Opioidergic Modulation of Voltage-Activated K+ Currents in Magnocellular Neurons of the Supraoptic Nucleus in Rat

Wolfgang Müller,2 Stephan Hallermann,1 and Dieter Swandulla1

 1Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Universität Erlangen-Nürnberg, D-91054 Erlangen; and  2Institut für Physiologie der Charité, AG Molekulare Zellphysiologie, D-10117 Berlin, Germany


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Müller, Wolfgang, Stephan Hallermann, and Dieter Swandulla. Opioidergic modulation of voltage-activated K+ currents in magnocellular neurons of the supraoptic nucleus in rat. Opioidergic modulation plays an important role in the control of oxytocin and vasopressin release by magnocellular neurons (MCNs) in the supraoptic and paraventricular nuclei of the hypothalamus. We have used whole cell patch-clamp recording in acute slices of the supraoptic nucleus (SON) of the hypothalamus to study opioidergic modulation of voltage-dependent K+ currents in MCNs that are involved in release activity. The µ-receptor agonist D-Ala2, N-Me-Phe4, Gly5-ol-enkephalin (DAMGO, 2 µM) affected K+ currents in 55% of magnocellular neurons recorded from. In these putative oxytocinergic cells, DAMGO increased the delayed rectifier current (IK(V)) amplitude by ~50% without significant effects on its activation kinetics. The transient A current (IA) was enhanced by DAMGO by ~36%. Its inactivation kinetic was accelerated slightly while the voltage dependence of steady-state inactivation was shifted by -6 mV to more negative potentials. All DAMGO effects were blocked by the preferential non-kappa -opioid antagonist naloxone (10 µM). The kappa -opioid agonist trans-(±)-3,4-dichloro-N-methyl-N(2-[1-pyrrolidinyl]cyclohexyl)benzeneacetamide (U50,488; 10 µM) strongly suppressed IK(V) by ~57% and evoked a 20-mV hyperpolarizing shift and an acceleration of activation in both, DAMGO-sensitive and -insensitive putative vasopressinergic MCNs. U50,488 reduced IA by ~29% and tau  of inactivation by -20% in DAMGO-sensitive cells. In contrast, in DAMGO-insensitive cells U50,488 increased IA by ~23% and strongly accelerated inactivation (tau  -44%). The effects of U50,488 were suppressed by the selective kappa -receptor antagonist nor-binaltorphimine (5 µM). We conclude that µ- and kappa -opioidergic inputs decrease and increase excitability of oxytocinergic MCNs, respectively, through modulation of voltage-dependent K+ currents. In vasopressinergic MCNs, kappa -opioidergic inputs differentially modulate these K+ currents. The modulation of K+ currents is assumed to significantly contribute to opioidergic control of hormone release by MCNs within the supraoptic nucleus and from the axon terminals in the neural lobe.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Oxytocin (OT) and vasopressin (AVP), synthesized in the magnocellular neurons (MCNs) of the supraoptic and paraventricular nuclei of the hypothalamus, are well recognized for their importance in parturition/lactation and water homeostasis, respectively. Oxytocin also may be involved in the control of sodium excretion and is released in response to various kinds of stress. In the supraoptic nucleus (SON), all MCNs either synthesize oxytocin or vasopressin. Release of these hormones from the axon terminals in the neural lobe appears to be controlled primarily by the frequency of action potential firing by these neurons, whereas feedback somato-dendritic release within the SON may rely rather on slow depolarization than on action potentials (di Scala-Guenot et al. 1987).

Opioid peptides, i.e., dynorphins, are coexpressed in vasopressinergic cells and also in some oxytocinergic cells in the magnocellular nuclei (for a recent review, see Russell et al. 1995). Most of these peptides exert their effects by activation of both, µ- and kappa -receptors (dynorphin A1-8), whereas the longer chain dynorphins act relatively selectively on kappa receptors (Kosterlitz 1985). Opioidergic inhibition of basal and stimulated firing activity of MCNs as well as of oxytocin release has been demonstrated using µ- and kappa -receptor agonists and antagonists in vivo (Russell et al. 1995). With intracellular recording, inhibitory effects of opiate-receptor activation in CNS neurons have been characterized as either presynaptic reduction of transmitter release or postsynaptic inhibition or a combination of both. It needs to be noted, though, that inhibition of inhibitory neurons or of release of inhibitory transmitters results in indirect excitation through disinhibition (Kim et al. 1997; Madison and Nicoll 1988).

