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The Journal of Neuroscience, February 1, 2003, 23(3):788
Oxytocin Regulates Neurosteroid Modulation of GABAA
Receptors in Supraoptic Nucleus around Parturition
Jan-Jurjen
Koksma1,
Ronald E.
van Kesteren2,
Thomas W.
Rosahl3,
Ruud
Zwart4,
August B.
Smit2,
Hartmut
Lüddens5, and
Arjen B.
Brussaard1
Departments of 1 Experimental Neurophysiology and
2 Molecular and Cellular Neurobiology, Vrije Universiteit
Amsterdam, 1081 HV Amsterdam, The Netherlands,
3 Neuroscience Research Centre, Merck Sharp and Dohme
Research Laboratories, Harlow, Essex, CM20 2QR, United Kingdom,
4 Research Institute of Toxicology, Utrecht University,
NL-3508 TD Utrecht, The Netherlands, and 5 Department of
Psychiatry, Clinical Research Group, University of Mainz, Mainz, 55131, Germany
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ABSTRACT |
In this study, we investigate how neurosteroid sensitivity of
GABAA receptors (GABAARs) is regulated. We
examined this issue in neurons of the supraoptic nucleus (SON) of the
rat and found that, during parturition, the GABAARs
become insensitive to the neurosteroid allopregnanolone
attributable to a shift in the balance between the activities of
endogenous Ser/Thr phosphatase and PKC. In particular, a constitutive
endogenous tone of oxytocin within the SON after parturition suppressed
neurosteroid sensitivity of GABAARs via activation of PKC.
Vice versa before parturition, during late pregnancy, application of
exogenous oxytocin brings the GABAARs from a
neurosteroid-sensitive mode toward a condition in which the receptors
are not sensitive. This indicates that there may be an inverse causal
relationship between the extent to which the GABAAR or one
of its interacting proteins is phosphorylated and the neurosteroid
sensitivity of the GABAAR. Neurosteroid sensitivity was not
affected by changes in subunit composition of GABAARs known
to occur concurrently in these cells.
Key words:
neurosteroid; GABAA receptor; supraoptic nucleus; PKC; phosphatase; oxytocin
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Introduction |
The neurosteroid allopregnanolone
[(5 -pregnan-3-ol-20-one (3-OH-DHP)] is a potent endogenous
allosteric modulator of GABAA receptors
(GABAARs) (Twyman and MacDonald, 1992 ) acting via
a specific binding domain (Turner et al., 1989; Lambert et al., 1995).
Variation in the efficacy of neurosteroids to modulate GABAARs, as observed in different types of
neurons (Harrison et al., 1987; Zhu et al., 1996; Brussaard et al.,
1997, 1999; Cooper et al., 1999), has been proposed to arise from
differences in subunit composition of the
GABAARs. Despite previous attempts to identify
the specific GABAAR subunits that mediate
neurosteroid effects, no adequate explanation for the apparent
correlation between diversity in receptor subtype and neurosteroid
sensitivity has been put forward (Belelli et al., 1996; Zhu et al.,
1996; Brussaard et al., 1997; Davies et al., 1997; Smith et al.,
1998a,b). Moreover, recent studies indicate that phosphorylation may
affect neurosteroid modulation of recombinant (Leidenheimer and
Chapell, 1997) and native (Fancsik et al., 2000)
GABAARs. The latter study suggested that
PKC-mediated phosphorylation is a prerequisite for neurosteroid
regulation by showing that, in supraoptic nucleus (SON) neurons, the
G-protein blocker GDP- -S and the PKC antagonist bisindolylmaleimide
both prevented an effect of subsequent 3-OH-DHP treatment on the
decay of IPSCs.
However, additional research is necessary because (1) the
pharmacological agents used by Fancsik et al. (2000) to manipulate phosphorylation had an effect on the IPSC decay in the absence of
neurosteroid, (2) pharmacological activation of G-protein signaling or
PKC activity did not potentiate the effect of 3-OH-DHP on GABAARs (Fancsik et al., 2000), and (3) hitherto
it remained unclear what mechanism underlies the occurrence of
neurosteroid-insensitive GABAARs in oxytocin (OT)
neurons in the SON after parturition and of
GABAARs being highly sensitive to neurosteroid
during late pregnancy (Brussaard et al., 1997). The latter issue may be
relevant because robust regulation of the endogenous PKC activity in
SON neurons is likely to occur after, but not before, parturition (Brussaard et al., 2000) attributable to increased levels of oxytocin (Lambert et al., 1994; Leng et al., 1999). In addition, the oxytocin neurons in the SON were shown to shift between a major role for 1-
during late pregnancy toward 2-containing
GABAARs after parturition (Brussaard et al.,
1997, 1999). Thus, the exact role of PKC activity, or of any other
intracellular enzyme, in conditioning the neurosteroid sensitivity of
GABAARs in the SON is still unclear.
Therefore, using the SON during the female reproduction cycle as a
model, we aimed at explaining the endogenous shift between different
modes of neurosteroid sensitivity of GABAAR
activity. We also used a transgenic 1 / knock-out mouse (Sur et
al., 2001), which excluded a prominent role for changes in
GABAAR subunit composition in the modulation of
neurosteroid sensitivity. Instead, we provide a cellular mechanism that
explains how GABAARs may switch their mode of
neurosteroid sensitivity and that is also fully consistent with all
observations on neurosteroid sensitivity of
GABAARs that have been reported previously in the
SON under natural conditions (Brussaard and Herbison, 2000).
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Materials and Methods |
Recombinant expression of GABAARs
Transfection and membrane preparation. Expression
vectors for the , , and  2L subunits from rat were transfected
in triple combination into human embryonic kidney cells (HEK 293;
American Type Culture Collection CRL 1573) as described previously
(Lüddens and Korpi, 1995). For optimal receptor expression, final
concentrations (in µg of vector DNA per 15 cm tissue culture plate)
were as follows: 5 1, 12 2, 25 2, and 0.5 2S. The 2S
variant is abbreviated 2 in the remainder of the text. Cells were
washed 40 hr after transfection with PBS, pH 7.4, at 37°C,
harvested in ice-cold PBS, and centrifuged at 500 rpm. Cell pellets
were homogenized in an Ultraturrax homogenizer for 15 sec, pelleted at
20,000 rpm, frozen at 20°C, and recentrifuged. The membrane pellets
were resuspended in 50 mM Tris-citrate buffer,
pH 7.3.
