1Netherlands Institute for Brain Research, 1105 AZ Amsterdam, The Netherlands; 2Neurosciences, Loeb Research Institute, Ottawa Civic Hospital and University of Ottawa, Ottawa, Ontario K1Y 4E9, Canada; and 3Department of Anatomy and Embryology, Academic Medical Centre, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands
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ABSTRACT |
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Hermes, M.L.H.J.,
J. M. Ruijter,
A. Klop,
R. M. Buijs, and
L. P. Renaud.
Vasopressin Increases GABAergic Inhibition of Rat Hypothalamic
Paraventricular Nucleus Neurons In Vitro.
J. Neurophysiol. 83: 705-711, 2000.
This investigation
used an in vitro hypothalamic brain slice preparation and whole cell
and perforated-patch recording to examine the response of magnocellular
neurons in hypothalamic paraventricular nucleus (PVN) to bath
applications of vasopressin (VP; 100-500 nM). In 22/38 cells,
responses were characterized by an increase in the frequency of
bicuculline-sensitive inhibitory postsynaptic potentials or currents
with no detectable influence on excitatory postsynaptic events.
Perforated-patch recordings confirmed that VP did not have an effect on
intrinsic membrane properties of magnocellular PVN neurons
(n = 17). Analysis of intrinsic membrane properties
obtained with perforated-patch recording (n = 23)
demonstrated that all of nine VP-sensitive neurons showed a rebound
depolarization after transient membrane hyperpolarization from rest. By
contrast, 12/14 nonresponding neurons displayed a delayed return to
resting membrane potentials. Recordings of reversed inhibitory
postsynaptic currents with chloride-loaded electrodes showed that
responses to VP persisted in media containing glutamate receptor
antagonists but were abolished in the presence of tetrodotoxin. In
addition, responses were mimicked by vasotocin [Phe2,
Orn8], a selective V1a receptor agonist, and
blocked by
[-Mercapto-
,
-cyclopentamethylenepropionyl1,O-Me-Tyr2,
Arg8]-VP (Manning compound), a V1a/OT receptor
antagonist. Neither [deamino-Cys1,Val4,D-Arg8]-VP,
a selective V2 receptor agonist, nor oxytocin were
effective. Collectively, the results imply that VP acts at
V1a receptors to excite GABAergic neurons that are
presynaptic to a population of magnocellular PVN neurons the identity
of which features a unique rebound depolarization. Endogenous sources
of VP may be VP-synthesizing neurons in suprachiasmatic nucleus, known
to project toward the perinuclear regions of PVN, and/or the
magnocellular neurons within PVN.
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INTRODUCTION |
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Magnocellular neurons of the hypothalamic
supraoptic (SON) and paraventricular nuclei (PVN) secrete the
peptidergic hormones vasopressin (VP) and oxytocin (OT) into the
bloodstream from axon terminals in the neurohypophysis
(Brownstein et al. 1980). The magnitude of this
neurosecretory process can be correlated with both the frequency and
the pattern of action potentials generated at the cell somata, features
that are dependent on intrinsic membrane properties and synaptic input
(Armstrong 1995
; Poulain and Wakerley 1982
; Renaud and Bourque 1991
). Interestingly,
the neurohypophyseal peptides are believed to regulate their own
secretion by acting as neurotransmitters on magnocellular neurons
either following synaptic release from magnocellular axon collaterals
or nonsynaptically from somatodendritic regions of the magnocellular
cells (Landgraf 1995
; Ludwig 1998
;
Morris et al. 1993
).
Data from in vitro studies support the notion that OT enhances the
excitability of putative OT-synthesizing magnocellular cells
(Inenaga and Yamashita 1986). Although some of these
observations may represent a direct effect of the peptide
(Yamashita et al. 1987
), OT also has been noted to alter
cell excitability through a reduction of GABAergic inhibition
(Brussaard et al. 1996
) and by an influence on
excitatory afferents (Kombian et al. 1997
). In vivo, OT
appears to facilitate bursting activity in OT-synthesizing neurons
during suckling (Freund-Mercier and Richard 1984
). By contrast, reports of the effects of VP on VP-synthesizing magnocellular cells vary with data from extracellular studies in vivo or in vitro
implying facilitatory, depressant, or no influence on cell excitability
(Abe et al. 1983
; Carette and Poulain
1989
; Inenaga and Yamashita 1986
; Ludwig
and Leng 1997
). However, a recent analysis suggests that these
combined actions of VP augment its hormonal release by promoting the
expression of phasic firing among putative VP-synthesizing neurons
(Gouzènes et al. 1998
).
