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The Journal of Neuroscience, October 15, 1998, 18(20):8133-8144
Postsynaptic Mechanisms Underlying Responsiveness of Amygdaloid
Neurons to Nociceptin/Orphanin FQ
Susanne
Meis and
Hans-Christian
Pape
Institut für Physiologie,
Otto-von-Guericke-Universität, D-39120 Magdeburg, Germany
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ABSTRACT |
Effects of nociceptin/orphanin FQ (N/OFQ), the endogenous
ligand of the opioid-like orphan receptor (ORL), were investigated in
the rat lateral (AL) and central (ACe) amygdala in
vitro. Approximately 98% of presumed projection neurons in the
AL responded to N/OFQ with an increase in inwardly rectifying potassium
conductance, resulting in an impairment in cell excitability.
Half-maximal effects were obtained at 30.6 nM; the Hill
coefficient was 0.63. In the ACe, 31% of the cells displayed responses
similar to that in the AL, 44% were nonresponsive, and 25% responded
with a small potassium current with a linear current-voltage
relationship. Responses to N/OFQ were reduced by 100 µM
Ba2+, were insensitive to 10 µM
naloxone, and were blocked by a selective ORL antagonist,
[Phe1 (CH2-NH)Gly2]NC(1-13)NH2
(IC50 = 760 nM). Involvement of G-proteins was
indicated by irreversible effects and blockade of action of N/OFQ
during intracellular presence of GTP- -S (100 µM) and
GDP- -S (2 mM), respectively, and prevention of responses
after incubation in pertussis toxin (500 ng/ml). These mechanisms may
contribute to the role of N/OFQ in the reduction of fear responsiveness
and stress that have recently been suggested on the basis of
histochemical and behavioral studies.
Key words:
nociceptin; orphanin FQ; amygdala; electrophysiology; pharmacology; potassium inward rectifier
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INTRODUCTION |
A heptadecapeptide, termed
nociceptin (Meunier et al., 1995 ) or orphanin FQ (Reinscheid et al.,
1995), has recently been identified as the endogenous agonist for an
opioid receptor-like protein (ORL) (Bunzow et al., 1994; Chen et al.,
1994; Fukuda et al., 1994; Mollereau et al., 1994; Nishi et al., 1994;
Wang et al., 1994; Wick et al., 1994; Lachowicz et al., 1995). Despite
its structural resemblance to opioid peptides, nociceptin/orphanin FQ (N/OFQ) selectively binds to the ORL receptor but not to
µ-,  -, and  -opioid receptor subtypes, whereas opioid
peptides were found to exert no action on the ORL receptor (Mollereau
et al., 1994; Lachowicz et al., 1995; Reinscheid et al., 1995). At the cellular level, the actions of N/OFQ appear similar to those of opioid
peptides (Standifer and Pasternak, 1997). The ORL receptor is coupled
to G-proteins (Meunier et al., 1995; Reinscheid et al., 1995), whose
activation resulted in inhibition of adenylyl cyclase activity (Meunier
et al., 1995; Reinscheid et al., 1995), modulation of calcium (Knoflach
et al., 1996) and potassium conductances (Connor et al., 1996a; Vaughan
and Christie, 1996; Ikeda et al., 1997; Vaughan et al., 1997), and
regulation of transmitter release (Liebel et al., 1997; Neal et al.,
1997; Vaughan et al., 1997). At the behavioral level, systemic
application of N/OFQ elicited a unique range of responses, including
pronociceptive (Meunier et al., 1995; Reinscheid et al., 1995) or
antinociceptive (Xu et al., 1996; Erb et al., 1997; King et al., 1997;
Yamamoto et al., 1997) effects, and impairment of or increases in
locomotion (Reinscheid et al., 1995; Florin et al., 1996), depending on
the concentration that was administered. A more general role of N/OFQ seems to be related to blockade of stress and reversal of
opioid-mediated antinociception (Mogil et al., 1996). A recent study
demonstrated N/OFQ anxiolytic-like effects in mice and rats that were
consistent across several behavioral paradigms generating different
types of fear-like responses by exposure to various stressful
environmental conditions (Jenck et al., 1997).
One important central structure involved in the integration of fear and
anxiety is the amygdala (Davis, 1992; LeDoux, 1995; Maren and Fanselow,
1996). In addition, amygdaloid mechanisms have been shown to be
involved in analgesic processes (Helmstetter et al., 1995; Manning and
Mayer, 1995; Pavlovic et al., 1996). ORL receptors, N/OFQ and its
precursor protein, are expressed in relatively high densities in the
various subnuclei of the amygdala, as has been demonstrated through
in situ hybridization and immunohistochemical and
autoradiographic procedures (Bunzow et al., 1994; Mollereau et al.,
1994; Wick et al., 1994; Lachowicz et al., 1995; Anton et al., 1996;
Nothacker et al., 1996; Schulz et al., 1996; Florin et al., 1997). In
addition, autoradiographic studies showed a particularly strong effect
of N/OFQ-mediated receptor-activation of G-proteins in the amygdaloid
complex (Shimohira et al., 1997; Sim and Childers, 1997).