Voltage-activated potassium currents, namely the transient A current (IA) and the delayed rectifier current (IK(V)), have been recognized as important players in the control of electrical activity in neurons and dendritic subcompartments. This electrical activity results from the interaction of these K+ currents with synaptic current input and excitatory Na+ and Ca2+ currents (Hoffman et al. 1997; Müller and Lux 1993). In supraoptic MCNs of the hypothalamus, potassium channels critically affect the characteristic firing patterns linked to release of hormones (Müller and Swandulla 1995). Segregation of K+-channels to sites of excitatory current flow appears to be an important component in the local control of excitability (Müller and Lux 1993). Subcellular segregation of K+ channels has been demonstrated in various types of neurons and is controlled by binding domains of the PSD-95 family of membrane-associated putative guanylate kinases (Kim et al. 1995; Sheng et al. 1994). In hypothalamic vasopressinergic MCNs, the A current appears to be segregated largely to the dendrites (Widmer et al. 1997b).

In the present study, we address direct postsynaptic opioidergic modulation of K+ currents by µ- and kappa -receptor specific agonists in supraoptic magnocellular neurons in acute rat hypothalamic slices using whole cell patch-clamp recording. A brief account of these results has been published in abstract form (Hallermann et al. 1998).


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METHODS
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Preparation

Sprague Dawley rats aged 15-21 days were anesthetized with ether and decapitated. Within 1 min after decapitation, the brain was removed carefully from the skull without damaging the optic nerves or the optic chiasm. The whole brain was placed in a chamber filled with a modified ice-cold artificial cerebrospinal fluid (ACSF) containing (in mM) 120 NaCl, 2.5 KCl, 1 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, 2 CaCl2, 5 HEPES, and 15 D-glucose (pH 7.4, 305 mOsmol, carboxygenated with 5% CO2-95% O2). A coronal tissue block including the optic chiasm (3-4 mm) was dissected and mounted on the stage of a Campden manual vibratome slicer (Loughborough, UK) using cyanoacrylate glue. Whole brain coronal slices (300 µm) were cut and three to four slices encompassing the optic chiasm were transferred into oxygenated Ringer solution in a glass beaker. The total preparation period took not more than 15 min. After incubation at room temperature (23°C) for >= 2 h, a single slice was transferred to a submerged slice recording chamber on the stage of an upright microscope (Axioskop, Carl Zeiss, Jena, Germany) and cells were visualized using a ×40 water immersion objective (Achroplan ×40/0.75 W, Carl Zeiss). The SON was identified by its location adjacent to the lateral parts of the optic chiasm and the occurrence of large cell bodies >= 25 µm in diameter that are typical for MCNs. The chamber was perfused with ACSF at a rate of 1-2 ml min-1 at room temperature.

To reduce space clamp errors, MCNs were acutely isolated from brain slice sections encompassing the supraoptic nucleus (Lambert et al. 1994). In brief, dissected tissues were dissociated enzymatically by incubation in ACSF supplemented with 0.21% trypsin EDTA (GIBCO) for 1 h. The cell suspension was centrifuged for 5 min at 100 g. The pellet was resuspended in ACSF containing 0.05% trypsin inhibitor type 1-S (Sigma) and centrifuged as above three times before repeated trituration with a pipette. The final pellet was resuspended in ACSF, and cells were allowed to adhere to the bottom of the recording chamber before recordings.

Electrophysiological recordings

Electrophysiological recordings were made from MCNs of the SON. Patch-clamp electrodes were pulled from borosilicate glass (Kimex-51, Kimble, Vineland, NJ) in three steps using a horizontal puller (DMZ-Universal, Zeitz Instruments, Germany). Filled with an internal solution containing (in mM) 10 NaCl, 130 KCl, 1 CaCl2, 10 EGTA, 2 Na-ATP, 0.1 Na-GTP, and 10 HEPES (pH 7.3), patch pipettes had resistances of 2-4 MOmega . Patch pipettes were positioned onto MCN somata under visual control, applying positive pressure to keep the tip of the pipette clean. After sealing and obtaining the whole cell configuration, currents were recorded with a switched voltage-clamp amplifier (Swandulla and Misgeld 1990) (20-kHz switching frequency, SEC 1 l, NPI Electronic, Tamm, Germany), digitized, and stored on a computer data-acquisition system (Apple Macintosh Quadra 700, ITC 16 interface, Instrutech, Elmont, NY; data-acquisition and analysis software: Pulse, HEKA, Lambrecht, Germany). Voltage-clamp gain was adjusted to obtain >= 98% clamp accuracy within 0.5 ms. Membrane input resistance was measured during the experiment by 20-ms hyperpolarizing steps to -90 mV. Immediately after obtaining the whole cell configuration, input resistance of magnocellular neurons was 553 ± 249 (SD) MOmega (n = 38). This value dropped to 263 ± 161 MOmega within 10 min and stabilized thereafter. All data presented are after 10 min. Currents were sampled at 10 kHz and leakage corrected and analyzed with the IgorPro 3.0 software (Wavemetrics, Lake Oswego, Oregon). All currents were measured in the presence of 1 mM NiCl2, 0.5 µM TTX, and, except initial experiments, 10 mM tetraethylammonium chloride (TEA). Where indicated, 2-10 mM 4-aminopyridine (4-AP) was added to reduce A currents. Drug-containing solutions [D-Ala2, N-Me-Phe4, Gly5-ol-enkephalin (DAMGO), trans-(±)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]cyclohexyl)benzeneacetamide (U50,488), naloxone, nor-binaltorphimine dihydrochloride (nor-BNI)] were applied by bath perfusion at a rate of 5-10 ml min-1. In some experiments, the slices were preincubated in naloxone and/or nor-BNI for >= 2 h.

