid-sensitive currents reversed near EK and were associated with an
increase in membrane conductance, consistent with the activation of
Girk (Fig. 4).
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Figure 3.
Effects of the µ-opioid agonist DAMGO (1 µM) and the GABAB agonist baclofen (60 µM) on a stratum oriens interneuron. A,
Time course of DAMGO- and baclofen-induced outward currents in a neuron
voltage-clamped at 66 mV. B, Individual current
responses obtained before and during baclofen application
(arrows) at the indicated voltage steps.
C, I/V curve (250 msec steps between 66
and 136 mV) constructed before (Control),
during, and ~7 min after (Wash) DAMGO application.
Note the larger effect of DAMGO on currents produced at more
hyperpolarized steps and the complete washout of this effect.
D, I/V curve constructed before
(Control), during, and after
(Wash) baclofen application. The reversal potential for
this baclofen-induced current (100 mV) is near
EK (96 mV) in these cells. All data are
derived from the same neuron, and the gaps in the records in
A are attributable to the construction of the
I/V curves.
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Figure 4.
The -agonist DPDPE (1 µM)
activates an apparent K+ current in a stratum oriens
interneuron lacking Ih. A,
Time course of DPDPE-induced outward current in a neuron
voltage-clamped at 56 mV. B1, Voltage and current
traces obtained ~3 min before DPDPE application. B2,
Current responses obtained before and during DPDPE
(arrow) application at the largest voltage step (126
mV). Note the absence of the inward current sag (compare with Fig. 2)
and the larger current response during DPDPE application. The holding
current change was subtracted from the trace obtained in DPDPE to
demonstrate more clearly the change in conductance. C,
Current-voltage relationships determined in the same neuron as
described in A and B. The DPDPE-sensitive
current reversed near EK (predicted = 96 mV; measured = 95.5 mV) and was associated with an increase
in whole-cell slope conductance. Only 8 of 94 (8.5%) stratum oriens
neurons showed a similar absence of inward sag in the current response,
and only 3 of these 8 neurons responded to opioid agonists with a
similar apparent increase in K+ conductance.
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Characteristics of Ih in stratum
oriens interneurons
The hyperpolarization-activated cation current,
Ih, is known to activate slowly and
displays virtually no time-dependent inactivation during a voltage step
(Mayer and Westbrook, 1983; Maccaferri and McBain, 1996; Watts et al.,
1996). In addition, in most preparations Ih is
not active at membrane potentials positive to approximately 55 to
75 mV, and it typically does not saturate at potentials as negative
as 155 mV (Maccaferri and McBain, 1996; Watts et al., 1996). The slow
activation kinetics of Ih suggest that this current should be larger at the end of a 2 sec voltage step, at which
point Ih is fully active (i.e., steady state,
Iss), compared with the current observed
near the beginning of a hyperpolarizing voltage step (i.e., the
instantaneous current, Iins), where the contribution of Ih is smaller. Similarly, the
voltage-dependent nature of Ih suggests that the
effects of modulators of this current should be seen at membrane
voltages within its range of activation (i.e., negative to 55 mV).
This can be seen in Figure 5 in which the
effects of extracellularly applied ZD 7288 (25 µM) and
cesium (3 mM), both blockers of
Ih, have been examined on the
instantaneous and steady-state currents. Further isolation of these
effects on Ih was achieved by subtracting
Iins from Iss at each
voltage step, in the presence and absence of the modulator, using the following equation:
![<IT>subtracted I</IT><SUB>h</SUB>=[(I<SUB><UP>ss</UP><SUB><UP>m</UP></SUB></SUB>−I<SUB><UP>ins</UP><SUB><UP>m</UP></SUB></SUB>)−(I<SUB><UP>ss</UP><SUB><UP>c</UP></SUB></SUB>−I<SUB><UP>ins</UP><SUB><UP>c</UP></SUB></SUB>)],](/content/vol18/issue18/fulltext/7084/img001.gif)
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(1)
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where Issm and
Iinsm are the steady-state and
instantaneous currents measured in the presence of the modulator, and
Issc and
Iinsc are these currents in the
control condition. Iss was inhibited to a
greater extent than Iins by both cesium and ZD 7288 (shown for ZD 7288 in Fig. 5B), and the cesium- and ZD
7288-sensitive currents were more prominent at step potentials more
negative than approximately 75 mV (Fig. 5C).
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Figure 5.
