Institute of Neurosciences, The Fourth Military Medical University, Xian 710032, People's Republic of China
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ABSTRACT |
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Liu, Qing-Song, Sheng Han, You-Sheng Jia, and Gong Ju. Selective Modulation of Excitatory Transmission by µ-Opioid Receptor Activation in Rat Supraoptic Neurons. J. Neurophysiol. 82: 3000-3005, 1999. Opioid peptides have profound inhibitory effects on the production of oxytocin and vasopressin, but their direct effects on magnocellular neuroendocrine neurons appear to be relatively weak. We tested whether a presynaptic mechanism is involved in this inhibition. The effects of µ-opioid receptor agonist D-Ala2, N-CH3-Phe4, Gly5-ol-enkephalin (DAGO) on excitatory and inhibitory transmission were studied in supraoptic nucleus (SON) neurons from rat hypothalamic slices using whole cell recording. DAGO reduced the amplitude of evoked glutamatergic excitatory postsynaptic currents (EPSCs) in a dose-dependent manner. In the presence of tetrodotoxin (TTX) to block spike activity, DAGO also reduced the frequency of spontaneous miniature EPSCs without altering their amplitude distribution, rising time, or decaying time constant. The above effects of DAGO were reversed by wash out, or by addition of opioid receptor antagonist naloxone or selective µ-antagonist Cys2-Tyr3-Orn5-Pen7-NH2 (CTOP). In contrast, DAGO had no significant effect on the evoked and spontaneous miniature GABAergic inhibitory postsynaptic currents (IPSCs) in most SON neurons. A direct membrane hyperpolarization of SON neurons was not detected in the presence of DAGO. These results indicate that µ-opioid receptor activation selectively inhibits excitatory activity in SON neurons via a presynaptic mechanism.
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INTRODUCTION |
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The magnocellular neurons of supraoptic nucleus
(SON) and paraventricular nucleus (PVN) synthesize oxytocin and
vasopressin and release these hormones from their terminals in the
neurohypophysis. A number of studies indicate that opioids may be
involved in controlling release of oxytocin and vasopressin, and that
both the terminals in neurohypophysis and the soma in hypothalamus are
important sites of opioid inhibition (Arnauld et al.
1983; Bicknell and Leng 1982
; Bicknell et
al. 1985
; Renaud and Bourque 1991
;
Wakerley et al. 1983
; Zhao et al. 1988
).
The SON and PVN receive opioid innervation from fibers originating from
other brain regions (Finley et al. 1981
;
Sawchenko et al. 1982
) and contain high levels of µ-
and
-opioid receptor-binding sites (Mansour et al.
1988
; Sumner et al. 1990
, 1992
).
Both in vivo and in vitro extracellular recordings have shown that
opioids inhibit firing of a portion of SON and PVN neurons via
activation of µ-,
-, or
-receptors (Arnauld et al.
1983
; Inenaga et al. 1990
; Leng and
Russell 1989
; Wakerley et al. 1983
).
Intracellular recordings have also revealed that the µ-agonist
D-Ala2,
N-CH3-Phe4,
Gly5-ol-enkephalin (DAGO) decreases or suppresses
spontaneous firing in less than one-half of the putative neurosecretory
magnocellular neurons in SON and PVN, but this inhibition is not
accompanied by an appreciable change of the resting membrane potential
or input resistance. In contrast, DAGO decreases or suppresses
spontaneous firing in most of the low-threshold
Ca2+ spike neurons (putative
nonneurosecretory) in PVN; this inhibition is accompanied by a marked
hyperpolarization (Wuarin and Dudek 1990
; Wuarin
et al. 1988
). Thus neighboring nonneurosecretory neurons are
more effectively inhibited by this opioid than magnocellular neurosecretory neurons per se. These findings suggest that modulation of synaptic inputs may be important in opioid-induced inhibition of the
neurosecretory neurons.
We tested the hypothesis that a presynaptic mechanism is involved in
µ-opioid inhibition of magnocellular neurosecretory neurons. Intracellular recordings have shown that the -agonist dynorphin suppresses evoked excitatory postsynaptic potentials (EPSPs) and inhibitory postsynaptic potentials (IPSPs) in a portion of the SON
neurons, whereas
-agonist D-Ala, D-Leu
enkephalin and µ-agonist morphine have little or no effect
(Inenaga et al. 1994
). In that study, the effect of
morphine was tested in only three neurons and in relatively low
concentrations. Here, using whole cell recordings from hypothalamic
slices, we examined the effects of DAGO, a highly selective µ-opioid
receptor agonist, on evoked and spontaneous excitatory postsynaptic
currents/inhibitory postsynaptic currents (EPSCs/IPSCs) in rat SON neurons.