Chemicals

Opioids and antagonists were dissolved in distilled water and stored in aliquots (10 mM) at -20°C. Fresh dilutions were made with standard external solution on the day of the experiment.

All inorganic salts were from Merck (Darmstadt, Germany). Naloxone, DAMGO, U50,488H, TTX, N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), ethylene glycol-bis(b-aminoethyl ether) N, N,N',N'-tetraacetic acid (EGTA), Na-ATP, Na-GTP, TEA, and 4-AP were from Sigma Chemie (Deisenhofen, Germany). Nor-BNI was from RBI (Natick, MA).

Statistics and curve fitting

Statistical analysis and curve fitting of K+ currents were performed by the Jandel Sigma Plot 2.0 software (Jandel GmbH, Erkrath, Germany). All statistical data are expressed as means ± SD. Statistical significance was evaluated with the use of unpaired t-test.

For (IK(V)), the peak current at each test potential was converted to peak conductance (g) using the following formula
<IT>g</IT><IT>=</IT><IT>I&cjs0823;  </IT>(<IT>V−E</IT><SUB>K</SUB>) (1)
where EK is the equilibrium potential (-99 mV) that was confirmed experimentally. Because [K+]i is clamped by perfusion of the cell through the patch pipette, this method does not allow assessment of possible effects of opioids onto EK. The peak conductance value for each test potential then was normalized to gmax (maximal g for the cell) and plotted against the test potential to produce an activation curve.

The current activation and inactivation relations were fitted using the following Boltzmann equations (Hlubek and Cobbett 1997). Activation
<IT>g&cjs0823;  g</IT><SUB>max</SUB><IT>=1&cjs0823;  {1+</IT>exp[−(<IT>V</IT><IT>−</IT><IT>V</IT><SUB><IT>0.5</IT></SUB>)<IT>&cjs0823;  </IT><IT>k</IT>]<IT>}</IT> (2)
Inactivation:
<IT>I&cjs0823;  I</IT><SUB>max</SUB><IT>=1&cjs0823;  {1+</IT>exp[(<IT>V</IT><IT>−</IT><IT>V</IT><SUB><IT>0.5</IT></SUB>)<IT>&cjs0823;  </IT><IT>k</IT>]<IT>}</IT> (3)


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
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REFERENCES

Upregulation of K+ currents by the µ-receptor agonist DAMGO

To isolate voltage-activated K+ currents, slices were perfused with external solutions containing 0.5 µM TTX to block Na+ currents and 1 mM Ni2+ to block voltage-activated Ca2+ influx and subsequent activation of Ca2+-activated currents. Application of DAMGO had no significant effect on input resistance (284 ± 222 MOmega vs. 263 ± 161 MOmega in control, n = 8).

In control, depolarizing voltage steps from a prepulse potential of -110 to 0 mV evoked large transient A currents (3.78 ± 0.95 nA) superimposed on hardly inactivating delayed rectifier currents IK(V) of 3.86 ± 0.85 nA (n = 25). To separate the delayed rectifier current, we used A-current inactivating prepulses from -110 to -30 mV for 50 ms, extracellular application of the preferentially IA-blocking compound 4-AP (2-10 mM), and a combination of both. In the SON, µ-receptor agonists selectively inhibit electrical activity of oxytocinergic but not of vasopressinergic MCNs (Pumford et al. 1993). In line with this finding, the µ-agonist DAMGO (2 µM) affected K+ currents only in 55% of MCNs recorded from, this fraction of MCNs most likely being oxytocinergic cells. Figure 1A, 1 and 2, demonstrates a significant augmentation of the delayed rectifier currents in these cells during superfusion of the µ-receptor agonist DAMGO (2 µM). To reduce possible space clamp problems due to extremely large delayed rectifier currents (Müller and Lux 1993), particularly after DAMGO application, we performed all but initial experiments in the presence of 10 mM TEA. At this concentration, TEA reduced delayed rectifier currents by 40-60% (Li and Ferguson 1996). On average, these reduced currents were augmented from 1.88 to 2.85 nA (+51 ± 14.6%, n = 8, t-test: P < 0.001, test potential: 0 mV, gmax was augmented by +21 ± 5.9%). Activation of the delayed rectifier current appeared to be slightly accelerated by DAMGO (tau activation reduced from 13.0 to 11.2 ms: -14.1 ± 17.4%, n = 7, Fig. 1A3, summarized data in Fig. 7). This was observed with the inactivating prepulse as well as with the prepulse in combination with 4-AP. Because of limited recording time in the whole cell configuration (<= 30 min) in relation to pharmacokinetics in brain slices (Müller et al. 1988), we only eventually observed a slight recovery of effects with wash out of the drugs (not shown).