Effects of extracellularly applied cesium (3 mM) and ZD 7288 (25 µM) on current responses
in stratum oriens interneurons. A, Current records
obtained during 2 sec hyperpolarizing voltage steps from 66 to 136
mV in the absence (Control) or presence of cesium
(A1) or ZD 7288 (A2). B,
I/V relationship demonstrating the effect of ZD 7288 on
the instantaneous (ins; Control Ins, ; ZD 7288,  )
and steady-state currents (ss; Control SS,  ; ZD 7288,  ). C, Cesium- and ZD 7288-sensitive
Ih obtained by subtracting the instantaneous
I/V relationship from the steady-state
I/V relationship in the absence and presence of the
blocking agent. Note that both cesium and ZD 7288 had a larger effect
on the steady-state versus the instantaneous currents and that both
inhibited a similar current with an activation threshold near 75 mV.
The symbols in B correspond to those
shown in A2.
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Voltage steps greater than 136 mV caused a rapid degradation of the
recordings and, therefore, were not used routinely. Overall, cesium
(3-9 mM) decreased the whole-cell conductance by ~60%
(control = 7.1 ± 1.1 nS; cesium = 4.3 ± 0.85 nS;
n = 6), indicating that the contribution of
Ih to the total whole-cell conductance was substantial. Consistent with previous reports, the block of
Ih by ZD 7288 began after 4-7 min of
superfusion, and a maximal blockade was achieved after 20-50 min
(Harris and Constanti, 1995; Maccaferri and McBain, 1996; Gasparini and
DiFrancesco, 1997). A complete block of Ih by
cesium was achieved only at concentrations >3 mM but was
seen much more rapidly (3-5 min) than with ZD 7288 (data not shown).
In addition to the alteration in whole-cell conductance, cesium also
generated outward currents ranging from 3.5 to 19 pA in amplitude
(mean = 7.5 ± 2.0 pA; n = 8), suggesting
that Ih may be activated partially in some
interneurons at a clamp potential of 66 mV. These effects of
extracellular cesium are consistent with previous reports demonstrating
the blockade of Ih in other preparations (Mayer
and Westbrook, 1983; McCormick and Pape, 1990; Bal and McCormick,
1996). The reversal potential of Ih
(Eh) was measured by using the method
described by Mayer and Westbrook (1983). Thus, instantaneous
I/V curves were generated by six to eight voltage steps (2 sec) from holding potentials of 56, in which
Ih is minimally active, and 96 mV, in which Ih is activated substantially. Then the
intersection of the extrapolated I/V curves was used as the
estimate of Eh. The estimate of
Eh obtained by using this method was 33.0 ± 2.0 mV (n = 7), which is similar to the value
previously reported in these interneurons (Maccaferri and McBain,
1996).
Opioids inhibit Ih in stratum
oriens interneurons
When the effects of DAMGO (1 µM) and DPDPE (1 µM) were examined on currents evoked by 2 sec
hyperpolarizing voltage steps, it was found that in some neurons,
similar to the effects of cesium and ZD 7288, Iss was inhibited to a larger extent than
Iins at voltage steps between approximately 76
and 136 mV (Fig.
6B,C). Thus, the
opioid-sensitive Ih currents were similar
to those that were sensitive to ZD 7288 and cesium (compare Figs.
5C, 6D).
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Figure 6.
The -opioid agonist DPDPE inhibits
Ih in a stratum oriens interneuron.
A, DPDPE-induced (1 µM) outward current in
a cell voltage-clamped at 66 mV. Gaps in the data record are
attributable to the construction of I/V curves.
B, Current responses evoked by selected 2 sec
hyperpolarizing voltage steps before and during DPDPE
(arrow) application. C, Effect of DPDPE
on instantaneous and steady-state I/V relationships.
Note that, similar to ZD 7288 and cesium (see Fig. 5), DPDPE caused a
greater reduction of the steady-state versus the instantaneous current
at voltage steps hyperpolarized to approximately 85 mV.
D, Effect of DPDPE on Ih
isolated by using the procedure described in Results. Note that DPDPE
caused a voltage-dependent reduction in this current and that the
opioid-induced inhibition was qualitatively similar to that observed
with cesium and ZD 7288 (see Fig. 5). Similar effects also were seen
with the µ-opioid agonist DAMGO.