Although results from behavioral and histochemical studies thus suggest
a role of N/OFQ within the integrative function of the amygdala, more
direct evidence supporting this hypothesis is missing. The present
study was therefore undertaken to investigate in detail the effects of
N/OFQ in the amygdala on the cellular level, using electrophysiological
and pharmacological techniques in the slice preparation of the rat
amygdala in vitro. The experiments were focused on the
lateral (AL) and central (ACe) nuclei, which represent the main input
and output structures of the amygdala, respectively (Pitkänen et
al., 1997).
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MATERIALS AND METHODS |
Preparation. Long-Evans rats of either sex
[postnatal days (P) 11-16] were anesthetized with halothane and
killed by decapitation. A block of tissue containing the amygdala was
rapidly removed and placed in ice-cold oxygenated physiological saline
containing (in mM): KCl 2.4, MgSO4 10, CaCl2 0.5, piperazine-N,N'-bis(ethanesulfonic acid) (PIPES) 20, glucose 10, sucrose 195, pH 7.35. Coronal slices (300 µm thick) were prepared on a Vibratome (Model 1000, Ted Pella, Redding, CA) and incubated in standard artificial CSF (ACSF) of the following composition (in mM): NaCl 120, KCl 2.5, NaH2PO4 1.25, NaHCO3 22, MgSO4 2, CaCl2 2, glucose 20, bubbled with
95%O2/5% CO2 to pH 7.3. Slices were
kept at 34°C for 20 min and for up to 8 hr at 24-25°C. A single
slice was transferred to the recording chamber and submerged in ACSF,
at a perfusion rate of ~2 ml/min. The tissue was fixed by a silk mesh
around a horseshoe-shaped platinum wire.
Recording techniques. Neurons located superficially at a
depth of ~50 µm on the dorsal surface of the slice were approached under visual control by differential interference contrast (Axioskop FS, Achroplan 40/w; Zeiss, Oberkochen, Germany) infrared
videomicroscopy (S/W-camera CF8/1, Kappa, Gleichen, Germany) (Dodt and
Zieglgänsberger, 1994). Electrophysiological recordings were
performed using the patch-clamp technique in whole-cell mode (Hamill et
al., 1981) with a patch-clamp amplifier (EPC-7, List Medical Systems,
Darmstadt, Germany). Patch pipettes were pulled from borosilicate glass
(GC150TF-10, Clark Electromedical Instruments, Pangbourne, UK). After
the whole-cell configuration was obtained, cells were held at  70 mV
unless indicated otherwise. In each slice, only one neuron was
recorded. Drugs were added to the external ACSF. In some experiments,
the extracellular KCl concentration was elevated to 5, 7.5, or 10 mM by substitution of an equimolar amount of NaCl. In 0 Ca2+ solution, CaCl2 was replaced by
MgCl2. Pipettes were filled with (in mM):
potassium gluconate 95, K3 citrate 20, NaCl 10, HEPES 10, MgCl2 1, CaCl2 0.1, BAPTA 1, MgATP 3, NaGTP
0.5, pH 7.2 with KOH. In some experiments, 2 mM GDP--S
or 100 µM GTP--S were included in the internal
solution in exchange for NaGTP. Typical electrode resistances were
2.5-3 M in the bath, with access resistances in the range of 5 to 7 M. Errors attributable to series resistance were <2.5 mV. A liquid
junction potential of 10 mV was corrected (Neher, 1992). Records were
low-pass-filtered at 2.5 kHz (eight-pole Bessel filter). Voltage-clamp
experiments were performed using pClamp software operating via a
Labmaster DMA interface (Axon Instruments, Foster City, CA) on a PC.
Experiments were conducted at 24-25°C. All substances were obtained
from Sigma (Diesenhofen, Germany), except for nociceptin/orphanin FQ
and
[Phe1(CH2-NH)Gly2]NC(1-13)NH2,
which were purchased from Gramsch Laboratories (München, Germany)
and Tocris (Langford, UK), respectively. Data are presented as
mean ± SEM.