To investigate the membrane actions of VP, we obtained whole cell and
perforated-patch recordings from magnocellular PVN neurons in
hypothalamic slice preparations. We here report that a subset of the
magnocellular cells demonstrates an increase in bicuculline-sensitive inhibitory postsynaptic events in response to bath applications of VP,
mediated by V1a-type VP receptors presumably
located on GABAergic neurons presynaptic to the PVN target cells. These
effects were selective for neurons that displayed a rebound
depolarization after transient membrane hyperpolarization, reminiscent
of a feature seen in identified OT-synthesizing neurons in SON
(Stern and Armstrong 1997). A portion of these
observations has been reported briefly (Hermes et al.
1996b
).
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METHODS |
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Slice preparation
Hypothalamic slices were prepared from male Long-Evans rats
30-70 days of age. In accordance with national guidelines, animals were decapitated without anesthesia to minimize unknown persisting actions of anesthetics on neural tissue. Brains were removed rapidly from the cranial cavity, cooled in ice-cold oxygenated (95%
O2-5% CO2) artificial
cerebrospinal fluid (ACSF), and sectioned with a vibratome. Slices
(coronal plane, 400-500 µm thickness) were maintained for 1 h in
oxygenated ACSF at room temperature (20-22°C) or at 36°C before recording.
Solutions and drugs
ACSF contained (in mM) 119 NaCl, 3.2 KCl, 2.4 CaCl2, 1.3 MgCl2, 26.2 NaHCO3, 1 NaH2PO4, and 10 glucose and
had an osmolality of 295-300 mOsm/kg, and a pH of 7.35-7.40. The
patch pipette contained a gluconate-based recording solution of the
following composition (in mM): 140 potassium gluconate, 10 KCl, 10 HEPES, and 1 EGTA. With these solutions, the chloride reversal
potential was 68.0 mV (at 35°C), approximating the values measured
for spontaneous or evoked bicuculline-sensitive inhibitory postsynaptic
potentials (IPSPs) recorded in SON or PVN magnocellular neurons using
potassium acetate-containing sharp electrodes (Hermes et al.
1996a
; Renaud and Bourque 1991
). For
voltage-clamp recording of reversed inhibitory postsynaptic currents
(IPSCs), the recording solution contained (in mM) 100 KCl, 35 potassium
gluconate, 10 HEPES, 1 EGTA, and 2 lidocaine N-ethyl bromide
(QX-314). Without ATP in the recording solution no rundown of
reversed IPSCs was observed for recording periods exceeding 2 h
(cf. Staley and Mody 1992
). The recording solutions had
an osmolality of 285-300 mOsm/kg and a pH of 7.30-7.40. For
perforated-patch recording, both amphotericin B (250 µg/ml) and
gramicidin (5 µg/ml) were added to the gluconate-based recording solution (Kyrozis and Reichling 1995
; Rae et al.
1991
). In some cases, only gramicidin (2 µg/ml), which forms
pores in the plasma membrane that are not permeable to chloride, was
added for an additional verification of the hyperpolarizing direction
of the bicuculline-sensitive IPSPs (Hermes et al. 1996a
;
Renaud and Bourque 1991
).
Drugs used in the study included bicuculline methochloride (BMC),
D()-2-amino-5-phosphonopentanoic acid
(D-APV),
D-(2-carboxypiperazine-4-yl)propyl-1-phosphonic acid
(D-CPP), and
6-nitro-7-sulfamoylbenzo(f)-quinoxaline-2,3-dione (NBQX)
from Tocris Cookson, Bristol, UK; bicuculline methiodide (BMI), VP, OT,
and
[
-Mercapto-
,
-cyclopentamethylenepropionyl1,O-Me-Tyr2,Arg8]-VP
or d(CH2)5[Tyr(Me)2]AVP (Manning
compound) from Sigma;
[deamino-Cys1,Val4,D-Arg8]-VP
(dVDAVP) from Bachem, Switzerland; vasotocin [Phe2,
Orn8] (PO-VT) from American Peptide, Sunnyvale, CA; and
tetrodotoxin (TTX) from Research Biochemicals Int., Natick, MA. Some VP
analogs were kindly provided by Dr. M. Manning (Toledo, Ohio).
