1Neurosciences, Ottawa Health Research Institute, University of Ottawa, Ottawa, Ontario K1Y 4E9, Canada; and 2National Institute on Drug Abuse, Intramural Research Program, Baltimore, Maryland 21224
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ABSTRACT |
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Oz, Murat,
Miloslav Kolaj, and
Leo P. Renaud.
Electrophysiological Evidence for Vasopressin V1
Receptors on Neonatal Motoneurons, Premotor and Other Ventral Horn
Neurons.
J. Neurophysiol. 86: 1202-1210, 2001.
Prominent arginine-vasopressin (AVP) binding and AVP
V1 type receptors are expressed early in the
developing rat spinal cord. We sought to characterize their influence
on neural excitability by using patch-clamp techniques to record
AVP-induced responses from a population of motoneurons and interneurons
in neonatal (5-18 days) rat spinal cord slices. Data were obtained
from 58 thoracolumbar
(T7-L5) motoneurons and
166 local interneurons. A majority (>90%) of neurons responded to
bath applied AVP (10 nM to 3 µM) and (Phe2,
Orn8)-vasotocin, a V1
receptor agonist, but not V2 or oxytocin receptor agonists. In voltage-clamp, postsynaptic responses in motoneurons were
characterized by slowly rising, prolonged (7-10 min) and tetrodotoxin-resistant inward currents associated with a 25% reduction in a membrane potassium conductance that reversed near 100 mV. In
interneurons, net AVP-induced inward currents displayed three patterns:
decreasing membrane conductance with reversal near
100 mV, i.e.,
similar to that in motoneurons (24 cells); increasing conductance with
reversal near
40 mV (21 cells); small reduction in conductance with
no reversal within the current range tested (41 cells). A presynaptic
component recorded in most neurons was evident as an increase in the
frequency but not amplitude (in motoneurons) of inhibitory and
excitatory postsynaptic currents (IPSCs and EPSCs), in large part
due to AVP-induced firing in inhibitory (mainly glycinergic) and
excitatory (glutamatergic) neurons synapsing on the recorded cells. An
increase in frequency but not amplitude of miniature IPSCs and EPSCs
also indicated an AVP enhancement of neurotransmitter release from axon
terminals of inhibitory and excitatory interneurons. These observations provide support for a broad presynaptic and postsynaptic distribution of AVP V1 type receptors and indicate that their
activation can enhance the excitability of a majority of neurons in
neonatal ventral spinal cord.
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INTRODUCTION |
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In mammals, the
nonapeptide arginine-vasopressin (AVP) is widely recognized for its
hormonal antidiuretic and vasopressor properties when released into the
circulation following activation of neurohypophysial-projecting
hypothalamic magnocellular neurosecretory neurons. However, the
immunocytochemical demonstration of additional hypothalamic and
extra-hypothalamic AVP-synthesizing parvocellular neurons and axonal
pathways in specific independent CNS sites implied that this molecule
also served as a possible neurotransmitter in brain (Buijs
1978). Indeed AVP has now been shown to influence a wide but
specific range of behavioral, memory, learning and autonomic functions
and to have neuromodulatory and neurotransmitter-like actions in
studies at the cellular level (reviewed in Urban et al.
1998
). Recently a novel form of local intracerebral release of
neurohypophysial peptides by exocytosis from somatodendritic membranes
has been observed in supraoptic nucleus (Pow and Morris 1989
), possibly serving a autocrine or paracrine role to alter firing patterns in the same AVP-synthesizing magnocellular neurons (Gouzenes et al. 1998
). Endogenous AVP is also released
into the extracellular space (Landgraf 1995
) as well as
into the cerebrospinal fluid (Pittman et al. 1984
;
Reppert et al. 1987
), thereby dispersing the peptide in
a hormonal-like fashion throughout the brain and spinal cord.