Histological procedures. In some experiments, 0.1% biocytin
was added to the intracellular solution. After recordings, slices containing a biocytin-filled cell were immersed in 4% paraformaldehyde in PBS (in mM: NaCl 120, NaH2PO4 10, K2HPO4 30, thimerosal 0.05, pH 7.4 with NaOH) for 48 hr and then cryoprotected in
a solution composed of 30% sucrose in PBS for 24 hr. Slices were then
sectioned at a thickness of 100 µm using a freezing microtome (Leica,
Benzheim, Germany) and rinsed three times (5 min each) with PBS.
Endogenous peroxidase reaction was by-passed by incubation in 0.3%
H2O2 in PBS for 30 min. Sections were then
treated with the avidin-biotin complex horseradish peroxidase (PK
4000, 1:100; Vector Laboratories, Burlingame, CA) in PBS supplemented
with 0.03% Triton X-100 and 2% bovine serum albumin overnight.
Between steps, sections were rinsed three times for 15 min each with
PBS. The immunohistochemical reaction was completed by adding
3,3'diaminobenzidine (0.5 mg/ml) and H2O2
(0.01%) to the incubation medium, including 0.02%
(NH4)2Ni(SO4)2 × 6 H2O and 0.025% CoCl2, and stopped
after 6 min by rinsing with PBS (two times for 5 min). Sections were
mounted on chromalaun gelatin-coated coverslips, dehydrated, and
coverslipped in DePeX (Serva Feinbiochemica, Heidelberg, Germany)
mounting media.
Pertussis toxin treatment. Slices were rinsed three times
with sterile medium of the following composition: 200 ml Eagle's basal
medium, 100 ml HBSS, 100 ml inactivated horse serum (all from
Life Technologies, Eggenstein, Germany), 2 mM glutamine, and 0.65% glucose, pH 7.3. The tissue was then affixed to coverslips with a chicken plasma (Cocalico, Reamstown, PA) clot coaggulated by
thrombin (25 U/ml; Calbiochem, Bad Soden, Germany) and cultured in a
roller incubator (Heraeus, Hanau, Germany) for ~18 hr at 34°C.
Slices were kept in medium supplemented with 500 ng/ml pertussis toxin,
and control slices were incubated in medium with no added pertussis
toxin. Control and treated slices were used for recording alternately
to confirm the viability of the neurons.
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RESULTS |
Recordings were derived from a total of 94 neurons in the AL and
55 cells in the ACe. Lateral amygdaloid neurons typically possessed a
pyramidal-like cell body with spiny dendrites, as revealed by
intracellular staining with biocytin (Fig.
1A) (n = 14). Central amygdaloid neurons had a heterogeneous morphological appearance with piriform to ovoid cell bodies and mostly spiny dendrites (Fig. 1B) (n = 11).
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Figure 1.
Typical morphology of neurons recorded in the
lateral (A) and central (B)
amygdaloid nucleus. Insets illustrate that dendrites
were covered with numerous spines. Scale bars in B also
apply to A. C, Typical response of an
amygdaloid neuron to N/OFQ. Under voltage-clamp conditions, application
of N/OFQ (as indicated above current trace) evokes a transient outward
current from a holding potential of 70 mV. Downward deflections
represent current responses to ramp-voltage commands from 70 to
140 mV (0.2 mV/msec) that were applied at 0.07 Hz to monitor the
membrane input conductance. Note that the N/OFQ-induced current is
associated with an increase in input conductance.
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Application of N/OFQ (1 µM) through bath perfusion
resulted in a transient outward current associated with an increase in membrane conductance (Fig. 1C) in 92 of 94 recorded AL
neurons. The average maximal amplitude of the outward current
deflection induced by 1 µM N/OFQ at a holding potential
of 70 mV was 53.3 ± 7.3 pA (n = 8). Similar
responses to N/OFQ were observed during blockade of synaptic
transmission in the presence of 1.5 µM TTX (61.1 ± 7.0 pA; n = 4) or in Ca2+-free
solution (55.9 ± 20.3 pA; n = 3), indicating a
direct postsynaptic response of the recorded neurons (data not
shown).
Increase in K+ conductance through N/OFQ in
AL neurons
Hyperpolarizing voltage steps of 350 msec duration applied between
80 and 140 mV in 10 mV increments elicited slowly developing currents in AL neurons (Fig.
2A). Application of
N/OFQ (1 µM) increased these membrane currents. The
effect of N/OFQ was isolated by subtracting currents obtained before
and during action of the peptide. The difference currents showed rapid
activation and no inactivation during the 350 msec voltage pulse.
Ramp-voltage commands (0.2 mV/msec) applied from 70 to 140 mV
revealed a membrane current with inwardly rectifying properties that
was increased by 1 µM N/OFQ (Fig. 2B).