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This is also evident in Figure 7, in
which the current responses that were inhibited by ZD 7288 (25 µM), cesium (3 mM), and DPDPE (1 µM) are shown (Vhold = 66 mV).
Here it can be seen more clearly that the current inhibited by ZD 7288 and cesium is similar in its kinetic activation and voltage dependence
to that inhibited by DPDPE. Exponential time constants (see below) fit
to these currents at the largest hyperpolarizing voltage steps also
indicate that the time course of these inhibited currents was similar
(Fig. 7, legend). This suggests that all of these agents inhibited a current possessing the same temporal characteristics as
Ih. In contrast, the currents that were
modulated by baclofen (60 µM) are clearly different from
those modulated by the inhibitors of Ih (Fig.
7D).
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Figure 7.
Currents inhibited by ZD 7288, cesium, and DPDPE
share similar time and voltage dependence. The currents shown in
A-C were obtained by subtracting traces obtained during
the application of the indicated modulator from control traces, using 2 sec hyperpolarizing voltage steps to 86, 106, and 136 mV. Each
trace thus represents the current that was inhibited by ZD 7288 (25 µM), cesium (5 mM), or DPDPE (1 µM) at these step potentials. The currents obtained at
the 136 mV voltage steps then were fit by using exponential time
constants (see Results) and are indicated by the solid
lines. A, Currents inhibited by ZD 7288 exhibit
typical Ih voltage and time dependence ( = 269 msec). B, Currents inhibited by cesium ( = 242 msec). C, Currents inhibited by DPDPE ( = 266 msec).
D, Currents activated by baclofen (60 µM) were obtained by subtracting the traces obtained
during the drug from those obtained under control conditions. In this
experiment 500 msec hyperpolarizing voltage steps to 86, 106, and
136 mV were used. Note that the currents inhibited by ZD 7288, cesium, and DPDPE share
similar time- and voltage-dependent properties, whereas the traces
obtained in baclofen do not. The traces shown in A and
B were obtained from the experiments illustrated in
Figure 5. The data shown in C were derived from the
experiment shown in Figure 6, and those in D are from
Figure 3. All experiments were conducted at
Vhold = 66 mV.
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Because a previous report has suggested that the inhibition of
Ih by baclofen may be secondary to
Girk activation (Watts et al., 1996) and because
it was unclear whether the modulation of Girk or
Ih was responsible for the opioid-induced
outward currents in the present study, barium was used to block the
opioid activation of Girk and to permit an
assessment of opioid effects on Ih in relative
isolation. These experiments, and those using cesium (below), were
conducted in the presence of the Na+ channel blocker
tetrodotoxin (TTX) to enhance the stability of the recordings and to
determine whether the effects of DPDPE and DAMGO were direct. Outward
currents were generated reliably by the opioids in the presence of TTX,
whether administered with barium or cesium (below). Alone, bath
application of barium (1-2 mM) caused a decrease in
membrane conductance of 1.8 ± 0.5 nS (n = 8)
(Fig. 8B). The
Erev of the current that was inhibited by barium
(measured at steady state, using 2 sec voltage steps) was 91 ± 3.2 mV, suggesting that, in agreement with previous reports, barium
blocked voltage-dependent K+ channels (e.g.,
IIR) (Hille, 1992), and
Ih was, in large part, unaffected (Halliwell and
Adams, 1982; Mayer and Westbrook, 1983; Maccaferri and McBain, 1996).
To evaluate the effects of barium on the opioid response, we
established sensitivity to DPDPE or DAMGO; then we washed the
opioid from the preparation and applied it again in the presence of
barium (Fig. 8A). Wherever possible, the effect of
the opioid also was evaluated after barium was washed from the
preparation (Fig. 8A1). Barium was found to have two major effects on the opioid response. First, in every cell, barium significantly reduced the amplitude of the outward currents generated by DPDPE and DAMGO (Fig. 9A).
However, these currents were reduced only to 45.8 ± 8.8% of
control (p < 0.05; paired Student's
t test; n = 5) (see Figs.
8A1, 11). Second, when the effects of the opioids were evaluated by the use of I/V curves in the presence of
barium, it was found that they caused a decrease in the remaining
whole-cell conductance in every cell that was tested, with a mean ± SEM reduction of 24.4 ± 5.7% (n = 8) (Figs.