The observation that neuronal response to exogenous AVP varies among
different brain regions and among specific neurons within a given
region is an indication of regional and cell-specific expression of AVP
receptors. In neural tissue, data from binding and molecular biological
studies indicate that these actions are mediated predominantly by AVP
acting at V1 subtype receptors (Lolait et
al. 1995; Tribollet et al. 1997
). The spinal
cord is one area where AVP binding, presumably a reflection of receptor
expression, has been reported to be prominent and to undergo
developmental change (Tribollet et al. 1997
). In adults,
AVP binding in the area of the intermediolateral cell column of the
spinal lateral horn coincides with the distribution of vasopressinergic
fibers and axon terminals that arise from parvocellular neurons located in the hypothalamus, notably the paraventricular nucleus (Buijs 1978
; Hallbeck and Blomqvist 1999
). A likely
target for AVP's actions at this specific site is the sympathetic
preganglionic neurons whose response to exogenous AVP has already been
noted (Kolaj and Renaud 1998
; Ma and Dun
1985
). This is in accordance with evidence that AVP has a role
in central regulation of cardiovascular and renal function
(Crowley 1982
; Pittman et al. 1982
;
Riphagen and Pittman 1989
). However, the distribution of
AVP receptors in the adult and particularly the neonatal spinal cord
extends throughout the gray matter of the lateral and ventral spinal
horns (Tribollet et al. 1997
). In a recent patch-clamp
analysis in neonatal tissue (Kolaj and Renaud 1998
), we
noted that AVP induced membrane depolarization and inward current in a
majority of lateral horn neurons. Extending this investigation to
thoracolumbar motoneurons and interneurons, we now report evidence for
the presence of AVP V1 subtype receptors on their
somatodendritic membranes and also on presynaptic terminals of
inhibitory and excitatory interneurons that synapse on motoneurons.
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METHODS |
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Experiments used Sprague-Dawley rats of either sex (5-18 days
old) cared for in accordance with the principles and guidelines of the
Canadian Council on Animal Care. On the day of experiment, animals were
anesthetized with methoxyflurane, decapitated, the spinal cord excised
after dorsal laminectomy and a 10- to 15-mm section of thoracolumbar
spinal cord resected and placed in ice-cold (4°C) artificial
cerebrospinal fluid (ACSF). The latter contained in (mM) 127 NaCl, 26 NaHCO3, 3.1 KCl, 1.2 MgCl2,
2.4 CaCl2, and 10 D-glucose (pH 7.35;
osmolarity 290-305 mosmol.) and was gassed with 95%
O2-5% CO2. Transverse
350-450 µm sections from the Th7 to
L5 segments were cut on a vibrating blade
microtome (Leica, VT1000S, Germany), equilibrated in ACSF at room
temperature for 1 h and transferred to a recording chamber where they
were continuously superfused at 4-6 ml/min.
Using the blind whole cell patch-clamp technique, data were obtained
with borosilicate thin-walled micropipettes (BORO, BF150-110-10, Sutter Instruments, Novato, CA) made with a Flaming-Brown Puller (P-87,
Sutter Instruments) and directed to the ventral horn. Micropipettes were filled with (in mM) 130 K-gluconate, 10 KCl, 10 NaCl, 1 MgCl2, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (HEPES), 1 ethyl glycol-bis (-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA), 2 Mg-ATP adjusted
to a pH of 7.3 with Tris buffer. The addition of Lucifer yellow
(dipotassium salt 1 mg/ml; Sigma) provided a convenient intracellular
label. In recordings requiring amplification of spontaneous and
miniature IPSCs, 100 mM of K-gluconate was replaced with CsCl. Pipette
resistances measured 3-7 M
. A series/access resistance <25 M
was considered acceptable. Corrections of the liquid-junction
potentials were performed off-line on recorded membrane current and
voltage traces. Input resistance of the neurons was determined from the
slope of the current-voltage (I-V) relationships at the
range of
50 to
80 mV.
Both current- and voltage-clamp recordings were obtained with an
Axopatch 200A or Axopatch 1D amplifier (Axon Instruments, Foster City,
CA), and data were filtered on-line at 2 kHz. In whole cell
voltage-clamp mode, recordings were made at a holding potential
(VH) of 65 mV. Membrane currents and
potentials were continuously monitored on an oscilloscope and displayed
on a pressure ink pen recorder (Gould, Valley View, OH; Gould 2400S or
Gould RS3200) simultaneously. Recordings were also stored on videotape for later, off-line analysis. Digidata 1200 interface and version 7 of
pCLAMP software (Axon Instruments) were used on-line to generate current and voltage-clamp commands.