Current-voltage (I-V) relationships (Fig. 2C,D) obtained by plotting the N/OFQ-induced current
amplitude at the end of the hyperpolarizing step against voltage (Fig.
2C) or by the ramp protocol (Fig. 2D) were
similar in that they demonstrated moderate inward rectification. The
N/OFQ-induced current reversed at approximately 100 mV, i.e., very
close to the K+ equilibrium potential as calculated
by the Nernst equation (EK = 104.1 mV). To
confirm the contribution of K+ as the main charge
carrier of the N/OFQ-induced membrane current, the
K+ concentration of the perfusing saline was varied
(Fig. 3). Ramp-voltage commands were
applied at 0.07 Hz to monitor the I-V relationship during
the experiment. The reversal potential of the N/OFQ-evoked response
induced by a 10-fold change in the external K+
concentration had a slope of 53.7 mV, i.e., similar to that predicted by the Nernst equation for a pure K+ electrode (Fig.
3B). Furthermore, increases in the extracellular K+ concentration were associated with an increase in
the slope of the I-V relationship. Such behavior is typical
of inward rectifiers (Fig. 3A) (Isomoto et al., 1997).
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Figure 2.
Activation of an inwardly rectifying membrane
conductance through N/OFQ. All recordings were obtained from neurons in
the AL before (control) and during application of
1 µM N/OFQ. A, Families of inward currents
evoked by hyperpolarizing voltage steps in the range of 80 to 140
mV for 350 msec (holding potential 70 mV). The difference obtained
from recordings before and during action of N/OFQ represents the
N/OFQ-induced currents. Note the time-dependent inward rectification of
the currents. B, I-V relationships
obtained from voltage ramps (0.2 mV/msec) between 70 and 140 mV.
The difference conductance (subtraction of I-V
relationships during action of N/OFQ from controls) displays inward
rectification. C, D, Average I-V
relationships derived by plotting the N/OFQ-induced steady-state
current amplitude against voltage (C,
n = 5; voltage steps as in A) or by
using the ramp protocol (D, n = 6;
protocol as in B). Currents reverse near 100 mV, i.e.,
close to the presumed K+ equilibrium potential, and
display inward rectification.
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Figure 3.
K+ dependence of N/OFQ-evoked
currents in AL neurons. A, I-V
relationships of the N/OFQ-sensitive current monitored by ramp
protocols (0.2 mV/msec, 0.07 Hz) at various external
K+ concentrations (as indicated in mM
near traces). B, Shift of reversal potentials of the
N/OFQ-evoked current at various external K+
concentrations according to the Nernst equation with a slope of 53.7 mV. Data represent averages and SEM obtained from three different cells
at each concentration using ramp protocols as in A. The
line was fitted by linear regression.
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Pharmacological properties of the N/OFQ response
To obtain full recovery of N/OFQ responses during pharmacological
experiments, the concentration of the peptide was reduced to 50 nM. Because the response on the first application of N/OFQ was often larger in amplitude compared with those to further
applications, presumably because of desensitization phenomena (Ikeda et
al., 1997), the order of application of N/OFQ alone and N/OFQ in the presence of antagonists was varied in different cells. In a first series of experiments, possible effects of N/OFQ on opioid receptors were tested through the use of the nonselective opioid receptor antagonist naloxone (Fig.
4A) (Raynor et al.,
1994). N/OFQ elicited an outward current of 43.0 ± 3.7 pA
(n = 5), which was not altered by the addition of 10 µM naloxone (44.8 ± 6.6 pA; n = 5).
By comparison, the selective antagonist to the ORL receptor
[Phe1(CH2-NH)Gly2]NC(1-13)NH2
(Guerrini et al., 1998) reduced the response to 50 nM N/OFQ
in a concentration-dependent manner (Fig. 4B). The
antagonistic effect was quantified as reduction in the maximal
N/OFQ-induced current amplitude during the presence of
[Phe1(CH2-NH)Gly2]NC(1-13)NH2
compared with the control N/OFQ response in an individual cell.
Half-maximal inhibition was obtained at 760 nM
[Phe1(CH2-NH)Gly2]NC(1-13)NH2
with a Hill coefficient of 0.89 (n = 31) (Fig.
4B).
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Figure 4.
Pharmacological properties of the N/OFQ-sensitive
current. Responses were obtained in AL neurons at a holding potential
of 70 mV. Substance application as indicated above current traces.
A, Similar responsiveness to N/OFQ (50 nM)
in a given cell with and without presence of naloxone (10 µM). B, Substantial reduction of N/OFQ (50 nM) responses during presence of the ORL selective
antagonist
[Phe1(CH2-NH)Gly2]NC(1-13)NH2.