Binding assays. Resuspended cell membranes (50-200 µg of
protein per tube) were incubated in a final volume of 0.5 ml of 50 mM Tris-citrate buffer, pH 7.3, supplemented
with 0.2 M NaCl for [35S]t-butylbicyclo-phosphothionate
(TBPS) (2-6 nM) binding. GABA was diluted
from a 100 mM solution in
H2O. Nonspecific binding was determined by 20 µM picrotoxin. After 90 min at room
temperature, the assay mixtures were rapidly diluted to 5 ml with
ice-cold 10 mM Tris-HCl, pH 7.4, filtered
through glass fiber filters and washed once with 5 ml of 10 mM Tris-HCl, pH 7.4. Filters were immersed in 4 ml of Packard Ultima Gold scintillation fluid, and the radioactivity
was determined in a Beckman Instruments (Fullerton, CA) liquid
scintillation counter using external standardization. Statistical
calculations were performed using the Graph Pad Prism program (GraphPad
Software, San Diego, CA).
Receptor expression in Xenopus oocytes.
Expression, drug application, and two-electrode voltage-clamp
experiments were performed as described previously (Zwart and
Vijverberg, 1997). Rat GABAAR 1, 2, 2,
and 2L subunit cDNAs, ligated into the VMT expression vector, were
kindly provided by Dr. Paul Whiting (Merck, Sharp, and Dohme, Harlow,
UK). Plasmids coding for the , , and subunits of
GABAARs, dissolved in distilled water at a 1:1:1
molar ratio, were coinjected into the nuclei of stage VI oocytes.
Approximately 1 ng of each plasmid containing , , and cDNA
was injected in a total injection volume of 15-20 nl/oocyte.
Experiments were performed on oocytes after 2-5 d of incubation in
modified Barth's solution. Oocytes were voltage clamped at 80 mV.
Aliquots of freshly prepared, concentrated stock solutions of GABA in
distilled water and of 3-OH-DHP in dimethylsulfoxide (DMSO) were
added to the recording solution immediately before the experiments. Drug applications were alternated by 5 min of superfusion with agonist-free saline to allow the receptors to recover from desensitization.
Mouse model and quantitative PCR
Generation of 1 / mice. The
GABAAR 1 gene-targeting vector was constructed
from the genomic 129/SvEv  fixII clone, which has been used for the
introduction of the 1H101R mutation. However, for the complete gene
knock-out, exon 4 was disrupted at the MscI restriction site
by cloning the 1.2 kb BstBI+MscI and the 7 kb EcoRV+BamHI DNA fragment blunt ended into the
targeting vector (Sur et al., 2001). For this study, the F5 generation
was used.
Quantative PCRs. Primers for quantitative-PCR (Q-PCR)
analysis of various GABAAR subunit mRNAs were
designed using the Primer Express program (version 1.0; Applied
Biosystems, Foster City, CA). Total RNA was isolated from SON punches
obtained from slices of individual wild-type (WT) and 1 /
animals after the recording session, reverse transcribed into cDNA, and
used as template in a Q-PCR experiment. -Actin mRNA levels were
measured as an internal standard. The ABI 7700 Sequence Detection
System (Applied Biosystems) was used with cyber green as the reporter dye.
Whole-cell voltage-clamp recordings of SON neurons of mice
and rats
For this study, 202 neurons were recorded from the dorsomedial
SON, which contains mostly oxytocin cells (Hou-Yu et al., 1986). In
experiments involving oxytocin application, all cells responded to the
peptide. After parturition, all cells recorded under control conditions
were neurosteroid insensitive (Brussaard et al., 1997), possibly
indicative of an oxytocinergic nature. Adult female WT and 1 /
mice, as well as juvenile male (21-24 d postnatal) or adult female
Wistar rats, either after 20 d of pregnancy (P20), or on
postparturition day 1 (PPD1) were decapitated, and 400-µm-thick coronal hypothalamus slices incorporating the middle portion of the SON
were prepared using a Leica (Nussloch, Germany) vibratome slicer. The
external solution contained the following (in mM): 125 NaCl, 25 NaHCO3, 3 KCl, 1.2 NaH2PO4·H2O,
2.4 CaCl2·2H2O, 1.3 MgSO4·7H2O 1.3, and 10 D(+)-glucose (304 mOsm, carboxygenated in 5%
CO2-95% O2, pH 7.4). DNQX
and AP-5 (both at 20 µM; Research Biochemicals,
Natick, MA) were continuously present. The pipette solution contained
the following (in mM): 140.5 CsCl, 10 HEPES, 2 MgATP, and
0.1 GTP (acid free), adjusted to pH 7.2 using CsOH (296 mOsm) and no
EGTA or Ca2+ (i.e., "unbuffered"
pipette medium), unless mentioned otherwise. Spontaneous GABAergic
synaptic currents (sIPSCs) were recorded at room temperature (20°C),
unless mentioned otherwise, at a holding potential of 70 mV with an
Axopatch 200A amplifier (Axon Instruments, Foster City, CA) in the
whole-cell voltage-clamp mode. For recording criteria for selection of
cells, see Brussaard et al. (1996). Recorded sIPSCs were not sensitive
to TTX or nominal zero extracellular Ca2+,
and bicuculline (20 µM; Research Biochemicals) blocked
all remaining synaptic activity (Brussaard et al., 1996, 1999). The
sIPSCs had a decay that was best fitted with a single-exponential
function. Applications were performed via a home-made so-called Y-tube
microperfusion system (Brussaard et al., 1996). Oxytocin effects were
effectively blocked by a specific oxytocin antagonist,
d(CH2)5-OVT
(dOVT) (Bachem, Bubendorf, Switzerland). 3-OH-DHP was obtained from
Research Biochemicals, dissolved in DMSO at 10 mM, and further diluted in external solution
before application (10 µM). The lipophylicity of this substance prevents accurate estimates of the final
concentration of 3-OH-DHP at the site of action, as has been
extensively discussed previously (Brussaard et al., 1997, 1999).
Recorded cells were in the second layer of neurons below the slice
surface (~50 µm deep). The effect of 3-OH-DHP in all recordings
was determined during a 3 min period of recording starting 2 min after
neurosteroid application.