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METHODS |
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Preparation of brain slices and whole cell recordings were
performed as described previously (Liu et al. 1997).
Briefly, Sprague-Dawley rats (4-6 wk old) were deeply anesthetized
with ketamine and chloral hydrate and decapitated, and the brains were
quickly removed and placed in ice-cold artificial cerebrospinal fluid
(ACSF) for a few minutes. The ACSF was well oxygenated with 95%
O2-5% CO2 and consisted of
(in mM) 125 NaCl, 5 KCl, 1.25 Na2HPO4, 2 CaCl2, 1.3 MgSO4, 25 NaHCO3, and 10 glucose. The hypothalamus was
blocked out, and 350-µm slices were cut coronally using a Vibratome
(TPI, St. Louis, MO). After sectioning, the slices were hemisected and allowed to recover for at least 1 h in warm ACSF (30-32°C). The slice was transferred into a submerged chamber perfused with warm ACSF
(30-32°C) at 1-2 ml/min. Tight-seal (2-10 G
) whole cell patch-clamp recordings (Blanton et al. 1989
) from SON
neurons were obtained using an Axopatch 200A amplifier (Axon
Instruments). Membrane currents were amplified at 5-50 mV/pA, filtered
at 1-2 kHz, and digitized at 5-10 kHz using pClamp software and
DigiData 1200 interface (Axon Instruments). Borosilicate glass
capillaries (1.6 mm OD, 1.2 mm ID, with filament) were pulled in two
stages to a resistance of 3-5 M
. For most experiments the pipette
solution was (in mM) 120 potassium gluconate, 5 KCl, 2 MgCl2, 10 HEPES, 10 EGTA, and 2 Mg-ATP, pH 7.2 with KOH. For recording of spontaneous miniature IPSCs, 120 mM
potassium gluconate and 5 mM KCl were replaced by 125 mM KCl. Evoked
EPSCs and IPSCs were elicited with a bipolar tungsten stimulation
electrode (WPI, Sarasota, FL) placed outside the dorsolateral border of
SON. Square pulses were delivered at a duration of 0.2-1 ms, intensity
of 0.1-1 mA, and frequency of ~0.1 Hz. These parameters were
adjusted to produce evoked EPSCs and IPSCs ranging from 80 to 300 pA.
Series resistance was routinely compensated by 80-85% in all
experiments except recording of mEPSCs. Series resistance (<20 M
)
was carefully monitored during experiment, data were not included if a
significant increase in series resistance (
15%) was detected. The
junction potential was nullified before attempting to form giga-seals.
The evoked EPSCs and IPSCs were averaged, measured, and fitted
using Clampfit. Data are given as means ± SE. The paired or unpaired t-test as appropriate was used to compare the
differences between these data. The statistical criterion for
significance was P < 0.05. Spontaneous miniature EPSCs
(mEPSCs) and IPSCs (mIPSCs) were analyzed off-line with a synaptic
current detection software provided by Dr. P. Vincent. The details for
this event-detection procedure were described elsewhere (Vincent
and Marty 1993). Synaptic events were detected with an
adjustable threshold, often set at 6-10 pA and kept constant for the
same group of data (control, treatment, and recovery). The analysis of
mEPSCs and mIPSCs was performed with cumulative probability plots
(Van der Kloot 1991
). The cumulative probability of
amplitude and interevent interval were compared using the
Kolmogorov-Smirnov test (K-S test), which estimates the probability
(P) that two distributions are similar. With this test, two
cumulative sets of data were considered significantly different only
when P < 0.01.
Cys2-Tyr3-Orn5-Pen7-NH2 (CTOP) and (±)-2-amino-5-phosphonopentanoic acid (AP-5) were from RBI (Natick, MA). All other reagents were from Sigma.
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RESULTS |
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Effect of DAGO on evoked EPSCs
SON neurons were voltage clamped at 70 mV, and
EPSCs were evoked following at least 20 min pretreatment with the
GABAA receptor antagonist bicuculline (20 µM).
