Neuroscience Research Group and Department of Physiology and Biophysics, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada
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ABSTRACT |
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Chen, Xihua and
Quentin J. Pittman.
Vasopressin and Amastatin Induce V1-Receptor-Mediated
Suppression of Excitatory Transmission in the Rat Parabrachial
Nucleus.
J. Neurophysiol. 82: 1689-1696, 1999.
We examined actions of arginine vasopressin (AVP) and
amastatin (an inhibitor of the aminopeptidase that cleaves AVP) on
synaptic currents in slices of rat parabrachial nucleus using the
nystatin-perforated patch recording technique. AVP reversibly decreased
the amplitude of the evoked, glutamate-mediated, excitatory
postsynaptic current (EPSC) with an increase in paired-pulse ratio. No
apparent changes in postsynaptic membrane properties were revealed by
ramp protocols, and the inward current induced by a brief application
of -amino-3-hydroxy-5-methylisoxazole-4-propionic acid was unchanged
after AVP. The reduction induced by 1 µM AVP could be blocked by a
V1 AVP receptor antagonist,
[d(CH2)51-O-Me-Tyr2-Arg8]-vasopressin
(Manning compound, 10 µM). Bath application of an aminopeptidase
inhibitor, amastatin (10 µM), reduced the evoked EPSC, and AVP
induced further synaptic depression in the presence of amastatin.
Amastatin's effects also could be antagonized by the Manning compound.
Corticotropin-releasing hormone slightly increased the EPSC at 1 µM,
and coapplication with AVP attenuated the AVP response. Pretreatment of
slices with 1 µg/ml cholera toxin or 0.5 µg/ml pertussis toxin for
20 h did not significantly affect AVP's synaptic action. The
results suggest that AVP has suppressant effects on glutamatergic
transmission by acting at V1 AVP receptors, possibly
through a presynaptic mechanism involving a pertussis-toxin- and
cholera-toxin-resistant pathway.
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INTRODUCTION |
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Arginine vasopressin (AVP) has profound effects in
the CNS on behavior, body temperature, and cardiovascular regulation,
sympathetic and neuroendocrine activation, and cognition (for reviews,
see de Wied et al. 1989,. 1993
; Herbert
1993
; Pittman et al. 1998
; Swanson and Sawchenko 1980
). These effects are mediated
mainly by the V1-type AVP receptors in keeping with
observations that the V1 subtype is most abundant in the
brain (Ostrowski et al. 1992
; Tribollet et al.
1988
). Distintive AVP cell groups have been identified to
regulate central functions with, for example, the paraventricular
nucleus activating the neuroendocrine and autonomic systems
(Antoni 1993
; Plotsky 1991
) and the bed
nucleus of stria terminalis promoting antipyresis (Pittman and
Thornhill 1990
; Pittman et al. 1998
). With
respect to possible regulation of autonomic function, it is
demonstrated that a reciprocal innervation exists between various
hypothalamic nuclei and the parabrachial nucleus (PBN) in the pons
(Jhamandas et al. 1991
; Krukoff et al. 1992
; Moga et al. 1990
). In keeping with its
role in relaying sensory and visceral information to higher centers,
the PBN has been implicated in cardiovascular regulation. Stimulation
of the baroreceptor afferents causes alteration in glutamate release in
the PBN (Saleh et al. 1997a
) and stimulation of the PBN
causes a pressor response (Chamberlin and Saper 1992
;
Sved 1986
) attributable to increased AVP release from
the hypothalamus (Sved 1986
).
As AVP is thought to act as a central neurotransmitter involved
in cardiovascular regulation (Pittman and Bagdan 1992)
and AVP immunoreactive fibers have been detected in the PBN
(Block and Hoffman 1987
), we were interested in defining
the cellular action of AVP in this nucleus. In the brain, AVP-induced
direct excitation of the postsynaptic cells has been observed in the suprachiasmatic nucleus, amygdala, area postrema, facial, and dorsal
vagal motorneurons (Ingram and Tolchard 1994
;
Lowes et al. 1995
; Lu et al. 1997
;
Mihai et al. 1994
; Mo et al. 1992
;
Widmer et al. 1992
). AVP also has been found to increase
excitatory synaptic transmission in a number of brain regions including
the hippocampus and the dorsolateral septum (Chen and Du
1993
; Chepkova et al. 1995
; Miura et al.