12-O-Tetradecanoylphorbol-13-acetate (TPA) (PMA is phorbol 12-myristate 13-acetate), okadaic acid, microcystin, and chelerythrine were all from Research Biochemicals; C2-ceramide (N-acetyl-d-erythro-sphingosine) was obtained from
Calbiochem (La Jolla, CA). For experiments using okadaic acid,
microcystin, and chelerythrine, which were in the pipette solution
only, 3-OH-DHP was applied after 8 min of "pretreatment"
recording. TPA and C2-ceramide were applied extracellularly 3 min after
the whole-cell configuration was established and after this 8 min
pretreatment period 3-OH-DHP was applied.
Analysis of synaptic currents
Digital detection of sIPSCs, amplitude analysis, and
curve-fitting procedures to quantify the decay rate of individual
sIPSCs have all been described previously (Brussaard et al., 1996,
1997, 1999). Per experiment and per condition, an unimodal lognormal function was fitted to histograms of binned data (either amplitude or
decay time constants) obtained from a minimum of 250 sIPSCs per
histogram. Average values thus obtained for control-pretreatment conditions and after neurosteroid application were compared using the
paired t test. Control and pretreatment conditions of
different experiments were compared using the unpaired t
test. In some experiments, nonparametric testing was performed using
the Mann-Whitney U test. In all summary figures in which
overall changes in sIPSC decay have been quantified, statistical
significance has been indicated using *p < 0.05 and
**p < 0.01. Data are reported as the mean ± SEM.
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Results |
Recombinant expression of GABAARs
To compare the sensitivity of 1- versus 2-containing
GABAARs we expressed recombinant
GABAARs in HEK 293 cells and in oocytes
In HEK 293 cells, we found that GABA at 1 µM is slightly
more potent in inhibiting the binding of
[35S]TBPS to 122L than to
222L receptors (reduction to 45 ± 7 and 70 ± 13%
of control [35S]TBPS binding,
respectively) (Fig. 1), which is in line
with previous results (Lüddens and Korpi, 1995). In addition, the potency of 3-OH-DHP to block
[35S]TBPS binding differed between the
two receptor subtypes in the absence of exogenous GABA
(Kd of 820 ± 220 and 2460 ± 160 nM respectively; p = 0.024). The latter result suggests that the affinity of 1-containing GABAARs for allosteric modulation with
3-OH-DHP is significantly higher than that of 2-containing
receptors. However, to study 3-OH-DHP modulation under more
physiological conditions, we also measured the potency of 3-OH-DHP
to block [35S]TBPS binding in the
presence of 1 µM exogenous GABA. Under these conditions, no significant difference was observed between the effects
of 3-OH-DHP on the two receptor subtypes (390 ± 110 and 610 ± 260 nM, respectively;
p = 0.4) (Fig. 1).
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Figure 1.
Neurosteroid affinity of recombinant
GABAARs containing 1 or 2 subunit. Two distinct
receptor compositions of the rat GABAAR were tested for
their apparent affinity for 3-OH-DHP using a
[35S]TBPS binding assay. 3-OH-DHP inhibited
binding of the radioactive ligand in a concentration-dependent manner.
In the absence of GABA (top 2 traces), the apparent
affinity for 3-OH-DHP was significantly higher for 1-containing
receptors. However, in the presence of GABA (1 µM;
bottom 2 traces), there was no such difference. All data
were normalized to the control [35S]TBPS binding
in the absence of GABA and 3-OH-DHP.
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To test the effects of 3-OH-DHP on GABAAR ion
channel activity, we expressed the same receptor subtypes in
Xenopus oocytes. In these tests, no difference was detected
between the two receptor subtypes in the potentiation of GABA-induced
(10 µM) whole-cell ionic currents by 100 nM 3-OH-DHP (14-fold increase in both cases; n = 4; data not shown). These data suggest that rat
GABAARs containing either the 1 or the 2
subunit are similarly sensitive to the neurosteroid allopregnanolone.
GABAARs in 1 subunit knock-out mice
To conclusively define whether in vivo the 1 subunit
does contribute to a higher affinity for neurosteroids than the 2
subunit, the effect of neurosteroids on GABAARs
needs to be studied within the context of its subcellular environment
(i.e., the postsynaptic density at the GABA synapse) (Kneussel and
Betz, 2000). To this end, we studied the effects of allopregnanolone on
GABAARs in the dorsomedial part of the SON of WT
and 1 knock-out (1 /) mice (Sur et al., 2001).
Real-time PCR analysis of 1 / versus WT SON samples showed a
complete lack of 1 subunit mRNA and no significant compensatory regulation of any of the other subunits (Fig.
2A). As a result, the
relative contribution of 2 and 3 is larger in the SON of 1
/ animals than in WT animals (Fig. 2B). Hence, in
1 / mice, the 2 and 3 subunits may have a prominent role
in GABAAR formation. In situ
whole-cell voltage-clamp recordings of dorsomedial SON neurons in adult
female mice showed that the decay time constant of sIPSCs in 1 /
mice was 35% longer than in WT mice (Fig. 2C,F). This is in line with observations
on cerebellar neurons in a different 1 / mouse (Vicini et al.,
2001). We found that both WT and 1 / mouse SON neurons express
neurosteroid-sensitive GABAARs, i.e., in both WT
and 1 / animals, 3-OH-DHP significantly increased sIPSC decay
time constants (Fig.
2D,E,G). Thus, in mouse SON neurons, lack of 1 subunit does not reduce the efficacy of allosteric modulation of GABAARs by
3-OH-DHP.
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Figure 2.
Effect of 3-OH-DHP in transgenic mice
lacking GABAAR 1 subunit. A, subunit
mRNA expression levels in the SON of 1 / animals, normalized for
the expression of -actin mRNA and plotted relative to the expression
of the WT levels (n = 4). None of the subunits in
1 / mice was significantly different from WT. B,
Relative contribution of 1 to 6 to the total subunit mRNA
expression in 1 / and WT mice. C,
Left, Average sIPSCs obtained in SON neurons from WT and
1 / mice. Traces are averages of 100 sIPSCs per
recording. The decay of sIPSCs was fitted with a single-exponential
function. Right, Histogram of the sIPSC decay time
constants of experiments shown on the left.