8D, 9B). The reduction in
Ih caused by the opioids in barium was manifest
as a 55.2 ± 7.2% decrease at 86 mV and a 29.3 ± 9.8%
decrease at 136 mV (n = 8). In addition, the shape of
the subtracted opioid-sensitive current was similar to that obtained
for Ih, as defined in the ZD 7288 and
cesium experiments (see Fig. 5C).
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Figure 8.
Blockade of Girk by
barium reveals the opioid inhibition of Ih
in stratum oriens interneurons. A, Time course of DAMGO-
and DPDPE-induced (both at 1 µM) outward currents and
inhibition by barium (2 mM). A1, A single
experiment in which DAMGO and DPDPE generated outward currents. Barium
reversibly reduced the outward current produced by DPDPE application.
A2, Another neuron in which both DAMGO and DPDPE
produced outward currents. The effect of DAMGO was nearly eliminated by
barium in this cell. B, Effects of barium
(arrows) on current responses at selected 2 sec voltage
steps. The holding current changes were subtracted to illustrate the
effect of barium on the whole-cell conductance. The apparent reduction
in the steady-state current produced by barium reversed near
EK. C, Effect of DAMGO on the
steady-state I/V relationship in the absence of barium
(same cell as A2). D, Effect of DAMGO on
the steady-state I/V relationship during the application
of 2 mM barium. Note the reduction in the whole-cell
conductance caused by DAMGO. Similar results were observed with
DPDPE.
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Figure 9.
Opioid effects on stratum oriens interneurons
after the blockade of Girk by barium.
Experiments were conducted by using the protocol described in Figure 8.
A, Opioid-induced (DAMGO or DPDPE, pooled data) outward
currents in each neuron before (Control) and
during barium application. Mean responses are indicated by  .
B, Mean ± SEM effects of DAMGO and DPDPE (each at
1 µM, pooled data) on Ih
normalized to the largest voltage step (136 mV) and isolated by using
the procedure described in the Figure 5 legend. Note that the
subtracted current is similar to that shown in Figure 5. In each
experiment barium was applied for 20-50 min before opioid
application.
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The effect of the opioid agonists on the time constant of
Ih activation also was determined in a subset of
opioid-sensitive interneurons. The activation of
Ih was studied by evoking current responses to
voltage steps (2 sec) between 86 and 136 mV from a holding
potential of 66 mV. Then the inward sag associated with
Ih activation was fit by using exponential
functions of the form:
|
(2)
|
where It is the amplitude of the current at
time t, Iss is the steady-state
current measured at the end of a 2 sec voltage step,
Ih is Iins subtracted
from Iss, and is the time constant of
activation. The rate of Ih activation
demonstrated strong voltage dependence, with control time constants of
0.551 ± 0.134 sec at the 96 mV voltage step and 0.196 ± 0.036 sec at the 116 mV voltage step (n = 8). The
opioid agonists significantly (p < 0.001;
paired t test) slowed the activation of
Ih, such that the time constants were
1.36 ± 0.122 sec at 96 mV and 0.45 ± 0.059 sec at 116
mV. Collectively, these results suggest that, when
Girk was blocked by the addition of barium, the
opioids consistently decreased the whole-cell conductance and slowed
the rate of Ih activation. In addition, the
persistence of the opioid effect on Ih in the presence of TTX suggests that it resulted from the direct stimulation of opioid receptors located on the interneurons.
Opioids activate K+ channels in stratum
oriens interneurons
In an effort to determine whether the activation of
Girk by the opioids was obscured by the
concomitant inhibition of Ih, we examined
the effects of DPDPE and DAMGO on stratum oriens interneurons during
the blockade of Ih by extracellular cesium or ZD
7288. A protocol similar to the one described for the barium
experiments was used except that, in each case, the concentration of
cesium or ZD 7288 was increased until a complete block of
Ih was achieved (cesium, 3-9 mM; ZD
7288, 25-100 µM). At a voltage-clamp potential of 66
mV, cesium inhibited the opioid-induced outward current in every cell
that was examined (n = 6) (Fig.
10A). On average, cesium inhibited this response by 58.1 ± 10.8%
(p < 0.01; paired Student's t test)
(Fig. 11), suggesting that the
inhibition of Ih was partly responsible for the
opioid-induced change in holding current. In addition, as reported
above, the application of cesium alone generated outward currents that
were similar in magnitude to those caused by the opioids during barium
application. This observation suggests that the inhibition of inward
current associated with Ih by both cesium and
the opioids was partially responsible for the holding current changes.