Focal stimulation of the dorsolateral border of the SON elicited EPSCs
in a total of 38 SON neurons. In 28 of the 38 neurons, only "pure"
monosynaptic EPSCs were observed. In the remaining 10 neurons, the
monosynaptic EPSCs were followed by brief, high-frequency bursts of
polysynaptic activity. Both the monosynaptic EPSCs (Fig.
1A, n = 6) and
the burst of polysynaptic activity (n = 4) were
completely and reversibly blocked by 10 µM
6,7-dinitroquinoxaline-2,3-dione (DNQX), indicating that they were
mediated by non-N-methyl-D-aspartate (NMDA)
glutamate receptors. The monosynaptic EPSCs had a fixed latency of <4
ms, and fast rising and slow decaying phases. The average rise time and
decay time constants were 2.1 ± 0.1 ms and 4.8 ± 0.6 ms,
respectively (n = 28). Bath perfusion of µ-agonist
DAGO (0.1-1 µM) for 5 min consistently reduced the amplitude of the
evoked EPSC in each of the 22 cells tested (Fig. 1). This effect peaked
at 2-4 min after perfusion of DAGO and was reversed by 10-20 min wash
out (Fig. 1, B and D). In the continuous presence
of 0.5 µM DAGO, subsequent addition of the opioid antagonist naloxone
(5 µM, Fig. 1D) or selective µ-antagonist CTOP (1 µM,
Fig. 1, C and D) reversed the effect of DAGO.
DAGO had no significant effect on the kinetics of the evoked EPSCs. In
the presence of DAGO, the rising time of the evoked EPSCs was 97 ± 3% of control, and the decay time constant was 103 ± 6% of
control (P > 0.05, n = 22).
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We also tested the effects of DAGO on the burst of polysynaptic
activity. The burst generally lasted ~80 ms after termination of the
stimulation, and in shape resembled a collection of spontaneous EPSCs
at high-frequency in the same neuron (Fig.
2). Their amplitudes ranged from 6 to 10 pA (threshold for detection) to ~100 pA. Bath perfusion of DAGO (0.5 µM) decreased the frequency of the polysynaptic events from 160 ± 9 Hz to 36 ± 3 Hz (26 ± 4% of control;
P < 0.001; n = 6), and this effect was
reversed on wash out (123 ± 11 Hz; 85 ± 11% of control).
The frequency was calculated as the number of events from 0 to 80 ms
after the stimulation. In these neurons, DAGO also reduced the
amplitude of monosynaptic EPSCs, but it was not always possible to
measure the amplitude accurately because the burst of polysynaptic
events was superimposed on the monosynaptic EPSCs. Therefore these data
were not included for amplitude analysis. DAGO had no effect on the
holding currents at the holding potential of 70 mV.
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Effect of DAGO on spontaneous mEPSCs
To investigate whether the effect of DAGO on evoked EPSCs is
mediated by a presynaptic mechanism, its action on mEPSCs was analyzed.
After pretreatment of slices with 20 µM bicuculline and 1 µM TTX
for at least 20 min, mEPSCs were recorded in SON neurons voltaged
clamped at 70 mV. In agreement with a previous report (Wuarin
and Dudek 1993
), mEPSCs are mediated by non-NMDA glutamate
receptors, because they were blocked by DNQX (10 µM; n = 5). The frequency of mEPSCs ranged from 0.5 to 15 Hz, and the amplitude ranged from 6 to 10 pA (the threshold for
detection) to ~100 pA. Seven cells with relative high-frequency of
mEPSCs were tested with DAGO. DAGO (0.5 µM) decreased mEPSC
frequency from 6.2 ± 1.8 Hz to 3.1 ± 1.2 Hz (47 ± 5%
of control; paired t-test, P < 0.001;
n = 7). Subsequent addition of the selective µ-opioid
receptor antagonist CTOP (1 µM) in the continuous presence of DAGO
reversed the effect of DAGO on mIPSCs (5.7 ±. 2.1 Hz, Fig.
3A). Cumulative frequency plot
analysis showed that DAGO did not change the cumulative amplitude
distributions (K-S test, P > 0.01; Fig. 3C)
but shifted the cumulative distribution of intervals between successive
events to the right in each of seven cells tested (K-S test,
P < 0.001; Fig. 3B). DAGO also had no effect on the kinetics of mEPSCs (Fig. 3D).