D, Averaged sIPSCs recorded from WT in the absence and
presence of 3-OH-DHP and decay time constant histogram showing a
significant shift to the right in the presence of 3-OH-DHP.
E, Average sIPSCs in the absence and presence of
3-OH-DHP and histogram of sIPSC decay from an 1 / recording
showing a similarly large increase of decay by 3-OH-DHP.
F, Summary graph illustrating difference in sIPSC decay
time constants between WT and 1 / mice (WT, 19.5 ± 1.1 msec, n = 14; 1 /, 26.3 ± 1.3 msec,
n = 15; p < 0.01; unpaired
t test). G, Summary graph showing
3-OH-DHP effects on the decay time constants (WT mice, significant
effect in 6 of 6 cells, Mann-Whitney U test,
p < 0.01, 256 ± 111% of control; 1 /
mice, significant effect in 5 of 5 cells, Mann-Whitney
U test, p < 0.01, 181 ± 52%
of control). In none of these conditions was the sIPSC amplitude
significantly affected (data not shown). All other traces of average
sIPSCs were plotted normalized to the control average in
C. Calibration (of the control trace), 20 msec, 40 pA.
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Neurosteroid sensitivity is dependent on signal
transduction pathways
If subunit composition of GABAARs cannot
explain the endogenous shift toward neurosteroid insensitivity in the
SON, alternatively, signal transduction pathways may play a substantial
role. Two changes in signal transduction pathways occur around
parturition that can affect GABAARs. First, in
contrast to the pregnant stage, at parturition and during lactation, a
rise in the intracellular calcium concentration may occur (Lambert et
al., 1994) as a result of endogenous oxytocin release (Neumann et al.,
1995). This may have numerous effects on postsynaptic signaling routes
targeted to GABAARs, which possibly involve
Ca2+-dependent kinases and phosphatases
(De Koninck and Mody, 1996). For instance, PKC was shown to affect
GABAAR activity in SON cells after parturition
(Brussaard et al., 2000). Second, the role of serine/threonine
(Ser/Thr) phosphatases 1, 2A, and 2B (Price and Mumby, 1999), which are
logical counterparts of PKC in regulating GABAAR
properties, may be larger during pregnancy than after parturition.
To address the first issue, we manipulated the free intracellular
calcium concentration and tested the effect on neurosteroid sensitivity. Next, given the second issue, we also manipulated endogenous Ser/Thr phosphatase activity to test a presumed
prerequisitional role of this activity for conditional neurosteroid
sensitivity of GABAARs.
The role of Ca2+ was studied in juvenile
(i.e., nonreproductive) male rats. For ethical reasons, we did not use
reproductive adult rats in these initial experiments. We titrated the
free intracellular calcium concentration,
[Ca2+ ]i, to
desired values by altering the concentrations of
EGTA/Ca2+ ratio in the pipette solution.
When recording from dorsomedial SON neurons, in the absence of
exogenous Ca2+ buffer in the pipette
(i.e., the unbuffered condition: 0 mM EGTA, 0 mM Ca2+), the average decay
time constant of sIPSCs was 18.9 ± 1.5 msec, and 3-OH-DHP
significantly increased the sIPSC decay time constant (Fig.
3C,D). Remarkably,
when [Ca2+]i was
nominal zero by adding 1 mM EGTA (0 mM Ca2+), which by
itself did not affect the decay of sIPSC, there was no effect of
3-OH-DHP on the decay time of IPSCs (Fig.
3A,D). In contrast, when
[Ca2+]i was set at
10 nM (11 mM EGTA, 1 mM Ca2+),
3-OH-DHP strongly increased sIPSC decay time constants (Fig. 3B,D). Also, in the latter
condition, the sIPSC decay before application of
3-OH-DHP was not different from control recordings in the unbuffered
recording condition. Thus, neurosteroid sensitivity of
GABAARs in SON neurons appeared to be dependent
on [Ca2+]i.
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Figure 3.
Effect of 3-OH-DHP is dependent on
[Ca2+]i in juvenile male rats
(postnatal days 21-27). A, Left,
Superimposed average sIPSCs obtained in SON neurons with
[Ca2+]i at nominal zero before and
after 3-OH-DHP application. Right, sIPSCs decay time
constant histogram of this experiment. B, Average sIPSCs
obtained in [Ca2+]i held at 10 nM in the absence and presence of 3-OH-DHP and histogram
of sIPSC decay time constants. C, Average sIPSCs
recorded using unbuffered [Ca2+]i in
the absence and presence of 3-OH-DHP and histogram of the sIPSC
decay time constants. D, Summary graph illustrating the
relative effects of 3-OH-DHP on sIPSC decay time constants at three
dissimilar Ca2+ concentrations. Decay plotted as
percentage of control values. White bars, Controls;
gray bars, 3-OH-DHP. Relative neurosteroid effects
are as follows: [Ca2+]i at nominal
zero, 108 ± 3% of control, p > 0.05, n = 5; [Ca2+]i at
10 nM, 284 ± 119% of control, p < 0.05, n = 6; unbuffered
[Ca2+]i, 220 ± 54% of
control, p < 0.05, n = 7. In
none of these conditions was the sIPSC amplitude significantly affected
(data not shown). All other traces of average sIPSCs were plotted
normalized to the control average in A. Calibration (of
the control trace), 20 msec, 100 pA.
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Next, we recorded from SON neurons in nonreproductive rats while
Ser/Thr phosphatases were blocked using okadaic acid and microcystin
(specific blockers of phosphatases 1 and 2A) in the pipette solution.
These two drugs had no effect on decay time constants by themselves.
Unlike the endogenous condition of GABAARs at
this stage (Fig. 4A),
okadaic acid (50 nM) and microcystin (100 nM) rendered GABAARs
insensitive to subsequent neurosteroid treatment (Fig.
4B,C,E). This implies
that basal levels of Ser/Thr phosphatase activity are of crucial
importance for modulation of GABAARs by
3-OH-DHP. In contrast, when PKC was blocked using the specific PKC
inhibitor chelerythrine (1 µM) in the pipette, sIPSC decay time constants were increased during neurosteroid application to the same extent as in the control condition (Fig. 4D,E). PKC inhibition did not
reduce neurosteroid sensitivity at physiological temperature (33°C)
either (data not shown; decay time 252 ± 58% of control;
n = 8).