When the interneurons were treated with a combination of barium (2 mM) and cesium (3-9 mM), the opioid-induced outward currents were blocked nearly completely (14.6 ± 7.5% of control, n = 6) (Fig. 11), and there was no effect of
the opioids on the I/V relationship.
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Figure 10.
Opioid effects on stratum oriens interneurons
during the blockade of Ih by extracellular
cesium. In each neuron the opioid sensitivity was confirmed by
examining the effect of either DPDPE or DAMGO on holding current. Then
cesium (3-9 mM) was applied via bath superfusion until
Ih was visibly blocked (20-50 min) and the
effect of the opioid on holding current and the steady-state
I/V relationship was evaluated. A,
Opioid-induced outward current in each interneuron produced before
(Control) and during cesium application. The mean
effects are indicated by . B, Mean ± SEM
opioid-sensitive current obtained from the subtracted
I/V curves before () and during cesium superfusion
(). The Erev of the opioid-sensitive
current was 101 ± 6.1 mV, suggesting that it was carried by
K+ ions.
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Figure 11.
Effects of channel blockers on outward currents
produced by opioid application (pooled data from 1 µM
DPDPE and DAMGO applications). The opioid-induced outward currents
obtained in the presence of the blocking agent were expressed as a
percentage of those obtained under control conditions. In each case
there was a significant reduction in the outward current
(p < 0.01-0.05; paired Student's
t test), and the effect of the combined application of
barium and cesium was significantly larger than with each blocker alone
(p < 0.01). Barium, n = 5; cesium, n = 7; barium and cesium,
n = 6).
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As reported above, under control conditions the opioid agonists DPDPE
and DAMGO caused small decreases in the whole-cell membrane conductance
measured at a holding potential of 66 mV (see Figs. 2B, 10B). However, during the
cesium block of Ih this decrease in conductance
was converted to an increase (from 4.1 ± 1.0 nS to 5.3 ± 1.3 nS; n = 6) (see Fig. 10B).
Furthermore, this increase in conductance was associated with a current
that reversed near the predicted Erev for
K+ ions (measured Erev = 101 ± 6.1 mV; predicted EK = 96 mV), suggesting that in the absence of Ih the opioid
activation of K+ channels was evident. Similar
results were obtained in three interneurons when the selective blocker
of Ih, ZD 7288, was used. In these cells
the opioid-induced current also reversed near the predicted
EK (measured Erev = 99.7 ± 9.0 mV).
A previous study has suggested that extracellular cesium can reduce the
inward component of the baclofen-activated K+
current observed at step potentials more negative than approximately 110 mV without affecting baclofen-induced outward currents (Sodickson and Bean, 1996). Therefore, in the present study the magnitude of the
opioid-induced increase in Girk in the presence
of cesium probably was underestimated. Moreover, the observation that
cesium did not alter the outward currents produced by baclofen implies that the opioid-induced outward currents caused by
K+ channel activation similarly were unaffected by
cesium and that the cesium attenuation of the opioid-induced outward
currents likely was attributable to the blockade of
Ih. These findings, considered together, suggest
that the opioids activate a K+ conductance and
inhibit Ih in the same stratum oriens
interneurons and that both of these conductances contribute to
the holding current changes seen at a holding potential of 66 mV.
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DISCUSSION |
The results of this study demonstrate that opioid agonists
selective for either µ- or -receptors can generate outward
currents in hippocampal stratum oriens interneurons. This would result in membrane hyperpolarization and the inhibition of these cells, which
is consistent with the effects of opioids on neuronal populations throughout the CNS (Williams et al., 1982; Madison and Nicoll, 1988;
North, 1989; Wimpey and Chavkin, 1991; Johnson and North, 1992).
However, our results diverge from these previous studies in that this
effect is not associated with an increase in membrane conductance and
the apparent activation of K+ channels under control
conditions. Instead, our results indicate that the outward currents
produced by the opioid agonists in the absence of channel blockers are
associated with a small decrease in conductance. One explanation for
these disparate findings is that, in stratum oriens interneurons,
either the opioids modulate a different current than that described in
other systems or they simultaneously act at more than one ion channel.
The large contribution of Ih to the total
membrane conductance (Maccaferri and McBain, 1996) and the previously
described actions of the opioids on Girk made
these currents prominent candidates for further investigation. The
present results suggest that concomitant modulation of these ion
channels can explain the ambiguous effects of the opioids on the
whole-cell conductance and that the outward currents produced by these
agonists are the result of this modulation.