Drugs were applied from reservoirs connected to the ACSF perfusion line by manually operable three-way valves. For agonists, mentioned concentrations were those in the reservoirs, although radio-immunoassay measurements revealed that the levels in the recording chamber rose to maximally 50% of these values at the end of 1-min applications. Antagonists were bath-applied for 5-10 min, and their concentrations in the recording chamber were similar to those in the reservoirs.
Recording and stimulation
Whole cell and perforated-patch recordings were obtained from
submerged slices that were superfused continuously with gravity-fed oxygenated ACSF flowing at 5-8 ml/min, either at room temperature or
at 33 ± 1 °C. The "blind" recording technique was used
(Blanton et al. 1989). Patch pipettes were pulled from
thin-walled borosilicate glass capillaries (4-7 M
when filled with
a gluconate-based solution) and connected to an Axopatch-1D amplifier
(Axon Instruments, Foster City, CA). In current-clamp experiments, the
series resistance was estimated in brief voltage-clamp sessions from
the whole cell capacitive current in response to a voltage pulse
(Marty and Neher 1995
). With whole cell recording, the
estimated series resistance was generally <20 M
, whereas with
perforated-patch recording (using the combination of amphotericin B and
gramicidin), this value stabilized at 25-35 M
. In whole cell
voltage-clamp recording, cells were clamped at potentials between
45
and
60 mV. The series resistance ranged between 8 and 23 M
and
usually was compensated for 60%; current signals were filtered at 1 kHz (4-pole Bessel filter). All data were stored on videotape for later
analysis off-line.
To evoke postsynaptic responses, electrical stimulation was applied between monopolar tungsten electrodes (tip diameter, 20 µm) placed in the medial and lateral parts of the subparaventricular zone (subPVN), using an isolated stimulation unit (Digitimer, Welwyn Garden City, UK) that provided pulses of constant voltage (7-25 V) or current (10-300 µA; duration, 0.2 ms).
Cell identification
Previous investigators have recognized at least three categories
of neurons in PVN based on action potential waveform and responses to
transient positive and negative current injections (e.g., Hermes
et al. 1996a; Tasker and Dudek 1991
). Analogous with recordings in SON, magnocellular neurons can be recognized by a
characteristic shoulder on the repolarizing phase of their action
potentials, which contributes to activity-dependent action potential
broadening, and a depolarizing sag (i.e., time-dependent inward
rectification) and a delayed return to original membrane potential
(i.e., transient outward rectification) during and after transient
membrane hyperpolarization, respectively (Erickson et al.
1993
; Renaud and Bourque 1991
).
Data analysis and statistics
All numbers are expressed as mean ± SE. To assess possible
postsynaptic influences of VP, several membrane properties were evaluated with perforated-patch recordings at 33 ± 1 °C in ACSF containing BMC and NBQX to block synaptic potentials. Resting membrane
potentials (not corrected for the occurrence of a junction potential)
were calculated from the mean potential over representative 1-min
periods (sampled at 200 Hz). Input resistances were measured from the
instantaneous voltage deflections induced by negative current
injections (10-20 pA; 1-s duration). Because the firing frequency in
this preparation was low (Hatton 1990), the number of
action potentials evoked by 2-s depolarizing current injections (5-60
pA) was taken as an index of cell excitability. Statistical significant
changes after VP application were assessed using the Wilcoxon
matched-pairs test.
To assess the influence of VP on inhibitory synaptic input, 3- to 4-min
records (sampled at 5 kHz) of reversed IPSCs, recorded in ACSF
containing NBQX and D-CPP (D-APV), were
analyzed according to frequency, amplitude, time to peak, and time
constant of decay using software written in Unix environment. Similar
to methods described previously (Bergles et al. 1996),
detection of events was accomplished by setting a threshold of five
times the standard deviation of the noise in the derivative of the
original data filtered at 300 Hz (Gaussian filter). As judged by eye,
this method reliably detected synaptic events, including those located
on the falling phase of previous events. Artifacts in the recording were removed manually.