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Figure 4.
Effect of 3-OH-DHP is dependent on Ser/Thr
phosphatase activity in juvenile male rats (postnatal days
21-27). B, C, Average sIPSCs in
the absence and presence of 3-OH-DHP and decay time constant
histogram showing that inhibition of phosphatase activity by 50 nM okadaic acid (A) or 100 nM microcystin (B) prevents the
effect of 3-OH-DHP. D, Average sIPSCs and histogram
of sIPSC decay time constants obtained in the presence of 1 µM chelerythrine showing that PKC inhibition does not
prevent the effect of 3-OH-DHP. E, Summary graph
illustrating the relative effects of 3-OH-DHP on sIPSC decay time
constants in the four experimental conditions. Decay plotted as
percentage of control values. White bars, Controls;
gray bars, 3-OH-DHP. Relative neurosteroid effects
are as follows: endogenous condition, 220 ± 54% of control,
p < 0.05, n = 7; 50 nM okadaic acid, 115 ± 4% of control,
p > 0.05, n = 7; 100 nM microcystin, 105 ± 6% of control,
p > 0.05, n = 5; 1 µM chelerythrine, 239 ± 63% of control,
p < 0.05, n = 5. Okadaic acid
(104 ± 3%; n = 7), microcystin (97 ± 2%; n = 10), and chelerythrine (99 ± 8%;
n = 5) by themselves had no effect on sIPSC decay
(unpaired t test; p > 0.05). In
none of these conditions was the sIPSC amplitude significantly affected
(data not shown). All other traces of average sIPSCs were plotted
normalized to the control average in A. Calibration (of
the control trace), 20 msec, 100 pA.
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Ser/Thr phosphatases determine neurosteroid sensitivity during
late pregnancy
Because GABAARs at the juvenile stage become
insensitive to 3-OH-DHP when endogenous Ser/Thr phosphatases were
blocked, we hypothesized that a relative shift toward more
phosphorylation would also reduce neurosteroid responsiveness of
GABAARs in the adult rat SON during parturition.
To test this, we first mimicked such a shift at P20 by inhibiting
phosphatase activity (Fig. 5).
GABAARs at P20 are very sensitive to
neurosteroids when using unbuffered pipette medium, as shown by the
196 ± 31% increase in decay time constant induced by 3-OH-DHP (Fig. 5A,D), which is in line with
previous results (Brussaard et al., 1997). This effect was completely
absent during a pretreatment with 100 nM okadaic
acid (n = 6) (Fig.
5B,D). The efficacy of this
treatment was dose dependent, i.e., 50 nM okadaic
acid reduced the effect of 3-OH-DHP in only three of six cells (data
not shown). Thus, at late pregnancy, as in juveniles, the constitutive
activity of endogenous phosphatases is a prerequisite for neurosteroid sensitivity of GABAARs.
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Figure 5.
Experimental induction of neurosteroid resistance
at late pregnancy. A, Left, Average
sIPSCs obtained in SON neurons at P20 showing a large 3-OH-DHP
effect under endogenous conditions using unbuffered pipette medium.
Right, Histogram of the sIPSC decay time constants of
experiment shown on the left. B, Average
sIPSCs in the absence and presence of 3-OH-DHP and histogram showing
that phosphatase inhibition prevents 3-OH-DHP effect on sIPSC decay.
C, Average sIPSCs in the absence and presence of
3-OH-DHP and histogram showing that PKC activation also prevents
3-OH-DHP effect on sIPSC decay. D, Summary graph
illustrating the relative effects of 3-OH-DHP on sIPSC decay time
constants under the endogenous condition (to 196 ± 31% of
control; p < 0.01; n = 6),
after phosphatase inhibition (107 ± 2%; p > 0.05; n = 6), and after PKC activation (108 ± 6% compared with during TPA pretreatment; p > 0.05; n = 7). TPA application did not affect sIPSC
decay itself (104 ± 3% of control; p > 0.05; n = 6). However, it did significantly
suppress the average sIPSC amplitude to 65 ± 9% of control
values (p < 0.05; n = 6), which corresponds to previous findings in this cell system
(Brussaard et al., 2000). sIPSC decay was not different in okadaic acid
(n = 6) compared with in experiments without the
drug (n = 22; 108 ± 2%; unpaired
t test; p > 0.05). All other traces
of average sIPSCs were plotted normalized to the control average in
A. Calibration (of the control trace), 20 msec, 100 pA.
|
|
Subsequently, we tested whether, at P20, 3-OH-DHP insensitivity of
GABAARs could also be induced by boosting
endogenous PKC activity instead of by suppressing phosphatase activity.
To this end, we used the phorbol ester TPA, which was applied to the
outside of the cell 3 min after the whole-cell configuration was
established. TPA (25 nM) by itself induced a reduction of
the average sIPSC amplitude to 65 ± 9% of control, in line with
previous findings (Brussaard et al., 2000), but had no effect on the
sIPSC decay rate. More importantly, 3-OH-DHP no longer affected
sIPSC decay time constants after 8 min of TPA pretreatment, showing
also that an increase in endogenous phosphorylation activity renders
GABAARs insensitive to neurosteroids (Fig.
5C,D).
Neurosteroid-insensitive mode of GABAARs
during parturition
Our next step was to test whether, after parturition, stimulating
dephosphorylation would restore 3-OH-DHP sensitivity of GABAARs, to show that the determinant mechanism
for neurosteroid effects on GABAARs is not
endogenous shifts in subunit composition but rather a conditional
alteration in signal transduction pathways. At PPD1, the decay of the
sIPSCs was 24.6 ± 1.1 msec (n = 16) compared with
19.8 ± 0.8 msec (n = 22) at P20
(p < 0.01; unpaired t test with
Welch correction). This is in line with previous work and is explained
by the idea that at parturition a subunit switch from 1 to 2
occurs (Brussaard et al., 1997).
Treatment with 3-OH-DHP had no effect on sIPSC decay at PPD1 (Fig.