1997
; Van den Hooff and Urban 1990
; Van
den Hooff et al. 1989
). In the hippocampus, however, it has
been reported that excitation of pyramidal cells is reduced due to
AVP's primary excitation of the local circuit inhibitory interneurons
(Albeck et al. 1990
; Smock et al. 1990
).
Recently work from our lab demonstrated that AVP reduces the evoked
excitatory postsynaptic current (EPSC) in the magnocellular cells of
the supraoptic nucleus without affecting the postsynaptic membrane
properties (Mouginot et al. 1998
). Regardless of pre- or
postsynaptic site of action, these reports concur that V1
receptors are implicated because of effective blockade by the V1 antagonist. V2-receptor-mediated effects,
although not as commonly seen as V1-mediated ones, have
been observed on acutely dissociated diagonal band of Broca cells
(Easaw et al. 1997
).
Corticotropin-releasing hormone (CRH) is another neurotransmitter
implicated in central cardiovascular regulation (Fisher 1993), and the PBN has intrinsic CRH-containing neurons and
also receives an extrinsic CRH innervation (Merchenthaler et al.
1982
; Otake and Nakamura 1995
; Palkovits
et al. 1985
). CRH has been found to increase spontaneous
discharge rate of locus coeruleus cells in vivo (Borsody and
Weiss 1996
) and excite dissociated or cultured neurons in vitro
(Aldenhoff et al. 1983
; Fox and Gruol 1993
). CRH-induced excitation is reflected by its actions on
calcium and potassium channels. It is reported that CRH increases whole cell Ca2+ currents in dissociated amygdaloid cells
(Yu and Shinnick-Gallagher 1998
) and intracellular
Ca2+ in corticotrophs (Lee and Tse 1997
),
and decreases K+ conductances in hippocampal and cerebellar
Purkinje cells (Aldenhoff et al. 1983
; Fox and
Gruol 1993
).
Neurons that coexpress AVP and CRH show a remarkable plasticity in the
synthesis and release of these peptides (Sawchenko 1987), and these peptides exert synergistic actions on anterior pituitary cells where their receptors are colocalized (Antoni 1993
; Leong 1988
). In light of similar
distribution of fibers immunoreactive for AVP and CRH in the PBN, we
therefore determined if there is a similar interaction between CRH and
AVP in terms of synaptic modulation.
This work was performed to determine: whether AVP modulates excitatory transmission in the PBN and, if so, which type of receptors are involved; whether AVP's action is accentuated by an aminopeptidase inhibitor that prevents AVP cleavage; whether AVP interacts with CRH; and whether alterations in G-protein activity change AVP's synaptic action.
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METHODS |
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Slice preparation
Male Sprague-Dawley rats (180-220 g) were anesthetized with
halothane. The brain was removed quickly and placed into ice-cold, carbogenated (95% O2-5%
CO2) artificial cerebrospinal fluid (ACSF; pH
7.3-7.4). Coronal slices (400-µm thick) were cut from a block of
tissue containing the PBN [bregma 9.1 to
9.8 mm (Paxinos and Watson 1986
)] in cold (4°C) carbogenated ACSF using a
vibratome. Slices were hemisected and incubated in ACSF at room
temperature (22°C) for
1 h before recording. A slice then was
transferred into a 500-µl recording chamber where it was submerged
and continuously perfused with prewarmed ACSF (28-30°C) at a rate of
2-3 ml/min. The composition of the ACSF was (in mM) 126 NaCl, 2.5 KCl,
1.2 NaH2PO4, 1.2 MgCl2, 2.4 CaCl2, 18 NaHCO3, and 11 glucose. To eliminate possible
GABAergic contamination of excitatory synaptic responses, 50 µM
picrotoxin was present in the ACSF at all times.