For each record, the frequency was calculated and a cumulative
amplitude distribution was constructed. The Wilcoxon matched-pairs test
was used to determine the presence of statistical significant (i.e.,
P < 0.05) differences in the mean frequency of
reversed IPSCs between two dependent experimental groups. Comparing
more than two dependent experimental conditions, the Friedman test, followed by a multiple comparison of groups, was applied. To assess changes in the amplitude distribution of reversed IPSCs after a certain
treatment, Kolmogorov-Smirnov (K-S) statistics were used to test
significant (i.e., P < 0.01) differences between two
cumulative amplitude distributions. To evaluate the influence over a
certain number of cells, P values of each K-S test were combined to one value (P combined K-S) using the method
described by Koizol and Perlman (1978).
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RESULTS |
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VP increases GABAA-receptor-mediated synaptic input
Observations are based on data, using conventional whole cell (at room temperature; n = 15) or perforated-patch (at 33 ± 1 °C, n = 23) recording methods, from PVN neurons that displayed properties attributable to magnocellular neurons. In 22/38 neurons a 1-min bath application of VP (100-500 nM) was followed by an increase in the frequency of IPSPs or IPSCs (Fig. 1A). The effect usually started within 2 min, persisted for a variable amount of time independent of the temperature of recording (0.5-18 min; mean of 9.50 ± 1.09 min; n = 22), and was repeatable after washout (6/6 cells tested). In spontaneously active cells recorded with the perforated-patch method, VP-induced increases in IPSPs were not associated with significant decreases in firing frequency (control, 1.01 ± 0.28 Hz; VP, 0.83 ± 0.20; n = 6, P = 0.22).
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VP-induced increases in IPSP(C) frequency were dependent on functional
GABAA receptors because the effects of a second application were blocked by preperfusion with BMC or BMI (10-20 µM;
n = 9; Fig. 1B). In the latter
circumstances, there was no detectable influence on the occurrence of
excitatory postsynaptic events (Fig. 1B). In 5/5 cells
tested, VP-induced increases in the frequency of inhibitory
postsynaptic events were equally large after the addition of
D-CPP (or D-APV) and NBQX, applied in
concentrations (10-20 and 2-5 µM, respectively) known to block fast
glutamatergic synaptic transmission in this preparation (a 2.24 ± 0.16-fold increase in control vs. a 2.21 ± 0.86-fold increase in
D-CPP/D-APV and NBQX; n = 5, P = 0.89; not illustrated) (cf. Hermes et
al. 1996a).
Because cytoplasmic dialysis of second-messenger pathways consequent to whole cell recordings might obscure other direct effects of VP on the neurons, the influence of VP also was studied with perforated-patch recordings, in ACSF containing BMC (10-20 µM) and NBQX (2-5 µM). Table 1 compares observations on seven cells where VP (500 nM) induced an increase in IPSP frequency in control ACSF, and 10 cells where VP had no such effect. Little or no significant influence was noted on resting membrane potential, input resistance or cell excitability (as defined by the number of current evoked action potentials) in either group.
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Responsiveness to VP is selective to a subpopulation of magnocellular neurons
Whereas whole cell recordings did not reveal any distinguishing property, current-clamp data from 23 cells recorded with perforated-patch techniques showed a feature unique to magnocellular cells that displayed a response to VP. In all of nine cells where VP induced an increase in IPSP frequency, the return from a transient membrane hyperpolarization (from resting levels) was characterized by a rebound depolarization (Fig. 1C, i and ii). By contrast, in 12/14 neurons that lacked the VP-induced effect, the return from hyperpolarized levels to resting membrane potential was delayed (Fig. 1Cii).