Diagram represents the concentration-response relationship of the
antagonist (see Results for further details). The number of
cells tested for each concentration is indicated near error
bars. The curve was drawn according to the equation
I = Imax/{1 + (EC50/A)n}, where
I represents the current response,
Imax the maximal current amplitude,
A the concentration of the agonist N/OFQ, and
n the Hill coefficient. The EC50 and Hill
values obtained from the curve were 762.8 nM and 0.89, respectively. C, Sensitivity of the N/OFQ-induced
current to external Ba2+. During prolonged
application of N/OFQ (1 µM), the induced current slowly
declines and persists after cessation of drug application. In a
different cell, addition of 100 µM
Ba2+ for 2 min reversibly reduces the N/OFQ (1 µM)-sensitive current.
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The effect of N/OFQ was substantially and reversibly reduced through
application of 100 µM Ba2+ (Fig.
4C). The effect of Ba2+ on N/OFQ
responses could not be analyzed in an individual cell, because
applications of N/OFQ for periods longer than 3 min led to a current
that did not return to baseline during the typical recording time of
60-80 min. This may be attributable to slow dissociation of the ligand
(Ardati et al., 1997) and/or slow wash-out of the drug during bath
perfusion (Muller et al., 1988). On average, Ba2+
blocked 39.3 ± 6.6 pA (n = 6) of the
N/OFQ-sensitive current, corresponding to a reduction of the maximal
amplitude by 87.4 ±. 6.6% (n = 6).
Concentration-response relationship of the N/OFQ response
Concentration-response relationships of N/OFQ were investigated
in an elevated extracellular K+ concentration (10 mM) at a holding potential of 80 mV, to increase the
driving force and hence the magnitude of the N/OFQ-induced current. In
an individual neuron, only one N/OFQ concentration was tested, and
concentration-response relationships were constructed from maximal
responses to a given concentration. The N/OFQ-induced current showed
clear concentration-dependency (Fig. 5).
The half-maximal effect (EC50) was elicited by 30.6 nM with a Hill coefficient of 0.63 (n = 36).
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Figure 5.
Concentration-response relationship of the
N/OFQ-induced current in AL neurons. Experiments were conducted in 10 mM external K+ concentration with a
holding potential of 80 mV, resulting in inward currents in response
to N/OFQ application. Examples for membrane currents examined in the
presence of 1 µM, 50 nM, and 1 nM
N/OFQ are presented as insets. Data are averages from
measurements in different numbers of cells, as indicated near error
bars. In a given neuron, only one N/OFQ concentration was tested. The
curve was drawn according to the Hill equation (as in Fig. 4). The
EC50 and Hill values obtained from the curve were 30.6 nM and 0.63, respectively.
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Involvement of G-proteins
Next, the possible mediation by G-proteins of the N/OFQ-induced
K+ current was tested (Fig.
6). The tip of the electrode was filled with standard pipette solution, and the remainder was backfilled with a
solution supplemented with 100 µM GTP--S, a
nonhydrolyzable GTP analog (Gilman, 1987). Under these conditions,
N/OFQ (50 nM) evoked an outward current shortly after
establishing the whole-cell configuration, with no indication of
recovery during the time course of the experiment (Fig.
6A, compare with Fig. 4A). A second application of N/OFQ had no further effect, whereas the standing current was significantly reduced by Ba2+ (Fig.
6A) (n = 3). Inclusion in the pipette
solution of 2 mM GDP--S, a nonhydrolyzable GDP analog
(Gilman, 1987), resulted in only a small outward current (9.4 ± 1.3 pA) in response to a first application of N/OFQ, whereas further
applications failed to evoke an effect (Fig. 6B)
(n = 3). Finally, after incubation of slices in a
medium containing pertussis toxin (500 ng/ml) for ~18 hr at 34°C,
N/OFQ effects could not be obtained in any neuron tested (Fig.
6C) (n = 3). In slices that had been
similarly treated, but with no addition of the toxin, all cells tested
responded to N/OFQ (1 µM) with the typical outward
current (maximal amplitude at a holding potential of 70 mV; 30.3 ± 4.2 pA; n = 4).
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Figure 6.
Mediation via G-proteins of the N/OFQ-sensitive
current in AL neurons. A, Inclusion of GTP--S (100 µM) in the recording pipette results in a sustained
outward current in response to a single application of 50 nM N/OFQ. A second application of N/OFQ has no further
effect, whereas the induced current is still sensitive to
Ba2+ (100 µM). B, With
GDP--S (2 mM) in the pipette, N/OFQ elicits a small
outward current, which cannot be repeated on subsequent application.