The properties of Ih, as it has been
described in the present study, are similar to those identified in this
and many different neuronal and cardiac preparations (DiFrancesco,
1981; Halliwell and Adams, 1982; Mayer and Westbrook, 1983; McCormick
and Pape, 1990; BoSmith et al., 1993; Ingram and Williams, 1994; Harris and Constanti, 1995; Maccaferri and McBain, 1996; Watts et al., 1996;
Gasparini and DiFrancesco, 1997) (for review, see Pape, 1996). Among
these characteristics are a reversal potential indicative of a mixed
Na+/K+ current, unique voltage
and time dependency, and sensitivity to the blocking agents ZD 7288 and
cesium. The evidence in support of the modulation
Ih by the opioid agonists in the present study is derived from several observations, including (1) the shape of the
I/V relationship for the opioid-inhibited currents was similar to that obtained for Ih, as
defined by cesium and ZD 7288; (2) the opioids reduced the magnitude of
Ih and the rate at which it activates; (3) the
opioids had a larger effect on the steady-state I/V
relationship, in which Ih is more fully active,
versus the instantaneous relationship; (4) extracellular cesium and ZD
7288 blocked Ih and eliminated the
opioid-induced decrease in membrane conductance. We also provide
evidence that K+ channels were modulated in the same
interneurons by the opioid agonists after Ih was
blocked by cesium or ZD 7288. Thus, the opioids clearly increased the
whole-cell conductance under these conditions, and this was found to be
the result of the activation of a current possessing a
Erev near EK. The
activation of Girk and the inhibition of
Ih by the opioids are similar to the effects of
adenosine on mesopontine cholinergic neurons (Rainnie et al., 1994),
suggesting that this dual modulation may be common to several inhibitory neuromodulators. Collectively, our data suggest that both
Ih and Girk are modulated
by opioids in the same interneurons and that the simultaneous
activation of Girk and the inhibition of
Ih by the opioid agonists results in outward
currents and little or no change in the whole-cell conductance.
In some cells the opioid modulation of Ih was
apparent in the absence of the blockade of Girk
by barium (e.g., Fig. 6), suggesting either that
Girk was not coupled to opioid receptors in
these neurons or that the coupling of the opioid receptor to
Ih was greater. However, in the majority of
interneurons the modulation of Ih was not
obvious until the competing K+ channel response was
blocked by barium. Similarly, the sole activation of a
K+ conductance was seen in a small number of cells
that had little apparent Ih (e.g., Fig. 4), but
rarely was seen in the absence of cesium blockade of
Ih. This was despite the fact that
K+ currents, presumably mediated by
Girk, could be activated in these same
neurons by the application of baclofen. This implies that, whereas the
opioids may modulate both Ih and
Girk, GABAB receptor
activation appears to modulate only Girk in
these cells. This contrasts with previous reports in which
Ih was inhibited by baclofen directly or as the
indirect result of Girk activation in
dopaminergic neurons (Jiang et al., 1993; Watts et al., 1996). In the
present study the opioid inhibition of Ih after
the blockade of Girk by barium suggests that
this action was direct, and not the consequence of
Girk activation. In addition, the observation that both GABAB and opioid receptors could activate
Girk, whereas only the opioids inhibited
Ih, suggests that these effects may occur
via coupling to different G-proteins. Alternatively, both GABAB and opioid receptors may activate
K+ channels via a shared set of G-proteins, but
opioid receptors may use a different pool of G-proteins to modulate
Ih.
Enkephalins are thought to be the endogenous opioid receptor ligands in
the CA1 region of the hippocampus, where they are synthesized by some
interneurons (Gall et al., 1981). Furthermore, axon terminals
containing leu-enkephalin have been found apposed to GABA-positive cell
bodies, dendrites, and axon terminals in this brain area (Commons and
Milner, 1996). Localization of opioid receptors, using antibodies
raised against the cloned µ-, -, and 
-opioid receptors, has
shown that only µ- and -receptors are expressed in the CA1 region
of the hippocampus in appreciable concentrations (Mansour et al., 1995,
1996; Commons and Milner, 1997). Moreover, both of these
receptors are distributed widely in this brain area, with the
-opioid receptor found to be associated with GAD-positive
interneurons at particularly high levels in stratum oriens (Commons and
Milner, 1997). The high selectivity of DAMGO and DPDPE for these
receptor subtypes at the concentrations used in this study is well
established (Mosberg et al., 1983; Cotton et al., 1985; Goldstein and
Naidu, 1989; Lupica, 1995). This information and the observation that
only 27% of the interneurons responding to either DPDPE or DAMGO also
responded to the alternate agonist suggest that these peptides were
selective for their respective receptor subtypes in the present study.