Nystatin-patch recording
Nystatin patch recordings from PBN neurons were made with glass
micropipettes (Garner Glass; tip resistance 4-10 M) first back-filled at the tip with a solution containing (in mM) 120 K-acetate, 40 HEPES, 5 MgCl2, and 10 EGTA and
then filled with the same solution containing nystatin 450 µg/ml and
Pluronic F127 (dissolved in DMSO). High-resistance seals (1-3 G
)
were made using an Axoclamp 2A amplifier. A
20-mV step (100-ms
duration) was applied to monitor the partitioning of nystatin into the
membrane. Access to the cell (series resistance of 15-30 M
) was
attained within 30 min after seal formation.
Data acquisition and analysis
After adequate access was attained, resting membrane potential
was measured and I-V characteristics were obtained by
current injections (in steps of 10 pA) in the current-clamp mode. All responses were filtered at 1 or 3 kHz. Cells then were voltage clamped
near the resting membrane potential at 65 mV (Zidichouski et
al. 1996
). Synaptic currents were evoked by applying single pulses via a bipolar stimulating electrode placed close to the ventral
tip of the superior cerebellar peduncle. A stimulus intensity that
yielded a response 50-60% of the maximum synaptic response was used
for the remainder of the experiment. Three successive synaptic
responses were taken 10 s apart, digitally averaged and stored for
off-line analysis. In all synaptic current experiments, a
20-mV,
100-ms square pulse was applied 150 ms after synaptic stimulation to
monitor input and series/access resistance. In addition to the
computer-assisted data acquisition, continuous records of membrane
potentials and currents were made on a pen chart recorder. In
toxin-treated slice experiments, slices were hemisected and one-half of
them was incubated in either 1 µg/ml cholera toxin (ChTX) or 0.5 µg/ml pertussis toxin (PTX) in a volume of 3 ml ACSF for 18-20 h
while the other halves were incubated in carbogenated ACSF of the same
volume for the same time period.
All acquired data were analyzed off-line using Clampfit (Axon Instruments, Foster City, CA). Data are expressed as means ± SE in either absolute values or normalized percentages. Statistical comparisons were performed on raw data using paired or unpaired Student's t-test or analysis of variance (ANOVA) where appropriate. Significance was accepted at the 0.05 level.
All drugs (except for ChTX and PTX) were bath applied by perfusion with
ACSF containing the final concentration of the drug. AVP, CRH, Manning
compound, 6-cyano-7-nitroquinoxaline-2,3dione (CNQX),
-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), and ChTX
were from RBI (Natick, MA); amastatin, nystatin, picrotoxin, and ACSF
components were from Sigma (St Louis, MO). Stock solutions of CNQX,
AMPA, AVP, and CRH were aliquoted and frozen at
20°C and diluted
into ACSF immediately before the experiment.
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RESULTS |
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Patch recordings were made from cells located in both
mesencephalic and pontine divisions of the lateral PBN and
predominantly from the dorsal lateral and external lateral subnuclei.
Cell characteristics were comparable with those reported previously
(Chen et al. 1999; Saleh et al. 1996
,
1997b
; Zidichouski et al. 1996
) and synaptic responses mediated by CNQX-sensitive,
non-N-methyl-D-aspartate (NMDA) receptors could
be evoked in a majority of cells by stimulating afferent fibers near
the ventral tip of the cerebellar peduncle (data not shown).
AVP depresses evoked EPSCs
In voltage-clamp mode, AVP depressed the evoked EPSC without
changes in holding current. This effect was readily reversible on
washout (Fig. 1A) and repeated
application on the same cell did not produce desensitization. On cells
that were treated with identical doses of AVP, the reductions in the
EPSC were similar (1st dose 23.6 ± 6.8%, 2nd dose 24.8 ± 5.4%, n = 3, P > 0.05 paired t-test). The AVP-induced EPSC depression was dose dependent
5 µM, and there was no significant difference in the amount of
depression at doses >1 µM (Fig. 1B). To differentiate
where AVP acts to depress the EPSC, we employed the paired-pulse
protocol where two identical afferent stimulations were given at a
fixed 50-ms interval and a change in the ratio of the two responses was
taken as an indicator for a presynaptic mechanism. AVP at a dose of 1 or 5 µM increased this ratio (Fig. 1C, averaged ratio for
control: 1.25 ± 0.1, for AVP: 1.51 ± 0.08, n = 4, P < 0.05 paired
t-test).