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Effect of DAGO on evoked IPSCs and mIPSCs
Next we tested whether IPSCs could also be modulated by
µ-receptor activation. After 20 min perfusion of slices with
glutamate receptor antagonists DNQX (10 µM) and AP-5 (100 µM),
focal stimulation evoked fixed-latency (<4 ms) monosynaptic IPSCs,
which were completely and reversibly blocked by 20 µM bicuculline
(Fig. 4A,
n = 7). Generally IPSCs could be evoked by lower
stimulating intensities than those required for EPSCs, and they were
not followed by a burst of polysynaptic activity. The evoked IPSCs
reversed at about 70 mV and appeared outward at more depolarized
holding potentials. In the present experiment, the neurons were voltage
clamped at
40 mV to record evoked IPSCs. The average rise time and
decay time constants of the evoked IPSCs were 2.2 ± 0.3 ms and
20.0 ± 4.2 ms, respectively (n = 15). Bath
perfusion of µ-agonist DAGO (0.5-1 µM) for 5 min had no
significant effect on the evoked IPSCs (Fig. 4B). In the
presence of DAGO, the amplitude of evoked IPSCs was 98.2 ± 4.2%
of control, the rise time was 106.1 ± 5.6% of control, and the
decay time constant was 104.3 ± 5.7% of control respectively
(n = 8). These values were not statistically
different from control (P > 0.05).
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We also tested the effect of DAGO on spontaneous mIPSCs, recorded
in the presence of glutamate receptor antagonists and also 1 µM TTX
to block spike activity. At a holding potential of 70 mV, mIPSCs
appeared inward when KCl-based pipette solution was used for whole cell
recording (Fig. 4C). They were also reversibly blocked
by bicuculline (10 µM) and thus mediated by activation of
GABAA receptors. Interestingly, TTX, applied at a
concentration that completely blocked the action potential or sodium
current, had no significant effect on the spontaneous IPSCs recorded
from the SON neurons, concurring with recent data (Brussaard et
al. 1996
; Kabashima et al. 1997
) and suggesting
that the spontaneous IPSCs in SON are produced by random release of
single vesicles from axon terminals, which are independent of
somatodendritic action potentials (Brussaard et al.
1996
). Overall, after TTX treatment, the frequency was
95.3 ± 5.6% of that control (n = 7, paired
t-test, P > 0.05); the amplitude
distribution was also not altered as determined by cumulative plot and
K-S test (P > 0.01). In the continuous presence of
TTX, bath perfusion of DAGO (0.5 µM) for 5 min had no significant
effect on the mIPSCs in five of seven cells tested (Fig.
4C). The mean frequency of mIPSCs before and after DAGO
perfusion was 5.2 ±1.6 Hz and 4.9 ± 1.5 Hz, respectively
(n = 5, paired t-test,
P > 0.05); the cumulative amplitude and interevent
interval distribution were not significantly changed (K-S test,
P > 0.01). In the remaining two cells, DAGO perfusion significantly decreased the mIPSC frequency by 35.3 and
43.6%, respectively, which was also accompanied by small but significant left shift (toward smaller values) of cumulative amplitude distribution. However, in contrast to the effect of DAGO on mEPSCs which was reversible, the inhibition of mIPSCs by DAGO showed partial
recovery on wash out of DAGO in only one of the two cells. This effect
could be due to either rundown of GABA responsiveness or a specific
action of DAGO on a minority of presynaptic terminals. The functional
significance is not clear. In each of these neurons, DAGO had no
significant effect on the holding current at the holding potential of
40 and
70 mV.
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DISCUSSION |
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Our data indicate that the µ-agonist DAGO selectively inhibits
excitatory transmission in SON in a receptor-mediated, presynaptic manner. DAGO is a highly selective µ-agonist, and its effect is reversible by addition of selective µ-antagonist CTOP and
nonselective opioid receptor antagonist naloxone. Therefore activation
of µ-opioid receptors accounts for the effects of DAGO. To determine
whether a presynaptic mechanism is involved in this inhibition, we have analyzed the effect of DAGO on spontaneous mEPSCs, recorded in the
presence of TTX to block spike activity. DAGO reduces the frequency of
mEPSCs without altering their amplitude distribution or kinetics. This
pattern of a decrease in the frequency of miniature synaptic events
without a significant change in other properties is typical of a
presynaptic effect (Bekkers and Stevens 1990; Del
Castillo and Katz 1954
; Van der Kloot 1991
).