VP response is mediated via V1a-type receptors located on GABAergic interneurons
To localize (pre- or postsynaptic) and characterize the receptor
that mediates the VP influence, voltage-clamp recordings using
chloride-loaded electrodes were obtained from magnocellular neurons
(recognized from their action potential waveform before inactivation by
QX-314). In responding cells, VP (500 nM) induced a significant mean
threefold increase in the frequency of reversed IPSCs (from 2.63 ± 0.48 to 7.69 ± 1.86 Hz, n = 17:
P < 0.01), but no change in the mean amplitude of
IPSCs (from 61.09 ± 6.84 to
68.16 ± 6.47 pA,
n = 17: P = 0.36). However, significant
(P < 0.01) rightward (toward larger amplitudes: in 10 cells) or leftward (toward smaller amplitudes: in 5 cells) shifts in
cumulative amplitude distributions were observed in 15/17 cells when
analyzed individually (Fig. 2). Combining
the K-S P values (Koizol and Perlman 1978
) indicated an overall significant influence of VP on the amplitude distribution (P combined K-S<0.01).
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The variable changes in the amplitude distribution of reversed
IPSCs suggest that the influence of VP on
GABAA-receptor-mediated synaptic transmission is
predominantly presynaptic. To address a possible influence of VP on the
release probability of GABA from synaptic terminals, we analyzed
miniature reversed IPSCs recorded in the presence of TTX (cf.
Bergles et al. 1996). In these conditions, bath
application of VP (500 nM) had no influence on the frequency of
miniature reversed IPSCs or on their amplitude distribution (Fig. 2,
Table 2).
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Selective VP agonists and antagonists were administered in random
order. In four cells where VP (500 nM) induced an increase in the
frequency of IPSCs, application of a selective
V1a receptor agonist (PO-VT: 500 nM) was 50%
as effective; this is in accordance with the reduced potency of PO-VT
as compared with VP (Barberis and Tribollet 1996
).
However, applications of a selective V2 receptor agonist (dVDAVP) as well as of OT (both at 500 nM) were ineffective (Fig. 3, Table 2). In addition, VP (500 nM) was without effect in four cells in the presence of the
V1a/OT receptor antagonist Manning compound (500 nM; VP increased the frequency of reversed IPSC 2.46-fold in control
conditions and 1.06-fold in the presence of Manning compound).
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DISCUSSION |
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The present study reveals that application of VP to hypothalamic
slices in vitro induces an increase in the frequency of
bicuculline-sensitive IPSP(C)s recorded in a subpopulation of
magnocellular neurons in PVN. Interestingly, the magnocellular neurons
that demonstrate this response display a characteristic rebound
depolarization after transient membrane hyperpolarization from rest.
This feature is reminiscent of a rebound depolarization noted in
OT-immunoreactive neurons in SON, where its demonstration requires that
the cells be held at depolarized membrane potentials (Stern and
Armstrong 1997). Although the selective presence in
OT-immunoreactive neurons of a depolarization-activated sustained
outward rectification may be responsible for this, differences in the
magnitude of transient outward rectification also may contribute to the
rebound depolarization (Fisher et al. 1998
; Stern
and Armstrong 1996
). Because in PVN the underlying ionic
mechanism seems partly dissimilar (because the rebound is detectable at
resting membrane potentials), it remains to be verified whether the
rebound depolarization is also a property unique to OT-synthesizing
magnocellular neurons in this nucleus.
Location and type of VP receptors
There are several reasons to propose that the targets for VP are
V1a-type receptors located directly on GABAergic
neurons that innervate a subpopulation of PVN magnocellular neurons.
First, responses persist in the presence of glutamate receptor
antagonists, indicating that VP is not acting through a glutamatergic
innervation of GABAergic neurons. Second, the VP-induced responses are
abolished completely in TTX-containing media with no remaining
influence of the peptide on the frequency or amplitude distribution of
miniature IPSCs. This indicates that the effect requires
neurotransmission in GABAergic neurons and that VP has little or no
influence on the presynaptic release mechanisms for, or postsynaptic
responsiveness to, GABA. Third, the effects are mimicked by a selective
V1a receptor agonist and blocked by a
V1a/OT receptor antagonist. And last, the
duration of the VP effect is mostly prolonged as might be anticipated
given the long depolarizing action of this peptide and of
V1a-type receptor activation on other central
neurons (e.g., Kolaj and Renaud 1998; Raggenbass
et al. 1989
).