Motoneurons were identified by their all-or-none antidromic responses to ventral rootlet stimulation applied with a concentric bipolar electrode (FHC, Bowdoinham, ME, tip diameter: 25 µm; 1-12 V, duration: 0.02 s) and/or by their morphology and evidence of an axon projecting toward the ventral root. Following the recording, slices were transferred to a fixation medium (4% paraformaldehyde with 0.1 mM phosphate buffer) and stored overnight at 4°C. After clearing for 60 min with dimethyl sulfoxide and fixation on microscope lamels, labeled cells were viewed and measured under epifluorescence.
AVP and drugs were dissolved in ACSF at their final concentrations and
applied by a computer-controlled, fast local pressure application
system (DAD-12; Adams and List Associated Scientific Instruments,
Westbury, NY). In some of the experiments, agents were delivered by
bath application at a perfusion rate of 4-6 ml/min.
Arg8-vasopressin (AVP),
desamino-[D-Arg8]-vasopressin
(DDAVP),
[-mercapto-
,
-cyclopentamethyleneporpionyl1,O-Me-Tyr2]
or Manning compound, (Phe 2, Orn
8)-vasotocin, [The4,
Gly7]-oxytocin, were from American Peptide (APC,
Sunnyvale, CA). Tetrodotoxin (TTX) was from Alomone Labs (Jerusalem,
Israel). Amastatin was from Sigma, St. Louis, MO.
Data were analyzed off-line with Clampfit software of pClamp versions 7 and 8 (Axon Instruments). For statistical evaluation, we used paired, unpaired Student's t-test, or ANOVA as it is indicated in the text (Origin version 6, Microcal Software, Northampton, MA). Results are presented as means ± SE. For assessment of spontaneous and miniature postsynaptic events, 2-3 min of recordings were sampled at 5 kHz and analyzed for frequency, amplitude, time to peak, and time constant of decay using commercially available Mini Analysis software (Synaptosoft, Leonia, NJ). Spontaneous and miniature events were defined as those recorded in the absence and presence of 1 µM tetrodotoxin (TTX), respectively. Synaptic events were detected with an adjustable threshold, often set at 8-15 pA and maintained at a constant level in a given neuron. The analysis of mIPSCs and mEPSCs was performed with cumulative probability plots. Frequencies of synaptic events were calculated as the reciprocals of inter-event intervals. Statistical comparisons of the frequency and/or amplitude of the synaptic currents before and after AVP were made using the Kolmogorov-Smirnov (K-S) test; P < 0.05 was considered significant.
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RESULTS |
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Motoneurons
Data were obtained from 39 cells antidromically identified as
motoneurons. Another 19 neurons were classified as motoneurons on the
basis of their location within Rexed laminae VIII and IX (Molander et al. 1984), similar electrical properties
and morphology. Collectively these cells measured 31.1 ± 1.7 µm
in diameter, with a mean resting membrane potential of
73.1 ± 1.2 mV and input resistance of 58.9 ± 5.6 M
. A majority
(>90%) of cells responded to exogenous applications of AVP and/or
V1 receptor agonists.
AVP INDUCES POSTSYNAPTIC MEMBRANE DEPOLARIZATION AND INWARD
CURRENT.
In 7/8 neurons tested while recording in current-clamp mode, bath
application of AVP (1 µM; 30 s) was followed by a slowly rising
(60-90 s to peak) membrane depolarization that reached a plateau of
14.7 ± 3.1 mV, sufficient to trigger a burst of action potentials
in three of the seven cells. Responses required several minutes to
recover (Fig. 1A). Since
washout intervals of 30 min were needed before regaining full recovery,
some desensitization seemed likely. In all instances, responses were
accompanied by a thickening of the baseline (see following text).