As reported previously (Dudek and Gribkoff 1987), focal
stimulation produced brief, high-frequency bursts of polysynaptic activity in SON neurons following the monosynaptic EPSP. This polysynaptic activity may result from repetitive firing of presynaptic neurons within or near the stimulation sites, which lie outside of the
SON. Although these polysynaptic events are present in the absence of
bicuculline (Dudek and Gribkoff 1987
), the presence of
bicuculline in the present study removes GABA-mediated tonic inhibition
and promotes firing of presynaptic neurons. DAGO-induced reduction of
the frequency of these polysynaptic events can be explained by its
ability to reduce firing from presynaptic neurons. This is in agreement
with the idea that DAGO inhibits firing of neighboring
nonneurosecretory neurons and thus reduces their excitatory input to
SON neurons.
As demonstrated in locus coeruleus and hippocampal neurons, DAGO
could affect the excitability postsynaptically by hyperpolarizing the
neuronal membrane via opening of K+ channels
(North and Williams 1985; Svoboda and Lupica
1998
). However, we have not detected any outward currents from
SON neurons by perfusion of DAGO, suggesting the lack of direct
membrane hyperpolarization. If DAGO had hyperpolarized the SON neurons,
it should have produced an outward current at holding potentials used
in the present experiment (i.e.,
40 and
70 mV). This result is in
agreement with previous intracellular work showing that DAGO has no
significant effect on the membrane potential of SON neurons
(Wuarin and Dudek 1990
). Together, these results suggest
that µ-receptor activation inhibits SON neurons primarily via a
presynaptic mechanism.
DAGO selectively inhibits EPSCs without affecting IPSCs in
most of the SON neurons tested. Selective modulation of synaptic transmission by µ-agonists has been well-documented in hippocampal neurons where µ-agonists reduce GABA release from interneurons, and
therefore excite pyramidal neurons through a mechanism of disinhibition
(Cohen et al. 1992; Copogna et al. 1993
;
Lupica 1995
). In addition, µ-agonists do not affect
EPSPs (Copogna et al. 1993
) or increase the amplitude of
evoked EPSPs via the reduction of GABAergic inhibition (Lupica
et al. 1992
). Although the common feature of µ-opioid's
acting on synapse transmission is a reduction of transmitter release,
they probably act selectively on either the excitatory or inhibitory
input, but not both. This specificity suggests that the functional
expression of opioid receptors is often limited to either the
excitatory or inhibitory terminals depending on the neuronal cell type.
Obviously this specificity may have important physiological implications.
The firing frequency and pattern of magnocellular neurosecretory
neurons can change dramatically in response to certain
physiological and pathological stimuli. For example, during the milk
ejection reflex and parturition, oxytocin neurons generate intermittent bursts of action potentials. The action potentials are synchronized among all oxytocin neurons, resulting in a periodic release of hormone
(Poulain and Wakerley 1982). These temporal patterns are well correlated with the function of the hormone. Thus magnocellular neurosecretory neurons are strongly dependent on synaptic inputs to
generate their output; modulation of the synaptic inputs may have great
impact on firing patterns and hormone secretion. Therefore µ-receptor
activation, by selectively reducing excitatory synapse inputs, will
inhibit firing of SON neurons and subsequent hormone release,
particularly in response to certain physiological and pathological
stimuli. This indirect effect could be more relevant for inhibition of
SON neurons than its rather weak action on the membrane potential.
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ACKNOWLEDGMENTS |
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We thank Drs. Darwin K. Berg and Jian-Tian Qiao for comments on the manuscript and Dr. P. Vincent for providing a program to analyze mEPSCs.
This work was supported by a grant from the National Natural Science Foundation of China.
Present address of Y.-S. Jia: Dept. of Neurobiology and Behavior, University of California, Irvine, CA 92697.
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FOOTNOTES |
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Present address and address for reprint requests: Q.-S. Liu, Dept. of Biology 0357, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093.
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 5 April 1999; accepted in final form 13 August 1999.
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REFERENCES |
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