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We further ruled out the postsynaptic involvement by examining the
passive membrane conductances and glutamate receptor sensitivity on the
postsynaptic cell. The AVP-induced depressant effect on the EPSC was
not accompanied by postsynaptic changes in input resistance at holding
potentials (65 mV). Furthermore steady-state I-V curves
generated by a slow ramp protocol (
120 to
30 mV) showed that AVP
did not change the I-V relationship over a wide voltage
range (Fig. 2A). The reduction
in the EPSC did not appear to be due to an interaction with the
postsynaptic glutamate receptors as inward currents induced by a brief
bath application of AMPA (5 µM, 30 s) in control and in the
presence of 1 µM AVP were also comparable (control: 61.5 ± 9.5 pA, in 1 µM AVP: 66 ± 9.6 pA, n = 4, P > 0.05, paired t-test, Fig.
2B). In the current-clamp mode, the action potentials evoked
by depolarizing steps and the I-V relationships generated by
a series of current injections were not altered by AVP (Fig. 2,
C and D). Collectively, these results suggest
that AVP reduces the EPSC through a presynaptic action.
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AVP depresses the EPSC by acting at V1 receptors
As AVP receptors in the brain are mostly of
V1 type (Ostrowski et al. 1992;
Tribollet et al. 1988
), we tested if the observed EPSC
effects were mediated by V1 receptors using
Manning compound, an AVP receptor blocker that is selective for the
V1 over the V2 receptors.
Cells were tested for AVP's synaptic effects, and when they were
recovered fully, Manning compound was perfused followed by AVP in the
presence of Manning compound. AVP (1 µM) alone induced a reduction in
the EPSC (18.7 ± 3.1%, n = 5), whereas the same
dose produced no significant EPSC reduction in the presence of 10 µM
Manning compound (6.9 ± 4.3%, n = 5, P < 0.05 vs. AVP alone, paired t-test, Fig.
3).
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Aminopeptidase inhibition induces V1-mediated synaptic effects
AVP is cleaved by aminopeptidases and blockade of these
aminopeptidases prevents AVP metabolism in vivo (Burback and
Lebouille 1983; Stark et al. 1989
). The PBN
previously has been found to be rich in peptidases, and their
inhibition greatly increases the efficacy of several of their peptide
substrates in the region (Saleh et al. 1996
). To test
whether inhibition of the aminopeptidase that cleaves AVP has any
effects on AVP-induced synaptic changes, cells were tested sequentially
for AVP (1 µM) and amastatin [an inhibitor of aminopeptidase that
cleaves AVP and oxytocin (Stancampiano et al. 1991
), 10 µM] plus AVP at the same dose. AVP alone resulted in an EPSC
reduction (17.4 ± 4.2%, n = 7). Amastatin also
caused a reduction in the EPSC (17.7 ± 2.1%, n = 7) comparable with that induced by AVP at 1 µM, and additional AVP,
in the presence of amastatin, further depressed the EPSC (28.6 ± 5.8%, n = 7, P < 0.05 vs. AVP alone,
paired t-test, Fig.
4A).
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Because amastatin prevents AVP degradation, we asked if the amastatin-induced change in the EPSC was a result of an action of endogenously released AVP in the PBN. Amastatin depressed the evoked EPSC on its own (16.7 ± 4.4%, n = 5), and this effect was blocked by 10 µM Manning compound (4.4 ± 5.1%, n = 5, P < 0.05 vs. amastatin alone, paired t-test, Fig. 4B). These results indicate that the action of endogenous AVP in the PBN is terminated by an amastatin-sensitive aminopeptidase.