Several observations imply that these effects were mediated via
V1 type receptors: application of a
V1 receptor antagonist (Manning compound, 1 µM), while without effect on resting membrane properties could block the AVP-induced responses in 5/5 cells tested (Fig. 1B);
similar membrane depolarizations (14.8 ± 2.3 mV) followed
applications of a specific V1 receptor agonist,
(Phe 2, Orn 8)-vasotocin in
4/4 cells tested (Fig. 1C); and cells lacked any response to
application of either a V2 receptor agonist
(DDAVP, 1 µM; 6 cells tested) or an oxytocin receptor agonist
[The4, Gly7]-oxytocin (1 µM; 7 cells tested; Fig. 1D). The latter would indicate that, at least in neonatal thoracolumbar motoneurons, vasopressin rather that oxytocin is the ligand for the effects observed at this
level. It is notable that neither Manning compound (5 cells) nor
amastatin, an aminopeptidase inhibitor (10 µM; 3 cells), induced significant changes in resting membrane properties or inward currents, features that might indicate an endogenous AVP action (cf. Chen and Pittman 1999).
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AVP ENHANCES PRESYNAPTIC EXCITATORY AND INHIBITORY INPUTS TO MOTONEURONS. As noted in the preceding text, AVP-induced responses were invariably accompanied by a TTX-sensitive increase in baseline thickening. The latter was due to a marked increase in the frequency of spontaneous postsynaptic currents (sPSCs). In standard ACSF, tests for a response to AVP 1 µM revealed an increase in sPSCs frequency from 1.3 ± 0.2 to 14.6 ± 2.3 Hz (P < 0.01, paired t-test, n = 7 cells). The major component of this response was clearly action potential dependent because it was abolished in the presence of TTX. Therefore neurons presynaptic to motoneurons were being activated by AVP. We next applied pharmacological antagonists to clarify whether this reflected input from excitatory or inhibitory sources.
After blockade of ionotrophic glutamate receptors with 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX, 5 µM) and D-2-amino-5-phosphonovaleric acid (D-APV, 50 µM), cells were tested for a response to 0.3 µM AVP, a concentration that was ~50% effective in inducing a postsynaptic response. We observed a significant increase in sIPSC frequency, from 0.6 ± 0.3 to 4.9 ± 1.6 Hz (P < 0.05, paired t-test, n = 6 cells; Fig. 3, A and B). In three cells tested, sIPSCs were completely abolished by addition of strychnine (2 µM) and bicuculline (20 µM; Fig. 3A). In three other cells tested, most sIPSCs were abolished with the addition of strychnine at a concentration of 1 µM, suggesting that most sIPSCs were glycinergic in nature.
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Interneurons
In view of the substantial enhancement of presynaptic events after
AVP, we further investigated AVP's actions on a population of 166 Rexed laminae VIII and IX cells that failed to demonstrate antidromic
activation and/or had a distinct morphology (smaller cell diameter,
i.e., 25.1 ± 1.2 µm; P < 0.05 vs. motoneurons; ANOVA). We label these as "interneurons" recognizing that this includes premotor and other unidentified ventral horn cells. As a
group, these cells displayed a lower resting membrane potential of
(59.7 ± 1.1 mV; P < 0.05, ANOVA) and higher
input resistance (167.1 ± 14.3 M
; P < 0.05, ANOVA). Responses to AVP included both postsynaptic and presynaptic components.
AVP INDUCES POSTSYNAPTIC RESPONSES.
In the presence of TTX, a majority of the tested neurons (86/93)
responded to AVP (1 µM) with membrane depolarization (14.9 ± 1.6 mV; n = 4 cells) or a mean inward current of
45.7 ± 8.6 pA (n = 86 cells; Fig.
5A). Responses were dose
dependent, with inward currents of 21.5 ± 5.4 and 6.7 ± 2.3 pA in response to AVP at 100 (n = 7 cells) and 10 nM
(n = 4 cells), respectively. Although of different
magnitude, the time course of these AVP responses were virtually
identical to the postsynaptic currents recorded in motoneurons and
resembled the time course of the AVP-induced changes in frequency of
their spontaneous and miniature postsynaptic currents. When we analyzed
voltage-current relationships to delineate net AVP current (as in Fig.
2B), the data collectively indicated only a minor
decrease in membrane conductance from a control value of
4.8 ± 0.6 to 4.4 ± 0.5 nS at the peak of the AVP response
(P > 0.05, paired t-test, n = 86). However, a comparison of the net AVP-induced currents for
individual cells revealed three significantly differing patterns (Fig.