In addition, the finding that outward currents were generated in the
majority of interneurons by either DPDPE or DAMGO suggests that most
stratum oriens interneurons express either µ- or -opioid receptors
and that some of these cells express both receptor subtypes. Together,
these data provide anatomical and pharmacological substrates for the
previously described physiological actions of the opioids in the
hippocampus, including the activation of
Girk, the activation of an outward
rectifier K+ current, and the presynaptic inhibition
of GABA release (Madison and Nicoll, 1988; Wimpey and Chavkin, 1991;
Cohen et al., 1992; Lupica, 1995). All of these opioid actions
ultimately diminish the release of GABA from these interneurons,
thereby increasing the excitability of the pyramidal cells on which
they impinge. The inhibition of Ih by opioid
receptors may provide an additional site to reduce the excitability of
the interneurons and alter the activity of the hippocampal network.
Functional significance of Ih and
Girk modulation by opioids
The most reliable effect of the opioid agonists was to induce
outward currents in the stratum oriens interneurons. This response could be produced via the activation of a current with a
Erev negative to the holding potential (e.g.,
Girk) or by the inhibition of a current
with an Erev positive to the holding potential
(e.g., Ih). The observations that barium
and cesium each reduced the opioid-induced outward currents to a
similar degree and that their combined application nearly completely
blocked these responses suggest that both Ih and
Girk contributed to the holding current changes.
Moreover, the observation that cesium alone could generate outward
currents similar in magnitude to those caused by the opioids in the
presence of barium suggests that Ih is activated
partially at the clamp potential of 66 mV and that the opioids may
generate outward currents via the inhibition of these ion channels.
Current-clamp experiments in several laboratories have indicated that
the blockade of Ih by ZD 7288 or cesium can
result in the hyperpolarization of neurons held near the resting
membrane potential (Harris and Constanti, 1995; Maccaferri and McBain,
1996; Gasparini and DiFrancesco, 1977). However, although resting
membrane potentials were not recorded routinely in the present study,
others have reported these values to range between approximately 52
mV and 66 mV in stratum oriens interneurons (Lacaille et al., 1987;
McBain et al., 1994; Bergles et al., 1996). Furthermore, Maccaferri and McBain (1996) demonstrated in these same interneurons that
Ih made a substantial contribution to the
membrane conductance when the cells were held at 60 mV, which is
likely close to rest. These data suggest the possibility that the
inward current contributed by Ih may help to set
the resting membrane potentials of these cells at more depolarized
levels.
In addition to its possible contribution to the membrane potential, the
inhibition of Ih by the opioids also would tend
to make these GABAergic interneurons less responsive to excitatory inputs, particularly at hyperpolarized potentials. Also, the unusual property of the activation of this inward current on hyperpolarization implies that one of its functions might be to oppose the influences of
inhibitory (i.e., hyperpolarizing) neuromodulators. Thus, the reduction
in neuronal excitability caused by the activation of K+ channels and the subsequent membrane
hyperpolarization initiated by the opioids and other inhibitory
modulators might be reversed substantially by
Ih. Because of this possibility we hypothesize that the concurrent modulation of Ih and
Girk in the same cells may help to maintain
decreased excitability by preventing Ih from returning the membrane potential closer to action potential threshold. These combined actions would ensure that the inhibition of interneuron activity and GABA release initiated by K+ channel
activation would not be diminished by the repolarizing influences of
Ih.
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FOOTNOTES |
Received April 10, 1998; revised June 18, 1998; accepted June 24, 1998.
This work was supported by National Institutes of Health Grant DA
07725, United States Public Health Service. We thank Drs. Thomas
Dunwiddie, Kevin Staley, and Jeffrey Weiner for their remarks on this
manuscript and Dr. Cathy Adams for assistance with the biocytin
assays.
Correspondence should be addressed to Dr. Carl R. Lupica, Department of
Pharmacology, Box C236, University of Colorado Health Sciences Center,
4200 East Ninth Avenue, Denver, CO 80262.
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Top
Abstract
Introduction