Lack of synergy between AVP and CRH
It is well established that AVP and CRH have synergistic effects
on the hypothalamic-pituitary-adrenal axis (Antoni 1993; Plotsky 1991
) and both peptides are present in the PBN
(Merchenthaler et al. 1982
; Palkovits et al.
1985
; van Zwieten et al. 1996
). We therefore
examined the effects of CRH on excitatory synaptic transmission and its
interaction with AVP. Bath application of 1 µM CRH slightly, but
significantly, increased the amplitude of the evoked EPSC (10.1 ± 6.8%, n = 6) without a change in I-V relationships generated by a ramp protocol (data not shown). AVP alone
depressed the EPSC by 16.1 ± 4.5% (n = 6), and
coapplication of AVP and CRH resulted in an attenuated action of AVP
(7.1 ± 6.6%, n = 6, P < 0.05 vs. AVP alone, paired t-test; Fig.
5). There was therefore no synergy
between these two peptides on the excitatory synaptic transmission in
the PBN.
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AVP depresses EPSC via a ChTX- and PTX-insensitive mechanism
AVP receptor transduction is accomplished via G-protein-coupled
second-messenger systems (Moriarty et al. 1989;
Nabika et al. 1985
). We therefore used ChTX and PTX to
determine if there was G protein involved in the signaling process.
Slices were incubated in control ACSF or one of the toxins in ACSF
(ChTX: 1 µg/ml; PTX: 0.5 µg/ml) for ~20 h before we began
recording. ChTX incubation irreversibly activates the Gs and therefore
occludes the effects of an agent that also uses Gs coupling. PTX
irreversibly disables the Gi protein, hence uncoupling the agonist from
all the downstream effectors. AVP-induced EPSC depression was 26.2 ± 7.8% in control slices (n = 5), 16.5 ± 5.5%
in PTX-treated slices (n = 5), and 30.4 ± 3.6%
in ChTX-treated slices (n = 4, Fig.
6). One-way ANOVA revealed no
significance among the three groups, rejecting the seemingly attenuated
response to AVP in PTX-treated slices. There were no changes in the
resting membrane potential (RMP) or input resistance
(IR) in control, ChTX- or PTX-treated
slices (control: RMP
62.7 ± 1.7 mV,
IR 580 ± 89 M
,
n = 5; ChTX-treated: RMP
63.2 ± 0.8 mV,
IR 597 ± 51 M
,
n = 4, and PTX-treated: RMP
63.4 ± 2.1 mV,
IR 535 ± 89 M
,
n = 5, one-way ANOVA, P > 0.05).
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DISCUSSION |
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The results presented in this work demonstrate that AVP depressed the evoked EPSC in the PBN, possibly through an action at V1 AVP receptors on presynaptic terminals. Aminopeptidase inhibition caused a reduction in the EPSC that could be blocked by a V1 receptor antagonist, suggesting the peptide is released (and degraded) in the PBN. AVP produced further EPSC depression in the presence of amastatin. AVP's synaptic effects were reduced in the presence of CRH and were mediated by a ChTX- and PTX-insensitive signaling pathway.
AVP depresses EPSC by acting on the V1 receptors presynaptically.
It is reported that AVP reduces the population spikes of the
pyramidal cells in the hippocampus by exciting local GABAergic interneurons (Albeck et al. 1990; Smock et al.
1990
). Although such a mechanism could account for our
findings, we have never seen a postsynaptic effect of AVP on any neuron
in the vicinity of the PBN which would be expected if it were to excite
a GABAergic interneuron. Furthermore as AVP reduced the EPSC even
though GABAA receptors were blocked with
picrotoxin, at least this subclass of receptors would not appear to
have been involved. Although we cannot eliminate the participation of
GABAB receptors, in our previous work
(Chen et al. 1999
), the magnitude and onset of
GABAB receptor activation was very different from
what we saw in the present experiments. We believe that the most likely
explanation for our effects is that AVP was acting at presynaptic
glutamatergic terminals to reduce glutamate release. Although the
mechanism of the inhibition is not yet determined, a likely scenario
would be inhibition of a calcium current in the presynaptic terminal such as has been suggested for a number of other presynaptic receptors (Colmers and Bleakman 1994
; Shapiro and Hille
1993
). Alternatively, increases in K+
conductances in axon endings would reduce equally the likelihood of
transmitter release, as is the case for opioid peptides (Simmons and Chavkin 1996
; Vaughan et al. 1997
).