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Fig. 1. Effects of the µ-agonist D-Ala2, N-Me-Phe4, Gly5-ol-enkephalin (DAMGO; 2 µM) on the delayed rectifier current (IK(V)) in supraoptic magnocellular neurons (MCNs). Currents were activated in whole cell patch-clamp by voltage steps to potentials between -70 and +80 mV in increments of 10 mV from a holding potential of -80 mV and after a prepulse to -30 mV for 50 ms to inactivate the transient current IA. A, 1 and 2: current recordings in control conditions and during superfusion of DAMGO demonstrate a strong potentiation of the delayed rectifier currents by DAMGO (at -20, 0, +20, +40, and +60 mV). A3: after rescaling to the same peak amplitude an acceleration of current activation by DAMGO becomes evident (test potential = 0 mV). B and C: current-voltage and conductance-voltage relations are shifted by ~10 mV to the left by DAMGO. ---, fits to the Boltzmann relation (see METHODS).

DAMGO also had effects on the voltage dependence of activation of IK(V). Figure 1, B and C, demonstrates that DAMGO shifted the voltage dependence of activation to the left by about -10 mV (-10.1 ± 7 mV, t-test: P < 0.01, from V0.5 = 5.0 ± 3.5 mV to V0.5 = -5.1 ± 10.6 mV, k = 20.5 ± 1.7 mV and k = 22.6 ± 3.4 mV, respectively, n = 8, Fig. 1C), a shift that accounts for roughly half of the amplitude potentiation of the delayed rectifier currents at the intermediate test potential (0 mV).

To isolate the A current, we recorded the total current without inactivating prepulse and subtracted the delayed rectifier current obtained after A-current inactivation by the 50-ms prepulse. Both pulse protocols always were applied in pairs to account for changes in both currents. On average, DAMGO increased the amplitude of the A current from 2.66 to 3.62 nA (+36 ± 23.2%, P < 0.01, n = 6, complete removal of inactivation at -100 mV, test potential +60 mV, Fig. 2A, 1 and 2) and accelerated its inactivation. The time constant of inactivation was reduced from 28.1 to 23.8 ms (-16 ± 8.2%, t-test P < 0.001, n = 6, Fig. 2B). The voltage dependence of activation was not significantly affected by DAMGO (+3.3 mV right shift from V0.5 = -6.3 ± 12.1 mV, k = 13.2 ± 1.8 mV to V0.5 -3.0 ± 12.6 mV, k = 15.5 ± 2.9 mV, respectively, n = 6, Fig. 2, C and D). Although the currents recorded in the presence of 10 mM TEA showed no signs of imperfect voltage clamp, we further tested for possible space clamp problems (Müller and Lux 1993). For control purposes, we obtained recordings from acutely dissociated cells that were electrotonically rather compact. In acutely dissociated MCNs, IA amplitude was on average only 45% of IK(V) amplitude as compared with about equal amplitudes in MCNs in brain slices, supporting a significant dendritic localization of IA. The relative effects of DAMGO on amplitude and kinetics of IA and of IK(V) were of similar magnitude as in brain slices, indicating good space clamp throughout (n = 4).



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Fig. 2. Effects of DAMGO (2 µM) on IA. A currents were isolated by subtracting delayed rectifier currents (see Fig. 1) from mixed currents recorded by direct voltage steps from -80 mV to the recording potentials. A, 1 and 2: DAMGO enhances IA. B: after normalization of the peak amplitudes an acceleration of current inactivation by DAMGO becomes evident (test potential = 0 mV). C and D: voltage dependence of activation ( and ) is not significantly affected by DAMGO, whereas steady-state inactivation (open circle  and ) is increased by DAMGO through a left shift of the conductance-voltage relation of, on average, -6 mV, that becomes particularly important through the steepness of the relation. Steady-state inactivation was evaluated by 100-ms prepulses to the test potentials, followed by a step to the recording potential (0 mV). ---, fits to the Boltzmann relation.

The voltage dependence of steady-state inactivation of A currents strongly affects membrane excitability (Connor and Stevens 1971; Hoffman et al. 1997). Steady-state inactivation was tested by using 100-ms prepulses to the test potentials followed by a voltage step to 0 mV for measurement of the A current. To record pure delayed rectifier currents to isolate A currents, again an inactivating prepulse preceded the test potential. Figure 2D shows that DAMGO caused a small shift of steady-state inactivation of -5.9 ± 5.2 mV to more negative potentials (from V0.5 = -61.0 ± 4.9 mV, k = -7.3 ± 0.5 mV to V0.5 = -66.9 ± 4.6 mV, k = -8.0 ± 0.8 mV, n = 8). This shift does not contribute to the observed potentiation of A currents, but it, in contrast, can reduce currents. Because of the steepness of this relation, this shift actually results in significantly reduced currents when activated from the potentials corresponding to the steep part of the I-V relation (-50 to -60 mV, cf. Fig. 2C). In conclusion, depending on resting potential, DAMGO either increases or decreases IA.