C, After incubation of the slices in a medium containing
pertussis toxin (500 ng/ml), application of N/OFQ has no effect,
whereas cells in slices that had been incubated with no toxin added
(Control) showed typical responses to
N/OFQ.
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N/OFQ responses in central amygdaloid neurons
In 17 of 55 neurons recorded in the ACe, N/OFQ (1 µM) elicited a current with properties similar to that in
AL cells, in that it possessed inwardly rectifying properties (Fig.
7A), it was reduced to
74.2 ± 5.4% by 100 µM Ba2+
(n = 7) (Fig. 7B), and its reversal
potential shifted in a Nernstian manner with the external
K+ concentration (Fig. 7E). The maximal
current amplitude at a holding potential of 70 mV was significantly
(p < 0.01) smaller in neurons of the ACe
(21.6 ± 4.1 pA; n = 9) compared with that in the
AL (53.3 ± 7.3 pA; n = 8). In 24 central
amygdaloid neurons, no responses to N/OFQ were detected (data not
shown). The remainder of the cells recorded in the ACe responded to
N/OFQ with a small current (8.6 ± 1.1 pA at a holding potential
of 70 mV; n = 14) (Fig. 7D), which
displayed no inward rectification as measured by the ramp protocol
(Fig. 7C) but was also blocked by 100 µM
Ba2+ (Fig. 7D).
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Figure 7.
N/OFQ responsiveness in ACe neurons. In two
different cells, N/OFQ elicits an inwardly rectifying current
(A, ramp protocol as in Fig. 2) and a small current with
no apparent rectification (C). The N/OFQ-evoked
currents are sensitive to external Ba2+ (100 µM) in both cells (B, D).
E, Reversal potentials of the N/OFQ-induced currents
shift on variation of the external K+ concentration
with a slope of 55.8 mV according to the Nernst equation
(E). For each K+
concentration, four cells were tested.
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Effect of N/OFQ on spike firing
Under current-clamp conditions, 50 nM N/OFQ induced a
membrane hyperpolarization from the resting membrane potential of 70 mV, with an average maximal amplitude of 10.3 ± 1.2 mV in AL
neurons (n = 7) (Fig.
8A). The input membrane
resistance decreased from 535.7 ± 58.3 to 326.4 ± 30.3 M
during the maximal response (n = 7). This
hyperpolarization and decrease in input resistance had a dampening
effect on cell excitability, as was indicated by a substantial
reduction in the number of spikes evoked by depolarizing current
responses before and during action of N/OFQ (Fig.
8B).
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Figure 8.
Reduced excitability of an AL neuron during action
of N/OFQ recorded under current-clamp conditions. A,
Application of 50 nM N/OFQ induces a transient
hyperpolarizing response from a membrane potential close to the resting
value (70 mV) associated with a decrease in apparent input resistance
(as indicated by responses to small hyperpolarizing test pulses).
Examples in B illustrate responses to depolarizing test
pulses (+150 pA, 500 msec, 0.1 Hz) and hyperpolarizing test pulses
(20 pA) at a faster time scale at times indicated. Note the reduction
in spike activity during action of N/OFQ. Spikes are truncated. Resting
membrane potential was 67.7 mV.
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DISCUSSION |
The results of the present study indicate that N/OFQ acts on ORL
receptors in the postsynaptic membrane of neurons in the rat lateral
and central amygdaloid nuclei, thereby inducing an increase in an
inwardly rectifying K+ conductance mediated via
pertussis toxin-sensitive G-proteins. This promotes significant
hyperpolarization of the resting membrane potential, an increase in
input conductance, and a decrease in cellular excitability. Responses
to N/OFQ were quantitatively different in various subnuclei of the
amygdala, in that almost every presumed projection class I neuron
encountered in the AL was responsive, whereas a typical response was
obtained in roughly one-third of neurons in the ACe.