6A,E)
(in line with Brussaard et al., 1997). To test whether we could restore
neurosteroid sensitivity, we first stimulated endogenous Ser/Thr
phosphatases in SON neurons with C2-ceramide (10 µM), an activator of phosphatases 2A and 1 (Yang, 2000). Before 3-OH-DHP treatment, we applied C2-ceramide for
8 min to allow dephosphorylation to take place at GABA synapses. There
was no effect of C2-ceramide on IPSC decay. However, 3-OH-DHP did
potentiate GABA current decay after C2-ceramide pretreatment, as shown
by an increase to 190 ± 45% of control values (Fig.
6B,E). Thus, by activation of
endogenous phosphatases in the SON after parturition, the
GABAAR responsiveness to 3-OH-DHP can be
restored.
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Figure 6.
Experimental rescue of neurosteroid
sensitivity after parturition. A, Left,
Average sIPSCs obtained in SON neurons at PPD1 showing no effect of
3-OH-DHP under endogenous conditions. Right,
Histogram of the sIPSC decay time constants of this experiment.
Filled circles are used for controls, and open
circles are for 3-OH-DHP. B, Average sIPSCs
in the absence and presence of 3-OH-DHP and decay time constant
histogram showing shift to the right with 3-OH-DHP after activation
of phosphatases by C2-ceramide. C, Average sIPSCs in the
absence and presence of 3-OH-DHP and histogram showing that PKC
inactivation by chelerythrine rescues 3-OH-DHP effect.
D, Recordings in 1 / mice as a model for the
postpartum stage in rats. Average sIPSCs and histogram from experiment
with okadaic acid showing that 3-OH-DHP sensitivity (compare with
Fig. 2E) is dependent on dephosphorylation. E, Summary
graph illustrating the relative effects of 3-OH-DHP on IPSC decay
time constants under the endogenous condition (109 ± 6% of
control; p > 0.05; n = 7),
after activation of phosphatases (190 ± 45% compared with during
C2-ceramide pretreatment; p < 0.05;
n = 9), and after PKC inhibition (207 ± 40%
of control; p < 0.05; n = 7).
In 1 / mice neurons treated with okadaic acid, the neurosteroid
effect on average was 103 ± 4% of controls
(p > 0.05; in 4 of 4 cells). We observed no
significant effect on the sIPSC decay of C2-ceramide (104 ± 2%
of control; p > 0.05; n = 9).
The sIPSC amplitude was unaffected by chelerythrine [275 ± 36 pA
endogenous condition (n = 21), 320 ± 64 pA in
chelerythrine (n = 7); p > 0.05]. The latter indicates that the constitutive release of OT within
the SON in not high enough to induce a suppression of the sIPSC
amplitude as was previously reported to occur with high
concentrations of synthetic OT (Brussaard et al., 1996). sIPSC
decay was not different in chelerythrine (n = 7)
compared with in experiments without the drug (n = 16) (unpaired t test; p > 0.05).
All sIPSC traces were normalized to the control amplitude level. In
none of these conditions was the sIPSC amplitude significantly affected
(data not shown). All other traces of average sIPSCs were plotted
normalized to the control average in A. Calibration (of
the control trace), 20 msec, 100 pA.
|
|
Next, we tested whether, at PPD1, PKC inhibition would also rescue
neurosteroid sensitivity. Chelerythrine (1 µM), did not affect sIPSC amplitude or decay but restored
GABAAR responsiveness to 3-OH-DHP (Fig.
6C,E), as shown by the 207 ± 40% increase
in decay time constant. This implied that, when PKC is blocked, basal endogenous phosphatase activity suffices to rescue 3-OH-DHP
sensitivity. This indicated that, under physiological conditions, an
increased level of phosphorylation activity is the causal factor to
determine 3-OH-DHP insensitivity in the SON during parturition.
Subsequently, we went back to record from 1 / mice SON neurons.
These mice have a putative receptor subunit composition in the SON
(Fig. 2B) that can be compared with that of rats at the postpartum state (Brussaard et al., 1997). However, they do not
show neurosteroid resistance and are in fact very sensitive to
3-OH-DHP (Fig. 2E,G). We argued
that, as in rats at the juvenile or adult pregnant stage, also in 1
/ mice a relatively higher level of endogenous dephosphorylation
activity may be responsible for the neurosteroid sensitivity. Hence, we
recorded SON neurons in 1 / mice while blocking Ser/Thr
phosphatases with okadaic acid. This treatment induced
GABAAR insensitivity to 3-OH-DHP (Fig.
6D,E). This conclusively showed
that, regardless of the GABAAR subunit
composition, i.e., with or without 1, it is the balance between
phosphorylation and dephosphorylation that determines their sensitivity
to neurosteroid.
Oxytocin causes neurosteroid-insensitive state
of GABAAR
To find the physiological trigger that caused the shift in the
balance between phosphatase and PKC activity at parturition, we tested
whether the neurosteroid-resistant mode of
GABAARs of oxytocin neurons in the SON is brought
about by activation of oxytocin autoreceptors. We applied oxytocin (5 µM) at P20 and the peptidergic receptor antagonist dOVT
(4 µM) at PPD1 during an 8 min pretreatment period.
Application of OT in experiments on late pregnant rats by itself
decreased sIPSC amplitudes as expected (Brussaard et al., 1996, 2000)
but had no significant effect on sIPSC decay (data not shown). However,
after oxytocin pretreatment, the normally observed effect
of 3-OH-DHP on sIPSCs was prevented (Fig.
7A,C).
Application of receptor antagonist dOVT during sIPSC recordings in PPD1
by itself had no significant effects on either sIPSC amplitude or decay
(Fig. 7 legend). Subsequent application of 3-OH-DHP increased decay
time constants (Fig. 7B), which is in sharp contrast to the
endogenous condition of the GABAAR neurosteroid
sensitivity at this stage (Fig. 7C). This implied that
constitutive OT release within the SON (Leng et al., 1999) after
parturition prevents 3-OH-DHP potentiation of GABA currents via
activation of the oxytocin receptor. This effect occurred already at
basal extracellular concentrations of OT after parturition and was
therefore independent of sIPSC amplitude reduction observed only at
higher concentrations of OT (Brussaard et al., 1996).
View larger version (30K):
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Figure 7.
Oxytocin autoregulation renders
GABAARs insensitive to neurosteroid. A,
Left, Superimposed average sIPSCs before and after
3-OH-DHP application obtained in SON neurons at P20 pretreated with
OT. Right, Decay time constant histogram of this
experiment. B, Average sIPSCs in the absence and
presence of 3-OH-DHP and decay time constant histogram showing large
3-OH-DHP effect at PPD1 after block of oxytocin autoreceptors.
