1Institut für Experimentelle und
Klinische Pharmakologie und Toxikologie,
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 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 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 In the present study, we address direct postsynaptic opioidergic
modulation of K+ currents by µ- and 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 To reduce space clamp errors, MCNs were acutely isolated from brain
slice sections encompassing the supraoptic nucleus (Lambert et
al. 1994 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 M Chemicals
Opioids and antagonists were dissolved in distilled water and
stored in aliquots (10 mM) at 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
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
6 mV to more
negative potentials. All DAMGO effects were blocked by the preferential
non-
-opioid antagonist naloxone (10 µM). The
-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
of inactivation by
20% in DAMGO-sensitive cells. In
contrast, in DAMGO-insensitive cells U50,488 increased
IA by ~23% and strongly accelerated
inactivation (
44%). The effects of U50,488 were suppressed by
the selective
-receptor antagonist nor-binaltorphimine (5 µM). We
conclude that µ- and
-opioidergic inputs decrease and increase
excitability of oxytocinergic MCNs, respectively, through modulation of
voltage-dependent K+ currents. In vasopressinergic MCNs,
-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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
).
). Most of these peptides exert their effects by activation of both, µ- and
-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
-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
).
; 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
).
-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
).
METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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.
). 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.
. 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) M
(n = 38). This value dropped
to 263 ± 161 M
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.
20°C. Fresh dilutions were made with
standard external solution on the day of the experiment.
where EK is the equilibrium potential
(
(1)
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
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(2) |
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(3) |
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RESULTS |
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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 M vs.
263 ± 161 M
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 (
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).
|
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).
|
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 -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
activation of IK(V) and
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
-receptor antagonist
nor-BNI (5 µM), in addition to naloxone, reduced the presumably
-opioidergic inhibition of K currents by DAMGO by 70-90% (Fig.
3B).
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When DAMGO was not applied prior to naloxone, naloxone still
significantly affected K+ currents. It slowed activation of
delayed rectifier currents ( 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
(
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 -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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This effect of U50,488 was not inhibited by naloxone (10 µM) as
expected for a µ- and -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.
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 (
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
-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 ( 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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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--receptor antagonist naloxone (10 µM) but were suppressed effectively by the
-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
-2 receptor subtype (Schoffelmeer et al. 1997
) as
opposed to similar effects in a cell line that may be mediated by
-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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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 -opioid-receptor preferring blocker naloxone and the
-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
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
-opioidergic side effects of DAMGO (Emmerson et al.
1994
), probably via the
-2 receptor (see the following text).
In contrast to the µ-receptor agonist DAMGO, the selective
-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
-receptor antagonist Mr 2266 BS was
found to inhibit stimulated release of vasopressin (Iwasaki et
al. 1994
), supporting an excitatory
-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
-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
-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
-1 receptor and intermediate
affinity/efficacy at the
-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
-3 receptor
(Brooks et al. 1996
; Clark et al. 1989
),
all these data point toward a mediation by
-2 receptors.
In conclusion, voltage-dependent K+ currents are modulated
in MCNs in an antagonistic manner by µ- and -receptors, with
-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, - 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
-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
-opioidergic modulation of
K+ currents described in this paper could contribute
strongly to many of these effects on hormone release.
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ACKNOWLEDGMENTS |
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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.
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FOOTNOTES |
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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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REFERENCES |
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