Even at maximal doses, the effects of AVP on the evoked potentials
seldom exceeded a 30% reduction. This contrasts with the action of
some other peptides we have previously tested in the PBN such as
substance P (Saleh et al. 1996) and cholecystokinin (Saleh et al. 1997b
) where maximal doses of the peptides
can suppress completely the EPSC. The much less dramatic action of AVP
could be because of the fact that there are fewer receptors for this peptide, making it relatively ineffective in altering ionic
conductances in the terminals; an alternate possibility is that only a
very few terminals from a heterogeneous population are strongly
affected by the peptide. We cannot distinguish between these
possibilities as our afferent stimulation undoubtedly activates a
nonhomogeneous population of afferents.
In this study, AVP's synaptic action could be blocked by a
V1 receptor antagonist, which is consistent with
V1 receptors mediating the reduction in the EPSC.
The Manning compound at the dose used in this study also blocks
oxytocin receptors, and AVP has been shown previously to have activity
at oxytocin receptors in the brain (Muhlethaler et al.
1983). Preliminary results from a cell that was responsive to
AVP showed that oxytocin caused a reduction in the EPSC, therefore it
is possible that some of the AVP-induced synaptic effects we observed
could result from AVP acting at the oxytocin receptors. However, it
must be noted that neither oxytocin receptors (Tribollet et al.
1989
) nor oxytocin receptor mRNA have been localized in the PBN.
ChTX incubation, which occludes further activation of the Gs, did not
alter AVP-induced EPSC depression, suggesting that Gs-coupled V2 receptors are not involved.
V1 receptors are coupled to Gq and other G
proteins to activate phospholipase C (Moriarty et al.
1989; Nabika et al. 1985
; Naro et al.
1997
). This coupling has been reported to be PTX sensitive or
resistant depending on the expression system used (Lynch et al.
1986
; Moriarty et al. 1989
). It now is
appreciated that there are numerous subtypes of G proteins that are
differentially sensitive to PTX (Fields and Casey 1997
).
In particular, G
z, which is coupled to N-type Ca2+ channels is PTX resistant (Jeong and
Ikeda 1998
). We found that incubation of slices with PTX
resulted in a smaller, but insignificant, AVP-induced reduction in
EPSC. It should be noted that PTX treatment could differentially affect
the pre- or postsynaptic elements (Colmers and Pittman
1989
) and the mechanisms that mediate excitatory or inhibitory
synaptic events (Thompson and Gahwiler 1992
). PTX incubation does block pre- and postsynaptic effects of baclofen (Thompson and Gahwiler 1992
), so it was taken as
adequate PTX treatment when it blocked baclofen-induced
hyperpolarization. The dose and duration of PTX incubation appeared
adequate because parallel experiments showed that PTX treatment blocked
baclofen-induced hyperpolarization of the PBN cells (Chen et al.
1999
). This would at least suggest that the AVP receptors on
the terminals are coupled to a G protein that is either wholly or
partially resistant to PTX.
Aminopeptidase inhibition enhances AVP's synaptic effects
Released AVP is terminated by aminopeptidase cleavage
(Burback and Lebouille 1983; Stark et al.
1989
). Inhibition of this group of aminopeptidase has been
shown to increase AVP-induced synaptic responses in the hippocampus and
the supraoptic nucleus (Kombian et al. 1997
;
Miura et al. 1997
). There are abundant peptidases in the
PBN, and their inhibition leads to a remarkable shift in the dose
response of peptides. For example, phosphoramidon (an inhibitor of
endopeptidases that degrade substance P) greatly enhance the effects of
the peptide in the PBN (Saleh et al. 1996
). This
prompted us to explore whether AVP's action in the PBN is modulated by
ongoing degradation by peptidases. Pretreatment with amastatin, an
inhibitor of the peptidase that cleaves AVP and oxytocin
(Burback and Lebouille 1983
; Stancampiano et al.