C, Summary graph illustrating the dependence of the
neurosteroid effect on oxytocin receptor activity. Decay plotted as
percentage of controls. White bars, Control; gray
bars, 3-OH-DHP. Notice the differences with the endogenous
condition at both stages. After OT pretreatment, 3-OH-DHP did not
affect sIPSC decay time constants (104 ± 5% compared with during
OT pretreatment; p > 0.05; n = 6). OT by itself had a small but nonsignificant effect on sIPSC decay
time constants (114 ± 7% of control; p > 0.05; n = 8). As expected, OT decreased sIPSC
amplitudes (68 ± 13% of control; p < 0.05;
n = 8). At PPD1, 3-OH-DHP increased sIPSC
decay time constants after OT antagonist (224 ± 26% compared
with during OT antagonist pretreatment; p < 0.05;
n = 5). OT antagonist by itself had no effect on
sIPSC decay (109 ± 5% of control; p > 0.05;
n = 5). OT antagonist had no effect on sIPSC
amplitude (96 ± 19%; n = 5). All other
traces of average sIPSCs were plotted normalized to the control average
in A. Calibration (of the control trace), 20 msec, 100 pA.
|
|
| |
Discussion |
We propose that the naturally occurring reduction in neurosteroid
sensitivity of the GABAAR in SON neurons after
parturition is brought about by increased levels of phosphorylation and
not, as we proposed previously (Brussaard and Herbison, 2000), by
subunit composition of the GABAAR. Our data
indicate that the extent of oxytocin receptor activation is the main
determinant of PKC activity and GABAAR
sensitivity to allopregnanolone in the SON. In lactating females that
normally have high oxytocin levels and allopregnanolone-resistant GABAARs, we were able to restore neurosteroid
sensitivity by pretreating the SON with an oxytocin receptor
antagonist. Conversely, before parturition, when oxytocin levels in the
SON are low (Leng et al., 1999) and GABAARs are
sensitive to neurosteroid (Brussaard et al., 1997), application of
oxytocin rendered GABAARs completely insensitive
to allopregnanolone within minutes. These results are fully consistent
with the functional significance of GABAAR plasticity in the oxytocin neuron (Brussaard and Herbison,
2000).
Neurosteroid sensitivity of GABAARs in the SON does not
depend on subunit composition
Previously, it was reported that 1-containing
GABAARs are sixfold to 10-fold more sensitive to
allopregnanolone than 2-containing receptors in oocytes (Belelli et
al., 1996). However, neurosteroids do bind with high specificity to the
2 subunit (Rick et al., 1998). Similarly, inclusion of 6 appeared
to increase neurosteroid sensitivity (Hauser et al., 1995), whereas the
4 subunit is associated with a decrease in both neurosteroid and
benzodiazepine sensitivity (Smith et al., 1998a,b). In addition, the
reduced sensitivity of native GABAARs to
neurosteroids during neonatal development of granule neurons in culture
was partly attributed to a increase of  subunit expression (Zhu et
al., 1996). Also, our group has put forward the idea of subunit
composition dependence of GABAAR neurosteroid
sensitivity (Brussaard and Herbison, 2000). Nonetheless, at this
moment, there is no unifying theory of the involvement of
GABAAR subunit composition to explain the
physiological regulation of allosteric receptor modulation by
neurosteroids. Here we exclude a role for 1-2 subunit switching,
which is known to occur in the SON after parturition and which
correlates with a transient decrease in receptor neurosteroid
sensitivity (Brussaard et al., 1999). Our measurements in 1
knock-out animals show that receptor subunit switching of native
receptors is very unlikely to be the direct cause of a reduction in
neurosteroid sensitivity of the GABAAR in SON neurons.
Phosphorylation events determine neurosteroid sensitivity of
GABAARs in the SON
Most likely the sensitivity of GABAARs to
allopregnanolone in SON neurons during late pregnancy is brought about
by a constitutive high level of endogenous phosphatase activity. Vice
versa, after parturition, when GABAARs are
insensitive to neurosteroids, inhibition of PKC or stimulation of
endogenous phosphatases restored allopregnanolone sensitivity.
Moreover, a constitutive level of oxytocin appears to be maintained
under basal conditions at this stage, because blocking putative
oxytocin effects with an antagonist readily brought about the normal
sensitivity of GABAARs to allopregnanolone.
This implies that phosphatases and PKC may have converging effects,
possibly acting on the same Ser/Thr residue of the 2, 3, or 2
GABAAR subunit (McDonald and Moss, 1997).
Alternatively, PKC and phosphatases may act on different
phosphorylation sites or even on different proteins of the postsynaptic
density of the GABA synapse (Kneussel and Betz, 2000), including the
GABAAR-interacting protein RACK-1 (receptor for
activated C kinase-1) (Brandon et al., 1999; Feng et al., 2001).
In addition, phosphatase-targeting proteins like spinophilin
(Hsieh-Wilson et al., 1999) or the scaffold protein AKAP79 (A kinase
anchor protein 79) that binds both PKC and phosphatase 2B
(Klauck et al., 1996) may be involved in the local regulation of
phosphorylation cascades.
The putative role of the above proteins in or near the postsynaptic
density may explain the discrepancy between the regulation of
allopregnanolone sensitivity described in native synapses and of
GABAARs expressed in oocytes (Leidenheimer and
Chapell, 1997). In addition, in oocyte experiments, the effect of
neurosteroids is on slow desensitization of
GABAAR activity rather than on ion channel
mechanisms involved in the decay of synaptic currents, i.e., receptor
deactivation and fast desensitization (Jones and Westbrook,
1995). Because fast desensitization of the postsynaptic GABAAR channels can keep the receptor protein in
an agonist-bound state, thereby slowing the rate of final ion channel
closure during the synaptic decay, it is very likely that neurosteroids
slow the recovery of the GABAAR from the
desensitized state and in this way prolong synaptic current decays (Zhu
and Vicini, 1997). Hence, we cannot exclude that distinct neurosteroid
effects on GABAARs do occur that are different in
their sensitivity to endogenous shifts in the balance of phosphorylation.