Figure 3 shows a complete blockade of the DAMGO-evoked augmentation of IK(V) and IA in the presence of the preferentially µ- and delta -receptor antagonist naloxone (10 µM) (Vonvoigtlander et al. 1983). Surprisingly, DAMGO still affected K+ currents in the presence of naloxone probably because of activation of kappa receptors (see next section). In the presence of naloxone, DAMGO reduced both K+ currents, IK(V) from 4.17 to 3.63 nA (-13 ± 14.8%) and IA from 4.77 to 3.96 nA (-17 ± 10.7%, P < 0.05), on average (n = 3). The DAMGO-effects on tau activation of IK(V) and tau inactivation of IA as well as on the voltage dependence of activation and inactivation (see preceding text) were blocked completely by naloxone, and small, insignificant changes of opposite sign were observed (see Table 1). When naloxone was washed off in the continued presence of DAMGO, we observed the normal augmentation of both K currents. Incubation of slices with the kappa -receptor antagonist nor-BNI (5 µM), in addition to naloxone, reduced the presumably kappa -opioidergic inhibition of K currents by DAMGO by 70-90% (Fig. 3B).



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Fig. 3. Blockade of DAMGO (2 µM) action by opioid receptor antagonists. A, 1 and 2: in the presence of naloxone (10 µM), DAMGO reduces IK(V) and IA. After wash of naloxone, both currents are enhanced. B, 1 and 2: suppression of K+ currents by DAMGO in the presence of naloxone is reduced significantly by the kappa -opioid receptor antagonist nor-binaltorphimine dihydrochloride (nor-BNI; 5 µM).


                              
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Table 1. Mean values of all effects of opioid agonists and antagonists on the delayed rectifier current (IK(V)) and the A current (IA)

When DAMGO was not applied prior to naloxone, naloxone still significantly affected K+ currents. It slowed activation of delayed rectifier currents (tau  from 5.6 to 6.26 ms, +12 ± 16.6%, NS), whereas effects on the amplitude were variable (-1 ± 34%, n = 6). IA was reduced from 3.16 to 2.52 nA (-20 ± 13.9%), and inactivation was slowed (tau  from 25 to 32.4 ms, +29.7 ± 21.5%, n = 3). These effects of naloxone are most likely due to blockade of tonically activated µ-receptors in the hypothalamic slice. Unexpectedly like DAMGO, naloxone shifted steady-state inactivation of IA to the left (-7.3 ± 2.5 mV, t-test: P < 0.01, n = 3). Obviously, this effect cannot be explained by blockade of µ-receptors. Within the duration of stable recordings, effects of naloxone were not reversible during washout of the drug.

Downregulation of K+ currents by the kappa -receptor agonist U50,488

In contrast to DAMGO, U50,488 affected K+ currents in both DAMGO-sensitive and -insensitive cells. After U50,488 application, the sensitivity to DAMGO was tested during a very slow washout phase of U50,488. Alternatively, recording of U50,488 effects during washout of DAMGO, either during the slow recovery phase or when DAMGO had had no effect, gave similar effects of U50,488 (n = 4, not included in Table 1). Figure 4A shows that stimulation of kappa receptors by superfusion of U50,488 (10 µM) strongly suppressed the delayed rectifier current from 2.53 to 1.10 nA (-57 ± 14.3%, P < 0.001, n = 15, test potential +40 mV). Because this effect was indistinguishable for both types of MCNs, data were pooled (Table 1).



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Fig. 4. Effects of the kappa -agonist trans-(±)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]cyclohexyl)benzeneacetamide (U50,488, 10 µM) on the delayed rectifier current. A, 1 and 2: current recordings in control conditions and during superfusion of U50,488 demonstrate a strong suppression of the delayed rectifier currents by U50,488. B: non-kappa antagonist naloxone somewhat reduces the delayed rectifier current but does not block strong reduction of this current by U50,488. C: current activation is accelerated by U50,488, as seen after normalization of the peak amplitudes (test potential = 0 mV). D and E: current-voltage relation demonstrates voltage-dependent suppression of delayed rectifier currents (D). When the respective conductances are scaled to the same maximum, the relative conductance-voltage relation is shifted by ~20 mV to the left by U50,488 (E). ---, fits to the Boltzmann relation.