1991
), caused a reduction in the EPSC, and this effect could be
blocked by a V1 receptor antagonist. This suggests that the
endogenously released AVP in the PBN is dynamically terminated by peptidases.
If endogenous AVP is present, one would expect that application of an antagonist would increase the size of the EPSC by blocking AVP's depressant action. We saw no such effect, suggesting that there is efficient cleavage by the aminopeptidase so that the endogenous AVP does not reach a concentration high enough to reduce EPSC. This enzymatic activity could account for the relatively high threshold for the AVP-induced effects. Reduced responses at the highest dose we tested may be a result of either fast desensitization associated with high doses or an unknown mechanism whereby the activity of proteolytic enzymes is triggered. Because amastatin-induced EPSC reduction is a V1-receptor-mediated phenomenon, the fact that AVP and amastatin combined to produce EPSC reduction greater than the maximal AVP-induced depression supports our notion that AVP is actively removed in the PBN by aminopeptidase activity.
AVP and CRH do not synergize to suppress EPSCs in the PBN
CRH neurons in the PBN are found to regulate locus coeruleus cell
activity and AVP release in the hypothalamus in the intact animal
(Borsody and Weiss 1996; Carlson et al.
1994
) and to excite dissociated or cultured neurons in vitro
(Aldenhoff et al. 1983
; Fox and Gruol
1993
). Our results that CRH slightly increased the EPSC is
consistent with its increasing Ca2+ entry and/or
decreased K+ conductance (Aldenhoff et al.
1983
; Fox and Gruol 1993
; Lee and Tse
1997
; Yu and Shinnick-Gallagher 1998
). The
marginal increase could be explained by the finding that CRH receptors
in the brain are low in normal conditions and are inducible on
increased demand (Mansi et al. 1996
). It is also
possible that there are subtle actions of CRH on membrane conductances
that were not revealed in our analysis and could account for the small
increase in the EPSC. Further studies will be required to investigate
more fully the action of CRH in this nucleus.
In the parvocellular PVN, cells coexpressing CRH and AVP are
capable of switching transmitter synthesis in favor of one over the
other as demanded and the peptides have synergistic effects on the
corticotropes (Antoni 1993; Leong 1988
;
Sawchenko 1987
). Corticotropes primed by one of the
peptides respond to the other with potentiation, possibly as a result
of cross-talk between messenger molecules. Rather than a synergistic
interaction, we found a functionally antagonistic interaction. As the
mechanism of action of the CRH-induced increase in the EPSC size has
not been investigated in the present study, we are not able to
determine the basis for this antagonistic interaction. It could relate
to the different subtype of AVP receptors in the CNS and the pituitary or even to the possibility that the receptors may not be colocalized on
the same cells.
Physiological relevance
The PBN is implicated in cardiovascular and neuroendocrine
regulation. Injection of AVP directly into the PBN (Berecek et al. 1984) or stimulation of vasopressinergic cell bodies in the hypothalamus (Pittman and Bagdan 1992
; Pittman
and Franklin 1985
) changes cardiac performance, implicating AVP
as a one of the mediators in this modulation. The means by which this
could occur may be an action of AVP on glutamate transmission in the
PBN such as that we have demonstrated in the present report.
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ACKNOWLEDGMENTS |
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The authors thank Dr. J. H. Jhamandas for critical reading of the manuscript.
This work was supported by the Heart and Stroke Foundation of Canada and the Medical Research Council of Canada (MRC). X. Chen is an MRC Postdoctoral Fellow, and Q. J. Pittman is an Alberta Heritage Foundation for Medical Research and MRC Senior Scientist and a Neuroscience Canada Foundation Alberta Scholar.
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FOOTNOTES |
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Address for reprint requests: Q. J. Pittman, Neuroscience Research Group, University of Calgary, 3330 Hospital Dr., NW, Calgary, Alberta T2N 4N1, Canada.
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 3 February 1999; accepted in final form 18 May 1999.
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REFERENCES |
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