5B). One group of 24 neurons demonstrated a net AVP-induced
conductance that decreased from a control of 4.7 ± 0.5 to 3.6 ± 0.4 nS (P < 0.05, paired
t-test) and reversed close to 100 mV (
101.8 ± 4.1 mV). This value approximated the potassium equilibrium potential under
these conditions, suggesting that the action of AVP in these cells was
mediated via reduction in conductance for potassium ions. By contrast,
in another 21 cells, the slope of the net AVP current reflected an
increase in conductance (from 4.5 ± 0.9 to 5.1 ± 1.1 nS; P < 0.05, paired t-test) with
current reversal at about
40 mV (
38.4 ± 2.9 mV). The latter
suggests that the AVP action was mediated via an increase in a
nonselective cationic conductance. In the remaining 41 cells, the mean
net AVP-induced current displayed a slight reduction in membrane
conductance (from 5.0 ± 0.7 to 4.7 ± 0.8 nS;
P > 0.05, paired t-test) but did not
reverse within the voltage range tested.
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AVP INDUCES PRESYNAPTIC RESPONSES. In addition to a postsynaptic inward current, all interneurons exposed to AVP in normal ACSF displayed an increase in the frequency and the amplitude of TTX-sensitive spontaneous postsynaptic potentials (e.g., Fig. 6, A and B). In the presence of amino acid receptor antagonists, it was apparent that these were composed of both inhibitory and excitatory events (Fig. 6, C and D), implying that AVP receptors were present on the somata and/or dendrites of both inhibitory and excitatory neurons that synapsed on interneurons. For IPSCs (n = 4 cells), frequency increased from 0.9 ± 0.2 to 11.2 ± 2.8 Hz (P < 0.01) while amplitudes increased from 27.9 ± 4.3 to 48.7 ± 6.1 pA (P < 0.05). For EPSCs (n = 5 cells), frequency increased from 1.7 ± 0.2 to 14.3 ± 3.1 Hz (P < 0.01) and amplitude increased from 16.4 ± 1.1 to 24.7 ± 3.2 pA (P < 0.05).
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DISCUSSION |
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This study provides electrophysiological evidence for the
existence of AVP V1 receptors on a majority of
thoracolumbar motoneurons and other ventral horn neurons (collectively
termed interneurons) in the neonatal rat spinal cord. Responses to
bath-applied AVP (10 nM to 3 µM) and V1
receptor agonists featured a slowly rising and prolonged TTX-resistant
inward current. In motoneurons, the net AVP-induced currents were
associated with a reduction in a membrane potassium conductance that
reversed near 100 mV. By contrast in interneurons, three patterns of
AVP-induced inward currents were evident: decreasing membrane
conductance with reversal near
100 mV; increasing conductance with
reversal near
40 mV; small reduction in conductance with no reversal
within the current range tested. A presynaptic component recorded in
most neurons was evident as an increase in the frequency, but not
amplitude (in motoneurons) of spontaneous IPSCs and EPSCs, attributed
to AVP-induced activity in inhibitory (mainly glycinergic) and
excitatory (glutamatergic) neurons synapsing on the recorded cells. On
the basis of AVP-induced TTX-sensitive increases in both IPSC and EPSC
spontaneous activity onto motoneurons, it can be concluded that both
excitatory and inhibitory premotor neurons must be included among the
"interneuron" population that is activated by AVP. An increase in
frequency, but not amplitude of mIPSCs and mEPSCs, also indicated
another presynaptic action of AVP, i.e., to enhance neurotransmitter
release from axon terminals of inhibitory and excitatory interneurons.
This further supports the broad distribution of pre- and postsynaptic
AVP receptors among ventral horn neurons in this age group. These data
supplement earlier electrophysiological evidence for AVP receptors
among a majority of lateral horn neurons in the neonatal spinal cord
(Kolaj and Renaud 1998
; Ma and Dun 1985
).