Location of GABAergic neurons
The location of the GABAergic neurons mediating these responses
remains to be defined. Although many hypothalamic areas innervate PVN,
candidate regions in this slice preparation would include the
perinuclear region of PVN, including subPVN, and suprachiasmatic nucleus (SCN). These are areas that contain many GABA- or glutamic acid
decarboxylase (GAD)-synthesizing neurons (Moore and Speh 1993; Okamura et al. 1990
; Roland and
Sawchenko 1993
). Moreover, local stimulation in these regions
evokes GABAergic inputs in PVN magnocellular neurons (Boudaba et
al. 1996
; Hermes et al. 1996a
;
Tasker and Dudek 1993
). Interestingly, VP has been shown to excite neurons in SCN as well as in perinuclear regions of PVN, an
influence that probably is mediated by V1a-type VP
receptors (Carette and Poulain 1989
; Shibata and
Moore 1988
). However, GABAergic neurons in SCN presumably do
not mediate the observations reported here because we detected similar
VP-induced changes in IPSP(C)s in transverse hypothalamic slices that
did not contain SCN. Therefore we would suggest that it is the
GABAergic neuronal populations situated in perinuclear regions of PVN
(i.e., subPVN) that are the most likely origins of the VP-induced
responses in PVN magnocellular neurons.
Possible endogenous sources of VP
The SCN, a site responsible for generation of circadian
rhythmicity in mammalian brain, is an obvious source for a VP
innervation of GABAergic neurons located in the area cited in the
preceding text. VP-immunoreactive neurons in SCN project their axons
into the perinuclear region of PVN (Buijs et al. 1993).
Moreover, a pronounced circadian rhythm in plasma and pituitary levels
of OT and VP (Windle et al. 1992
) suggests that SCN
neurons indeed may be capable of regulating the activity of PVN
magnocellular neurons either directly, as implied from recent in vitro
electrophysiological data (Hermes et al. 1996a
), or
indirectly, as might occur through GABAergic interneurons. Whereas a
role of VP in SCN efferents requires further clarification, the present
observations suggest that an action via the GABAergic interneuron
pathway may be one of the possibilities.
Other possibilities are suggested by recent evidence that sources of
neurohypophyseal peptides also release them locally in brain (reviewed
in Landgraf 1995; Ludwig 1998
). Should
this occur via TTX-sensitive synaptic release from terminals of axon
collaterals of magnocellular neurons, the present observations suggest
that VP-synthesizing magnocellular neurons could be engaged in a
feedback inhibitory mechanism to other magnocellular neurons, operating through GABAergic neurons. In addition, VP and/or OT are likely released into the extracellular space via TTX-insensitive exocytosis from somatodendritic regions of the magnocellular cells
(Landgraf 1995
; Ludwig 1998
;
Morris et al. 1993
). Somatodendritic release is believed
to be responsible for many of the described peptidergic influences on
magnocellular neurons or on synaptic input to these neurons
(Brussaard et al. 1996
; Freund-Mercier
and Richard 1984
; Gouzènes et al. 1998
;
Kombian et al. 1997
). Most of these influences result
from a peptidergic action close to the site of (somatodendritic) release, i.e., on the neuron itself or on synaptic terminals
innervating the neuron, because extensive diffusion of active peptide
probably is limited (Kombian et al. 1997
). Because our
observations suggest that the GABAergic interneurons are probably
located at a distance from the magnocellular cells, a distant dendritic
source of the peptide seems less likely. Further investigations should
clarify some of these possibilities.
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ACKNOWLEDGMENTS |
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The authors thank Dr. C.M.A. Pennartz for contributing to the development of the software for analysis of postsynaptic currents.
This work was supported by postdoctoral fellowships from the International Human Frontier Science Program Organization, the Heart and Stroke Foundation of Canada, and a grant from Institut de Recherches Internationales Servier (PHA SCREEN-907-NLD).
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FOOTNOTES |
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Address for reprint requests: M.L.H.J. Hermes, Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ Amsterdam, The Netherlands.
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 26 October 1998; accepted in final form 15 September 1999.
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REFERENCES |
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