Mediation of N/OFQ responses through ORL receptors
The involvement of ORL receptors is indicated by three lines of
evidence. First, responses to N/OFQ were reversibly blocked by
the selective ORL receptor antagonist
[Phe1(CH2-NH)Gly2]NC(1-13)NH2
(Guerrini et al., 1998), whereas naloxone, a prototypical antagonist to
the µ-, -, and -subtypes of opioid receptors (Raynor et
al., 1994), was ineffective even at high concentrations. In the
guinea-pig ileum and in mouse vas deferens, 1 µM
[Phe1(CH2-NH)Gly2]NC(1-13)NH2
reportedly reduced the effect of 50 nM N/OFQ by ~80%, compared with a 61% reduction of N/OFQ-induced response in amygdaloid neurons. These results support the idea of a selective effect of N/OFQ
on ORL receptors in the present study. Second, the
concentration-response relationships of N/OFQ found in amygdaloid
neurons (EC50 = 30.6 nM) are comparable to
those reported earlier for various types of cellular responses to
N/OFQ, including increases in K+ conductance
(EC50 between 12 and 90 nM) (Connor et al.,
1996a; Vaughan and Christie, 1996; Ikeda et al., 1997; Vaughan et al., 1997; Wagner et al., 1998), reduction of calcium currents
(EC50 at 42-80 nM) (Connor et al., 1996b;
Knoflach et al., 1996), and inhibition of K+-evoked
glutamate release (EC50 = 51 nM) (Nicol et al.,
1996). Third, mRNA-expressing ORL receptors and ORL receptor binding sites were demonstrated to be highly enriched in the amygdala (Bunzow
et al., 1994; Mollereau et al., 1994; Wick et al., 1994; Lachowicz et
al., 1995; Anton et al., 1996; Florin et al., 1997). In addition, the
mRNA of the N/OFQ precursor protein (Houtani et al., 1996; Nothacker et
al., 1996) and immunoreactivity against the peptide (Schulz et al.,
1996) were detected in this limbic area. Therefore, it seems reasonable
to assume a functional role of the ORL receptor/N/OFQ peptide system in
the amygdala.
Involvement of G-protein-coupled
K+ channels
The primary structure of ORL displays the seven putative
membrane-spanning domains of a G-protein-coupled membrane receptor (Bunzow et al., 1994; Fukuda et al., 1994; Mollereau et al., 1994; Wick
et al., 1994; Lachowicz et al., 1995). Indeed, N/OFQ potently inhibited
forskolin-stimulated cAMP accumulation in transfected cells (Meunier et
al., 1995; Reinscheid et al., 1995) and modulated Ca2+ conductances via pertussis toxin-sensitive
G-proteins in acutely isolated hippocampal neurons (Knoflach et al.,
1996) and SH-SY5Y neuroblastoma cells (Connor et al., 1996b). By
comparison, the inhibition of the T-type Ca2+
channel in rat sensory neurons induced by N/OFQ occurred through a
G-protein-independent mechanism (Abdulla and Smith, 1997).
Three lines of evidence suggest that in amygdaloid neurons, the
activation of ORL through N/OFQ resulting in an increase in an inward
rectifier K+ conductance appears to be mediated by a
G-protein-linked pathway. First, during the intracellular presence of
GTP--S, a nonhydrolyzable GTP analog (Gilman, 1987), N/OFQ evoked
sustained responses, presumably attributable to irreversible activation
of the G-proteins. Second, intracellular GDP--S, the nonhydrolyzable
GDP analog (Gilman, 1987), significantly reduced the response amplitude
to a first application of N/OFQ and prevented further responses in
individual cells, presumably caused by functional inactivation of the
G-proteins. Third, incubation of slices overnight with pertussis toxin,
a blocker of the Gi/G0 class of
G-proteins (Hille, 1994), prevented responses to N/OFQ, whereas slices
that were similarly treated, but with no addition of the toxin, showed
qualitatively unaltered responsiveness to N/OFQ. Taken together, these
data indicated a coupling of ORL to the
Gi/G0 class of G-proteins (Hille,
1994).
In amygdaloid neurons, the targets of ORL activation appear to be the
inwardly rectifying types of K+ channels (Fakler and
Ruppersberg, 1996; Isomoto et al., 1997; Karschin et al., 1997), as was
indicated by the I-V relationship of responses to N/OFQ and
their sensitivity to low concentrations of extracellular
Ba2+. This hypothesis is supported by results from
in situ hybridization studies showing that the mRNA of
subunits of G-protein-coupled K+ channels, namely
Kir3.1, Kir3.2, Kir3.3, are expressed in high density in amygdaloid
nuclei (Karschin et al., 1996). By comparison, only low levels of mRNA
of "classical" inwardly rectifying K+ channels
belonging to the Kir2 family are present in the amygdala (Karschin et
al., 1996). The inwardly rectifying properties of membrane currents
observed under control conditions (Fig. 2A,B) may
thus represent the background conductance of G-protein-gated channels
(Dascal, 1997) or an H-current component rather than classical inward
rectifier activity (Womble and Moises, 1993). The exact subunit
composition of the G-protein-gated inward rectifier channels mediating
responses to N/OFQ remains to be elucidated, although the fast but not
instantaneous activation of the N/OFQ-induced current (Fig.
2A) suggests an involvement of Kir3.1 subunits.