In addition to a putative phosphorylation-dependent regulation of the
effect of an allosteric modulator, direct effects of phosphorylation of
residues of and subunit on desensitization properties of
GABAAR channels have been reported previously
(Krishek et al., 1994). Decay time constants of sIPSCs may be affected by alterations in protein kinase or phosphatase activity, such as
reported for hippocampal neurons (Jones and Westbrook, 1997; Poisbeau
et al., 1999).
Specificity of experimental interference with balance of PKC and
phosphatase activity
It was proposed previously that PKC activation rather than PKC
inhibition is required for allopregnanolone potentiation of the
synaptic currents in the juvenile SON (Fancsik et al., 2000). However,
we would argue that these previous experiments depended to some extent
on the specificity of one PKC inhibitor, bisindolylmaleimide. This
substance enhanced sIPSC decay already in the absence of neurosteroid
(132.5% of control) (Fancsik et al., 2000), complicating the
interpretation of the effect of subsequent neurosteroid treatment. We
also observed that bisindolylmaleimide at a concentration of 500 nM, in the absence of neurosteroid, significantly
potentiates the sIPSC decay (data not shown).
Instead, we used chelerythrine (1 µM), which did not
affect the sIPSC decay in SON neurons by itself. In the
neurosteroid-resistant condition after parturition, PKC inhibition by
chelerythrine clearly shifted the GABAARs toward
the neurosteroid-sensitive mode (Fig. 6). In contrast, in
neurosteroid-sensitive nonreproductive animals, chelerythrine did not
reduce neurosteroid effects, at neither room temperature (Fig. 4) nor
33°C (Fig. 4 legend).
To further substantiate our findings that PKC inhibition, rather than
activation, renders GABAARs in the SON sensitive
to allopregnanolone, we also performed the reverse experiment and blocked endogenous phosphatase activity in both juvenile and late pregnant rats. Again, we first tested the specificity of the
phosphatase inhibitors. Both okadaic acid (50-100 nM) and
microcystin (100 nM) did not affect the sIPSC decay in the
absence of neurosteroid, whereas at high concentrations such blockers
might affect GABA currents in other brain regions (Nusser et al.,
2001). Subsequently, as expected from the PKC inhibition results, we
found that, before parturition, both inhibitors shifted the
neurosteroid-sensitive GABAARs toward the
neurosteroid-insensitive mode (Figs. 4, 5). Moreover, activation
of endogenous phosphatase activity after parturition, using
C2-ceramide, rescued neurosteroid sensitivity of
GABAARs after parturition (Fig. 6).
A model of regulation of GABAAR activity before and
after parturition
During pregnancy, allopregnanolone levels are high (Concas et al.,
1998), there is little oxytocin secretion (Leng et al., 1999), and
basal oxytocin levels are low attributable to significant breakdown by
aminopeptidases (Kombian et al., 1997). GABAARs
at that stage are occupied by endogenous allopregnanolone, which results in a slower decay of synaptic current responses (Brussaard et
al., 1997), and prevents putative PKC modulation (Brussaard et al.,
2000). Intracellular [Ca2+] under these
conditions is ~43 nM (Lambert et al., 1994), which apparently favors endogenous phosphatase activity over PKC activity (this report). Thus, all conditions in the SON during pregnancy are
aimed at keeping oxytocin neurons silent.
Before parturition, allopregnanolone levels abruptly drop (Concas et
al., 1998), and the somatodendritic release of oxytocin goes up
dramatically. Oxytocin receptor activation then results in increased
PKC activity attributable to activation of the phospholipase C-IP3-DAG pathway, and release from allopregnanolone modulation will
allow GABAARs to be influenced by this PKC
activity (Brussaard et al., 2000). Moreover, an additional increase in
[Ca2+] may be expected from the increase
in firing rate and the concomitant transient calcium flux via
voltage-gated calcium channels. Thus, altered conditions around
parturition allow the SON system to be relieved from inhibition by at
least two complementary cellular mechanisms: (1) reduced allosteric
GABAAR potentiation attributable to removal of
endogenous allopregnanolone, and (2) reduced
GABAAR sensitivity toward allopregnanolone
attributable to increased phosphorylation at the postsynaptic density.
Physiological significance of allosteric
GABAAR modulation
Although the important functions of allopregnanolone become
particularly clear during the female reproduction cycle, levels of
allopregnanolone are also known to rise transiently in relation to
acute stress (Purdy et al., 1991). Neurosteroid modulation of
GABAARs has been associated with phenomena such
as memory formation, anxiety, sleep, depression, seizure-like activity
(Majewska, 1992), postpartum blues (Nappi et al., 2001), and mood
swings during the menstrual cycle (Smith et al., 1998a,b). Thus,
studying the molecular mechanism underlying regulation of neurosteroid
sensitivity of postsynaptic GABAARs is important
and may lead to the further development of neurosteroid-related
compounds for use as improved anxiolytic drugs with a reduced risk of
tolerance as an alternative to benzodiazepines (Rupprecht and Holsboer,
1999). In addition, GABAAR susceptibility to
ethanol is altered in transgenic mice lacking particular PKC isomers
(Hodge et al., 1999). Hence, metabotropic control over allosteric
modulation of GABAARs may be a general phenomenon
that regulates the efficacy of GABA synapses throughout the brain.
| |
FOOTNOTES |
Received June 17, 2002; revised Oct. 24, 2002; accepted Oct. 25, 2002.
We thank N. Burnashev, W. P. Geraerts (Vrije Universiteit
Amsterdam, Amsterdam, The Netherlands), H. D. Mansvelder (Columbia University, New York, NY), K. Wafford, R. McKernan, and P. Whiting (Merck Sharp & Dohme, Harlow, UK) for their comments on previous versions of this manuscript, and J. C. Lodder and T. Buse for technical support. This research was supported by Nederlandse Organisatie voor Wetenschappelijk Onderzoek-Aard en
Levenswetenschappen Grant 809.67.011.
Correspondence should be addressed to Arjen Brussaard at the above
address. E-mail: brssrd{at}bio.vu.nl.
R. Zwart's present address: Eli Lilly and Company, Lilly Research
Centre, Erl Wood Manor, Sunninghill Road, Windlesham, Surrey, GU20 6PH, UK.
| |
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TOP
ABSTRACT
Introduction