This effect of U50,488 was not inhibited by naloxone (10 µM) as expected for a µ- and delta -receptor antagonist (Fig. 4B). U50,488 considerably accelerated activation of the delayed rectifier current. This becomes particularly evident after rescaling amplitudes, as depicted in Fig. 4C. tau activation was reduced from 11.7 ± 4.7 ms to 6.9 ± 3.5 ms (-41 ± 15.8%, P < 0.001, n = 11). As shown in Fig. 4, D and E, U50,488 shifted activation of the delayed rectifier to more negative membrane potentials (-22 ± 7.7 mV, P < 0.001, from V0.5 = 4.9 ± 3.5 mV, k = 20.5 ± 1.7 mV to V0.5 -17.4 ± 12.6 mV, k = 24.0 ± 3.1 mV, n = 9). Interestingly, this shift is in the same direction as that induced by DAMGO and tends to increase fractional activation, whereas total currents and conductances were reduced by U50,488 (cf. Fig. 4, D and E). At the test potential (+40 mV) for suppression of IK(V), suppression of the total conductance is actually underestimated by some 10% because of some increase of fractional activation.

With respect to IA, U50,488 discriminated between the two types of MCNs. In DAMGO-sensitive oxytocinergic MCNs, U50,488 suppressed the transient IA from 4.60 to 3.46 nA (-24.8 ± 13.8%, P < 0.001, n = 7) and increased inactivation (tau  -19.8 ± 14.7%). In recordings of combined A current and delayed rectifier current, the suppression of the delayed rectifier often appeared to spare the initial peak contributed by IA, resulting in an apparently strong enhancement of the transient current. However, when the delayed current was subtracted, IA clearly was reduced. This is due to the strong kappa -opioidergic acceleration of activation of the delayed rectifier current that superimposes onto IA. As a control we obtained recordings from acutely dissociated cells that also were DAMGO sensitive. In these MCNs, the relative effects of U50,488 on amplitude and kinetics of IA and of IK(V) were of similar magnitude as in brain slices, indicating good space clamp throughout (n = 3).

In contrast to DAMGO-sensitive cells, U50,488 enhanced the transient IA in DAMGO-insensitive MCNs, from 3.71 to 4.55 nA (+22.6 ± 9.9%, P < 0.01, n = 4). In these cells, inactivation of IA was accelerated significantly (tau  from 36.9 to 20.9 ms, -43.8 ± 27.5%, P < 0.05, n = 4, Fig. 5, A and B).



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Fig. 5. Effects of the kappa -agonist U50,488 (10 µM) on the transient A current in a DAMGO-insensitive MCN. A, 1 and 2: U50,488 enhances IA and accelerates its inactivation. B: direct comparison after normalization of the peak amplitude demonstrates the acceleration of current inactivation by U50,488 (test potential = 0 mV). C and D: effects of U50,488 on conductance-voltage relation of activation ( and , right shift) and steady-state inactivation (open circle  and , left shift) were small and variable from cell to cell (average left shift -2.5 ± 3.1 mV and -4.6 ± 10.7 mV, respectively). ---, fits to the Boltzmann relation.

Mean effects of U50,488 on voltage dependence of steady-state inactivation and activation were variable (averages: -4.6 ± 10.7 mV, n = 4 and -2.5 ± 3.1 mV, n = 3, respectively, Fig. 5, C and D).

As expected, the effects of U50,488 were not affected by the non-kappa -receptor antagonist naloxone (10 µM) but were suppressed effectively by the kappa -receptor antagonist nor-BNI. In agreement with some reports but not others, only rather high concentrations of 1-5 µM effectively antagonized the effects of U50,488 (10 µM) (Baraban et al. 1995; Llobel and Laorden 1997). Nor-BNI antagonized the suppression of IK(V), acceleration of its activation as well as effects on A-current amplitude and kinetics (Fig. 6). The rather high nor-BNI concentration needed to antagonize the effects of U50,488 indicates a mediation by the kappa -2 receptor subtype (Schoffelmeer et al. 1997) as opposed to similar effects in a cell line that may be mediated by kappa -1 receptors (Baraban et al. 1995). The mean values of all effects of opioid agonists and antagonists are summarized in Fig. 7 and Table 1.



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Fig. 6. Blockade of U50,488 (10 µM) effects by opioid receptor antagonists. A, 1 and 2: in the presence of nor-BNI (5 µM, 1 h preincubation, kappa -opioid receptor antagonist), suppression of IK(V) by U50,488 (A1) is antagonized effectively (A2, different cell and slice, 10 µM naloxone to block µ-opioidergic effects). A3: normalization of the peak amplitude shows a blockade of acceleration of current activation by U50,488 (test potential = 0 mV, cf. Fig. 4). A4: in the presence of nor-BNI (5 µM) and naloxone (10 µM, both 1 h preincubation), suppression of IA and acceleration of inactivation by U50,488 (cf. Fig. 5) is blocked effectively.