In our recent analysis (Kolaj and Renaud 1998
), we
reported that AVP and V1 agonists applied to
spinal preganglionic neurons and the majority of unidentified lateral
horn neurons also induced a prolonged, G-protein-coupled membrane
depolarization and TTX-resistant inward current. While net AVP-induced
currents in 36% of these lateral horn neurons reversed about
100 mV,
reflecting reduction in one or more barium-sensitive potassium
conductances, the net AVP-induced current in another 20% of cells
reversed about
40 mV, suggestive of an increase in a nonselective
cationic conductance. This resembles the data obtained from the
"interneuron" population in the current analysis (see following
text). These differences aside, the fact that a majority of cells
respond to AVP and that [125I]vasopressin
antagonist binding and AVP V1 receptor mRNA
expression are prominent in these areas of spinal cord
(Tribollet et al. 1997
), one is left with the impression
that AVP receptors are present on most neurons in the lateral and
ventral horns in the neonatal rat spinal cord.
Responses identified as "postsynaptic" are deemed to arise from
activation of AVP receptors located on the somata and dendrites of both
motoneurons and interneurons, and are responsible for the TTX-resistant
membrane depolarizations seen in current-clamp recordings and the
inward currents seen under voltage clamp. Analyses of I-V
relationships and net-AVP-induced inward currents indicate that two
different conductances are likely to mediate these responses to AVP. In
motoneurons, and in a population of interneurons, the linearity and the
reversal points for the net AVP-induced currents indicate
reduction of voltage-independent potassium conductances that
contribute to resting membrane potential, often referred to as
"leak" conductances. The features of these conductances, also noted
in a population of lateral horn neurons (Kolaj and Renaud
1998), resemble a neurotransmitter-modulated conductance observed in brain-stem motoneurons that has recently been attributed to
a family of two-pore domain pH-sensitive potassium channels named
TASK-1 (Talley et al. 2000
). However the validity of
this comparison with channels responsible for AVP-induced inward
current in neonatal spinal motoneurons remains to be established. By
contrast, the AVP-induced inward currents in another population of
interneurons are associated with an increase in membrane
conductance that reverses about
40 mV, suggesting opening of
nonselective cationic channels. Should AVP receptors couple with
both conductances, their competition in the same neuron
could explain the "parallel shift" and lack of reversal in net AVP
currents noted in a third population of interneurons. Perhaps more
importantly is the fact that here, as in lateral horn neurons, these
two patterns of AVP-induced conductances reflect mechanisms that can be
established early in development. In lateral horn cells, we
(Kolaj et al. 1999
) have suggested that these
conductances may be developmentally regulated based on observations
that ~70% of cells from slices at ages P8-P10 demonstrate evidence
of opening of nonselective cationic channels, whereas ~90% of cells
from slices at ages P18-P20 reveal AVP-induced inward currents
associated with reduction in a potassium conductance. Among ventral
horn interneurons, the smaller sample from the older age group
currently precludes our definition of a similar trend.
AVP-induced conductances seem to vary among cell types. In hypoglossal
motoneurons, AVP induces a noninactivating inward current that reverses
around 15 mV (Palouzier-Paulignan et al. 1994
). In
subfornical organ, AVP inhibits both a delayed rectifier
IK and a transient outward current
IA (Washburn et al.
1999
). In facial motoneurons AVP induces depolarization and
inward current via a persistent voltage-dependent sodium current
(Ragenbass et al. 1991
). In horizontal limb of diagonal
band of Broca neurons, AVP modulates conductance through
IC (Easaw et al. 1997
).
In cultured aortic A7r5 cells, AVP modulates activity by acting on
different types of ionic conductances, including a late nonselective
cationic channel that reverses at 5 mV (Byron and Taylor
1995
; Thibonnier et al. 1991
; Van
Renterghem et al. 1988
). Activation of AVP receptors can
increase calcium influx through high-voltage-activated calcium channels, as observed in area postrema neurons (Hay et al.
1996
), cerebral cortex, and hippocampus (Chen et al.
2000
; Mihara et al. 1999
). Thus AVP receptors
may be coupled with different signaling mechanisms and ion channels in
different types of neurons. The present study did not undertake ion
substitution or pharmacological blockade to characterize in more detail
the AVP-induced conductances.
AVP responses that we refer to as presynaptic comprise two categories.