N/OFQ and "classical" opioid effects in the amygdala
At the cellular level, N/OFQ and opioid peptides seem to share a
number of common mechanisms, such as the increase in a
G-protein-coupled inwardly rectifying K+
conductance, whereas on the systemic level, N/OFQ can potently reverse
opioid analgesia (Darland et al., 1998). In the AL, an opioid-induced
hyperpolarization presumably mediated through an increase in
K+ conductance reportedly occurs in the nonpyramidal
type of cells (type 2), whereas cells similar in appearance to those
termed class I by McDonald (1992) were nonresponsive (Sugita and North, 1993). AL neurons that were labeled with biocytin in the present study
possessed morphologies indicative of class I neurons, suggesting that
the effects of N/OFQ and opioids are mediated by different subsets of
neurons. The observation that only a comparatively small percentage of
the morphologically heterogeneous group of neurons encountered in the
ACe was responsive to N/OFQ seems to be supportive of this hypothesis.
A more direct correlation between morphologically defined types of
cells and N/OFQ responsiveness will require immunocytochemical analyses
of the cellular distribution of receptors and K+
channels in identified types of cells in the various parts of the
amygdala.
Possible source and functional significance of N/OFQ
The expression of N/OFQ mRNA, ORL mRNA, and the ORL
protein, as determined through in situ hybridization and
immunocytochemical techniques, is very dense to dense in the amygdala
(Bunzow et al., 1994; Mollereau et al., 1994; Anton et al., 1996;
Houtani et al., 1996; Nothacker et al., 1996; for review, see Henderson and McKnight, 1997; Meunier, 1997; Darland et al., 1998). Although these data suggest that N/OFQ may act as a transmitter of the abundant
interneurons inherent to the amygdaloid complex (Nitecka and Frotscher,
1989; Smith and Paré, 1994), electrical stimulation at different
sites within the amygdala or the external capsule using different
stimulus protocols failed to evoke a postsynaptic potential sensitive
to the selective ORL antagonist in the present study (data not shown).
On the other hand, a dense expression of N/OFQ or its receptor, or
both, has been found to exist in various regions with axonal projection
to the amygdala, including the ventral tegmental area, the locus
coeruleus, and the dorsal raphe nuclei of the brain stem (LeDoux, 1995;
LeDoux and Muller, 1997), which may as well give rise to an
N/OFQ-releasing system. Further analyses, such as an electron
microscopic study, will be needed for the detection of N/OFQ at the
synaptic level and thus the identification of the substrates of
N/OFQ-mediated neurotransmission in the amygdala.
In any case, ~98 and 55% of the cells encountered in the AL and ACe,
respectively, responded to local application of N/OFQ. Morphological
identification of the N/OFQ-sensitive neurons with intracellular
injection of biocytin revealed characteristics indicative of projection
cells (McDonald, 1992, 1996), such as pyramidal-shaped spiny cells in
the AL and morphologically more heterogeneous types of cells in the
ACe, all of which possessed spine-rich dendrites. Following from this
is the conclusion that synaptic transmission mediated through N/OFQ is
not negligible in the amygdala. The hyperpolarizing effect and
associated decrease in excitability produced by N/OFQ, in turn,
indicates that N/OFQ promotes a net dampening effect on amygdaloid
output activity. In view of the known role of the amygdala for the
integration of sensory and aversive signals (Davis, 1992; LeDoux, 1995;
Maren and Fanselow, 1996), with the lateral and central nucleus serving
as the main input and output station, respectively (Pitkänen et
al., 1997), these conclusions support the speculation that the
N/OFQ-induced dampening of amygdaloid output activity contributes to
the reduction in fear responsiveness that has been observed recently
during systemic application of this neuropeptide in mice and rats
(Jenck et al., 1997). Studies should now be performed that combine
local application of the selective ORL antagonist
[Phe1(CH2-NH)Gly2]NC(1-13)NH2
in the amygdala, with behavioral paradigms generating various types of
fear responses, to understand the exact conditions under which N/OFQ is
acting as an anxiolytic.
| |
FOOTNOTES |
Received June 29, 1998; revised July 30, 1998; accepted July 30, 1998.
This work was supported by the Kultusministerium des Landes
Sachsen-Anhalt (FKZ 2279A/0085H) and the Deutsche
Forschungsgemeinschaft (SFB 426, TP B3). We thank Professor V. Höllt for stimulating the work on N/OFQ, and A. Ritter, R. Ziegler, and A. Reupsch for expert technical assistance.
Correspondence should be addressed to Hans-Christian Pape, Institut
für Physiologie, Medizinische Fakultät,
Otto-von-Guericke-Universität, Leipziger Strasse 44, D-39120
Magdeburg, Germany.
| |
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Top
Abstract
Introduction