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Fig. 7. Mean effects of DAMGO and U50,488 on current amplitudes and kinetics. Effects of DAMGO (A) and U50,488 (B and C) on the delayed rectifier current (IK(V)) amplitude and time constant of activation and on the amplitude and time constant of inactivation of the A current (IA) in putative oxytocinergic (OT; A and B) and vasopressinergic (AVP; C) MCNs.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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The present study demonstrates significant opioidergic modulation of transient (IA) and sustained (IK(V)) voltage-activated K+-outward currents in MCNs of the supraoptic nucleus. The biophysical parameters of these currents in control conditions, as repeated here, are in good agreement with published data (Hlubek and Cobbett 1997; Nagatomo et al. 1995; Widmer et al. 1997b).

K currents are important players in the electrophyisiological activity patterns that control release of oxytocin and vasopressin from MCNs. In this respect, it is important to note that these hormones are released somato-dendritically within the SON as well as from the axon terminals within the neural lobe into the systemic circulation. Within the SON, oxytocin and vasopressin act as local feedback signals that most likely contribute to the electrophysiological behavior of MCNs. Interestingly, somatodendritic release appears to be insensitive to blockade of action potentials by TTX but depends on slow depolarization (di Scala-Guenot et al. 1987).

IK(V) is available for activation at resting membrane potentials of MCNs of -50 to -60 mV (Erickson et al. 1993) and should contribute significantly to action potential repolarization (Widmer et al. 1997a) but particularly to the balance of inward and outward currents during slow burst depolarizations. Although a large fraction of IA becomes rapidly inactivated in this membrane potential range, the remaining current amplitude is still in the order of magnitude of IK(V). Action potentials and bursts in MCNs are followed by hyperpolarizing afterpotentials and slow afterhyperpolarizations mediated by Ca2+-activated K+ conductances (Bourque et al. 1985; Kirkpatrick and Bourque 1996). Both these processes remove inactivation of IA, resulting in enhanced A currents. Localized somato-dendritically, IK(V) and, depending on the temporal pattern, IA can strongly counteract somatodendritic depolarization and release of hormones and neurotransmitters within the SON. For long-lasting depolarizations, IK(V) is most likely more important for the control of somatodendritic depolarization and release than, because of rapid inactivation, IA.

Systemic release of hormones from the neural lobe appears to depend primarily on the frequency of action potentials there. Generation of high-frequency action potential activity, which is transmitted to the neural lobe, requires a lasting strong depolarization at the axon hillock in conjunction with strong spike repolarization for removing inactivation of sodium channels. Somatodendritic depolarization is supposed to affect strongly the axon hillock. IA, particularly, and initial IK(V) support spike repolarization and removal of inactivation of sodium channels. Both mechanisms are required for fast spikes and high-frequency discharge. In the axon terminals, on the other side, activation of Ca2+-channels and release of hormones increase with spike duration. In effect, potentiation of IK(V) in the somatodendritic membrane and IA in axon terminals would strongly inhibit release from the neural lobe and vice versa.

The µ-receptor agonist DAMGO potentiates the sustained delayed rectifier current as well as the transient A current, thereby exerting a strong inhibitory effect onto putative oxytocinergic cells. At synaptic terminals, spike duration controls Ca2+-influx and transmitter release. It needs to be verified, though, whether µ-opioidergic modulation of IA in axon terminals of the neural lobe is the same as in the soma. Opioidergic inhibition of glutamate release from hippocampal mossy fiber terminals has been shown to depend on IA (Simmons and Chavkin 1996). Although µ-opioidergic potentiation of IA amplitude is very likely to inhibit release of oxytocin and neurotransmitters caused by trains of action potentials, acceleration of inactivation would affect dendritic temporal integration of synaptic input. This might play a supportive role in fine tuning of oxytocin release. The overall effect of µ-receptor activation is clearly inhibitory and is likely to contribute to inhibition of basal and stimulated oxytocin release during pregnancy in addition to depression of excitatory synaptic input, a hyperpolarization through activation of G-protein-gated K+-channels (Inenaga et al. 1994), and a possible inhibition of Ca2+ channels, as observed in other brain areas (Kim et al. 1997).

The effects on IA and IK(V) appear to be mediated by µ- receptors; indeed the effects were blocked completely by the µ- and delta -opioid-receptor preferring blocker naloxone and the kappa -receptor agonist U50,488, at well-defined concentrations in vitro, evoked clearly distinct effects. There appears to be even an endogenous, tonic µ-receptor activation in slices, probably due to corelease of dynorphins from MCNs (Russell et al. 1995) as suggested by the effects of naloxone on IA and tau activation of IK(V) in slices not treated with DAMGO (cf. Table 1). The small U50,488-like, nor-BNI-sensitive effects in the presence of naloxone are most likely kappa -opioidergic side effects of DAMGO (Emmerson et al. 1994), probably via the kappa -2 receptor (see the following text).