In one situation, the term indicates synaptic inputs to the recorded
neuron from activated glycinergic/?GABAergic and glutamatergic
interneurons; these are responsible for the increase in baseline
"noise" and can be blocked with tetrodotoxin. A recent report
(Omura et al. 1999) noted that AVP can cause a reduction in the amplitude of glycinergic IPSPs in hippocampus, an effect not
observed in our spinal cord study. In the other situation, receptors
responsible for the AVP-induced increase in mIPSCs and mEPSCs are
considered as truly presynaptic and presumed to be located on the axon
terminals of inhibitory interneurons that synapse on motoneurons and on
axon terminals of glutamatergic and glycinergic/?GABAergic neurons that
synapse on interneurons. These distributions are schematically
summarized in Fig. 8.
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Presynaptic conductances are deemed important in the regulation of
transmitter release (Meir et al. 1999). Our data
indicate the presence of presynaptic AVP receptors that can change the frequency but not the amplitude of mIPSCs and mEPSCs, i.e., modulate release of inhibitory and excitatory transmitters. Mechanisms remain to
be identified but may include enhancement of calcium currents and/or
closing of potassium channels in presynaptic terminals. Verification
will pose a challenge.
An obvious question relates to the endogenous origin of the ligand
(AVP) for these V1 receptors. In the adult CNS,
one source could be through exocytosis from terminals of hypothalamic
AVP-synthesizing parvocellular neurons whose axons can be shown to
project to spinal lateral and ventral horn areas (Hallbeck and
Blomqvist 1999; Hallbeck et al. 1999
;
Wagner and Clemens 1993
). These pathways may mediate release of neurohypophysial peptides in spinal cord perfusates after
supraspinal stimulation (Pittman et al. 1984
). It
remains uncertain whether such neuronal connections are established or are sufficiently abundant in neonatal spinal cord. AVP has not been
detected in dorsal root ganglia neurons (Hallbeck et al. 1996
,
1999
), an argument against local synthesis and release. Not to
be overlooked, however, is AVP that circulates in cerebrospinal fluid
with a circadian rhythmicity that depends on the integrity of the
hypothalamic suprachiasmatic nucleus (Reppert et al.
1987
; Schwartz et al. 1983
). Reported levels
vary from 10 to 30 pg/ml in adult rat spinal subarachnoid space (e.g.,
Pittman et al. 1984
)
100 pg/ml in the preoptic recess
in fetal lambs in utero (Stark and Daniel 1989
),
sufficient to activate these receptors.
What role may such widely distributed AVP receptors have in spinal cord
function? Observations to date leave little doubt that their activation
can enhance neuronal excitability, thereby rendering neurons more
responsive to excitatory inputs. In neonatal tissue, this may have
important consequences for neuronal survival and development. Of note
are reports of impaired neural functions in the vasopressin-deficient
Brattleboro rat (reviewed in Bohus and de Weid 1998).
AVP does influence neuronal growth and development in various
preparations (reviewed in Carter et al. 1993
), including hippocampal and cortical neuronal cultures (Brinton et al.
1994
; Chen et al. 2000
; Tarumi et al.
2000
), and accelerates neurite outgrowth from cultured
embryonic neurons (Brinton and Gruener 1987
).
Interestingly, AVP, but not oxytocin, is reported to have a
neurotrophic action in cultured explants of spinal cord (Iwasaki et al. 1991
). AVP belongs to a family of vasoactive and
mitogenic peptides that participate in physiological and pathological
cell growth and differentiation (Van Biesen et al. 1996
)
and can activate a set of kinases that are known for their importance
in cell survival (Thibonnier et al. 2000
). The
observation that the expression of AVP binding and
V1 receptors can be regulated by sex steroids and
by nerve injury (Chritin et al. 1999
; Tribollet
et al. 1994
, 1997
) is perhaps an additional reflection of the
multiple roles that AVP receptors impart in target neurons. Studies of
the distribution pattern and responses to activation of AVP receptors
in the mature spinal cord and in response to injury (cf.
Tribollet et al. 1994
) may provide some insight on these issues.
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ACKNOWLEDGMENTS |
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This work was supported by the Canadian Institutes for Health Research and Canadian Heart and Stroke Foundation.
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FOOTNOTES |
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Address for reprint requests: L. P. Renaud, Neurology/Neurosciences, Ottawa Hospital Civic Campus, 1053 Carling Ave., Ottawa, Ontario K1Y 4E9, Canada (E-mail: lprenaud{at}ohri.ca).
Received 26 February 2001; accepted in final form 30 May 2001.
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REFERENCES |
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