Selective Modulation of Excitatory Transmission by µ-Opioid Receptor Activation in Rat Supraoptic Neurons

Qing-Song Liu, Sheng Han, You-Sheng Jia, and Gong Ju

Institute of Neurosciences, The Fourth Military Medical University, Xian 710032, People's Republic of China


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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.


    INTRODUCTION
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INTRODUCTION
METHODS
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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 kappa -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-, delta -, or kappa -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 kappa -agonist dynorphin suppresses evoked excitatory postsynaptic potentials (EPSPs) and inhibitory postsynaptic potentials (IPSPs) in a portion of the SON neurons, whereas delta -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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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 GOmega ) 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 MOmega . 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 MOmega ) 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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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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Fig. 1. Effects of µ-agonist D-Ala2, N-CH3-Phe4, Gly5-ol-enkephalin (DAGO) on evoked monosynaptic excitatory postsynaptic currents (EPSCs) recorded from supraoptic nucleus (SON) neurons. A: non-N-methyl-D-aspartate (NMDA) glutamate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX; 10 µM) reversibly blocked evoked monosynaptic EPSCs. Trace marked "wash" was taken after 20 min wash out of the DNQX. In this and subsequent traces, stimulation artifacts are blanked out. B and C: DAGO (0.5 µM) reduced the amplitude of evoked EPSCs. This inhibition was reversed by 10 min wash out of DAGO (B) or by subsequent addition of µ-antagonist Cys2-Tyr3-Orn5-Pen7-NH2 (CTOP; 1 µM, C). Each trace was obtained by data averaged from 10 consecutive stimuli. D: mean amplitude was expressed as a percent of that obtained in control. Each bar represents the means ± SE of 5-22 cells; the numbers inside the bars are numbers of cells tested. *P < 0.05, **P < 0.001.

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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Fig. 2. Effects of µ-agonist DAGO on evoked polysynaptic activity. A: DAGO (0.5 µM) decreased the frequency of polysynaptic activity. This effect was reversed by 10 min wash out. Arrows indicate the time at which stimuli were given. B: traces are the same as the lowest traces in A but with different vertical scale. Both frequency of polysynaptic events and amplitude of monosynaptic EPSCs were decreased by DAGO. C: summary of the effect of DAGO. Each bar represents the means ± SE of 6 cells. **P < 0.001. The total number of synaptic events from 0 to 80 ms after termination of the stimuli was used to calculate the frequency.

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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Fig. 3. µ-Agonist DAGO affected the frequency, but not the amplitude or kinetics of mEPSCs. A: bath application of DAGO (0.5 µM) decreased the frequency of mEPSCs from 13.7 to 7.3 Hz. This effect was reversed by subsequent addition of selective µ-antagonist CTOP (1 µM). B: DAGO shifted the cumulative frequency distribution to the right, i.e., toward longer interevent intervals (K-S test, P < 0.001). C: DAGO had no significant effect on the cumulative amplitude distributions. D: superimposed averages of 200 consecutive mEPSCs obtained during control and DAGO treatment. Dashed line represents a monoexponential fit to the control average. Note the similar amplitudes of both averages (28.5 vs. 28.7 pA), and the similar decay constants (tau  = 1.05 ms).

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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Fig. 4. µ-Agonist DAGO did not affect the evoked monosynaptic inhibitory postsynaptic currents (IPSCs) and miniature IPSCs (mIPSCs). A: GABAA receptor antagonist bicuculline (20 µM) reversibly blocked evoked IPSCs. Trace marked "wash" was taken after 15 min wash out of the bicuculline. B: bath application of DAGO (1 µM) for 5 min had no significant effect on evoked IPSCs. In A and B, each trace was obtained by data averaged from 5-10 consecutive stimuli. C: mIPSCs were recorded from the same cell before (left) and 5 min after 0.5 µM DAGO perfusion (right).

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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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.


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

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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