In contrast to the µ-receptor agonist DAMGO, the selective kappa -agonist U50,488 strongly suppressed the delayed rectifier currents and at the same time strongly accelerated activation of this current in both oxytocinergic and vasopressinergic cells in the SON. In effect, flow of IK(V) during few initial milliseconds hardly is affected by U50,488 in contrast to thereafter. Suppression of IK(V) can support an increase in firing activity (Pumford et al. 1993) and in release of hormones during burst activity with prolonged strong depolarizations. Indeed, in vasopressinergic neurons the kappa -receptor antagonist Mr 2266 BS was found to inhibit stimulated release of vasopressin (Iwasaki et al. 1994), supporting an excitatory kappa -opioidergic effect.

In DAMGO-sensitive, putative oxytocinergic cells, U50,488 reduced IA while it potentiated IA in DAMGO-insensitive, putative vasopressinergic cells. Because IA probably is located in the dendrites (Widmer et al. 1997b), we cannot exclude a potentiation of IA due to reduction of IK(V) and improvement of space clamp. Inactivation of IA was accelerated in both types of MCNs. Potentiation of IA in putative vasopressinergic neurons is supposed to be inhibitory, but because of its role in removal of inactivation of sodium channels, it can have also excitatory effects. The inhibitory effects of kappa -receptor activation on release of oxytocin and vasopressin may be mediated by potentiation of IA in axon terminals in the neural lobe, depression of excitatory synaptic input, a hyperpolarization through activation of G-protein-gated K+ channels, and a possible inhibition of Ca2+ channels (Inenaga et al. 1994). The reported effects on IA would probably rather refine the kappa -opioidergic modulation of MCNs.

The effects of U50,488 on IA and IK(V) were mediated by kappa receptors because they were not blocked by naloxone but by nor-BNI, in agreement with the respective binding constants and IC50s for kappa receptors (Baraban et al. 1995; Vonvoigtlander et al. 1983). Both U50,488 and nor-BNI have been reported to have highest affinity/efficacy at the kappa -1 receptor and intermediate affinity/efficacy at the kappa -2 receptor (Clark et al. 1989; Schoffelmeer et al. 1997; Zukin et al. 1988). These intermediate affinities correspond well to the concentrations used in this in vitro study. Considering that U50,488 and nor-BNI have been found to be ineffective at the kappa -3 receptor (Brooks et al. 1996; Clark et al. 1989), all these data point toward a mediation by kappa -2 receptors.

In conclusion, voltage-dependent K+ currents are modulated in MCNs in an antagonistic manner by µ- and kappa -receptors, with kappa -receptor action being particularly suited to support high-frequency burst discharge in vasopressinergic MCNs. In this way some differential control of somatodendritic feedback and release from the neural lobe may be achieved (di Scala-Guenot et al. 1987), particularly in conjunction with modulation of IA in the neural lobe. No evidence was found for inhibition by G-protein-activated inward rectifying K+ channels (GIRK) in MCNs as opposed to other brain areas (Madison and Nicoll 1988; Williams et al. 1982).

In the brain stem, kappa - and µ-receptors are each localized on physiologically different types of neurons. In the SON of the hypothalamus, µ-receptors have been shown to be functional on oxytocinergic MCNs only (Pumford et al. 1993). It is an attractive speculation that activation of µ- and kappa -receptors on these neurons are controlled by distinct and independent presynaptic opioidergic neurons.

In late pregnancy, naloxone rapidly increases oxytocin but not vasopressin secretion, while release of both hormones is inhibited in water-deprived rats by µ agonists (Douglas et al. 1995; Hartman et al. 1986a,b; Van de Heijning et al. 1991). Stimulated vasopressin secretion has been found to be inhibited strongly by U50,488 in a nor-BNI-sensitive way in nonpregnant rats and weakly toward the end of pregnancy (Douglas et al. 1993; Wells and Forsling 1991). The µ- and kappa -opioidergic modulation of K+ currents described in this paper could contribute strongly to many of these effects on hormone release.


    ACKNOWLEDGMENTS

W. Müller and S. Hallermann contributed equally to this work. We thank S. Gabriel and A. Düerkop for excellent technical assistance and Dr. R. Klee for helpful discussions.

This work was supported by Deutsche Forschungsgemeinschaft Grant Mu 809/6-2 and a Heisenberg fellowship to W. Müller and a Sonderforschungsbereich 353 grant to D. Swandulla.


    FOOTNOTES

Address for reprint requests: W. Müller, AG Molekulare Zellphysiologie, Institut für Physiologie der Charité, Tucholskystr. 2, D-10117 Berlin, Germany.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 21 August 1998; accepted in final form 2 December 1998.


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ABSTRACT
INTRODUCTION
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DISCUSSION
REFERENCES

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