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INTRODUCTION |
The nonapeptide arginine vasopressin (AVP) is commonly recognized as a hypothalamic neurohypophysial peptide synthesized by magnocellular neurons of the supraoptic and paraventricular nuclei and released into the plasma from their posterior pituitary axon terminals to function as a circulating pressor agent and antidiuretic hormone. However, a differential expression of AVP receptors in brain (see Tribollet et al. 1991
), detection of AVP in the cerebrospinal fluid (reviewed in Reppert et al. 1987
), evidence of an influence of AVP and related peptides on behavior (reviewed in deWied et al. 1991
), and demonstration of AVP-immunoreactive neurons and axons in CNS regions unrelated to posterior pituitary control (Buijs 1978
) fostered the notion that vasopressin has a role in neural function, in particular neurotransmission. Supporting data come from electrophysiological evidence that AVP has an influence on neuronal excitability in a variety of CNS sites, including hippocampus (Mühlethaler et al. 1982
), lateral septum (Raggenbass et al. 1988
), brain stem (Mo et al. 1992
; Palouzier-Paulignan et al. 1994
; Raggenbass et al. 1991
; Sun and Guyenet 1989
), area postrema (Lowes et al. 1995
), and spinal cord (Backman and Henry 1984
; Ma and Dun 1985
; Sermasi and Coote 1994
). In confirmation of binding and molecular biological studies, these responses are mediated by AVP receptors of the V1 subtype (Lolait et al. 1995
; Ostrowski et al. 1994
; Tribollet et al. 1991
, 1997
).
In spinal cord, AVP is implicated in central regulation of cardiovascular and renal function (Crowley 1982
; Noszczyk et al. 1993
; Pittman et al. 1982
; Riphagen and Pittman 1985a
,b
; 1989
). Anatomical tracer and immunocytochemical studies indicate that a population of parvocellular AVP-synthesizing neurons in the hypothalamic paraventricular nucleus, a center for autonomic regulation, project to the brain stem and spinal cord where fibers in the thoracolumbar cord have a distribution around the central canal and intermediolateral column, the principal location of sympathetic preganglionic neurons (SPNs) (Hosoya et al. 1991
; Saper et al. 1976
; Sawchenko and Swanson 1982
). Thus, because SPNs and other lateral horn neurons are physiologically relevant targets for an AVP innervation, there is interest in defining the properties of their AVP receptors. Although previous studies in adult cat in vivo (Backman and Henry 1984
) and neonatal rat in vitro (Ma and Dun 1985
; Sermasi and Coote 1994
) reported excitatory or depolarizing actions of AVP on SPNs and lateral horn cells, ionic mechanisms were not defined. With the patch-clamp technique applied in neonatal spinal cord slices, we now report that AVP triggers a G-protein-mediated inward current in a majority (90%) of lateral horn neurons, including all cells identified as SPNs. Additionally, our analysis of I-V relationships indicates that two ionic mechanisms may participate in these AVP-induced responses.
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METHODS |
Preparation
Transverse slices of lower thoracic spinal cord from neonatal (11-22 days) Sprague-Dawley rats of either sex were prepared as follows. Under methoxyflurane-induced anesthesia, a dorsal laminectomy was performed to access and remove a 10- to 15-mm segment of the thoracolumbar spinal cord, which was then immersed in oxygenated (95% O2-5% CO2) cooled (4°C) artificial cerebrospinal fluid (ACSF) of the following composition (in mM): 127 NaCl, 3.1 or 1.9 KCl, 0 or 1.2 KH2PO4, 1.3 MgCl2 or MgSO4, 2.4 CaCl2, 26 NaHCO3, 10 glucose, pH 7.3, 300 mosmol. Slices were cut at 400 µm on a vibratome and transferred to an incubation chamber filled with oxygenated ACSF at room temperature and stored for
1 h before transfer to a recording chamber where individual slices were continuously superfused with oxygenated ACSF (3-6 ml/min) at room temperature (23-26°C). In some experiments, slices were incubated for
18 h in either ACSF (control) or in ACSF containing pertussis toxin (PTX) at a concentration of 4-5 µg/ml, previously shown to inhibit G-protein-mediated responses in other slice preparations (Hsu 1996
).
Recording
With the blind technique, recordings were obtained with borosilicate thin-walled micropipettes (BORO, BF150-110-10, Sutter, Novato, CA) pulled on a Flaming-Brown puller (P87) and filled with an internal solution containing (in mM) 130 K-gluconate, 10 KCl, 10 NaCl, 1 MgCl2, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 1 ethyl glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 2 Mg-ATP, 0.3 guanosine-5'-triphosphate (GTP), 2 Lucifer yellow, and tris(hydroxymethyl)aminomethane (Tris) base at pH 7.3; electrodes had resistances of 3-7 M
. In some experiments, GTP was omitted from the solution. In others, GTP was substituted with 0.2 mM GTP-
-S, or 0.4 mM GMP-PNP, both nonhydrolyzable activators of G-proteins, or 0.8 mM GDP-
-S, which inhibits G-protein binding (Gilman 1987
). Correction of liquid junction potential was applied to recorded membrane currents and voltages. An access resistance <15 M
was considered acceptable. Input resistance was determined from the linear slope (i.e., between
50 and
80 mV) of the current-voltage (I-V) relationships.
Whole cell current- and voltage-clamp recordings (holding potential
65 mV for all voltage-clamp recordings) obtained from lateral horn neurons were filtered at 2 kHz with an Axopatch-1D amplifier (Axon Instruments, Foster City, CA). Membrane current and membrane potential were continuously monitored on an oscilloscope, displayed on a pen recorder, and stored on videotape for later analysis. A TL-1 or Digidata 1200 interface and p-CLAMP (version 6) software (Axon Instruments) were used on-line to generate current and voltage commands.
Application of agents
Agents were dissolved in ACSF at known concentrations and applied to the slice by a computer-controlled superfusion system (DAD-12, ALA Scientific Instruments Inc., Westbury, NY). Agents included CsCl, BaCl2, GTP, N-ethylmaleimide (NEM), Mg-ATP, Lucifer yellow, AVP, strophanthidine, and TTX from Sigma (St. Louis, MO), [(Phe2,Orn8)-vasotocin], DDAVP, and [
-mercapto-
,
-cyclopentamethylenepropionyl1, O-Me-Tyr2]-AVP, or Manning compound (Manning et al. 1993
) from American Peptide (Sunnyvale, CA), glibenclamide, 4AP, and TEA from RBI (Natick, MA), GTP-
-S, GMP-PNP, and GDP-
-S from Calbiochem-Novabiochem (San Diego, CA), or RBI and PTX from List Biological (Campbell, CA) or RBI. AVP was applied by either bath perfusion or local pressure ejection, both methods yielding similar results. Except for experiments with agonists and antagonists, G-protein manipulations, and potassium channel blockers, a majority of the cells and slices was exposed to the peptides only once as a precaution against possible receptor desensitization.
Data analysis
Off-line analyses were performed with Clampfit version 6 (Axon Instruments). Statistical comparisons between control and experimental values were determined with both the paired or unpaired Student's t-test and analysis of variance. Results are expressed as the means ± SE.
Cell identification and morphology
Slices containing cells injected with Lucifer yellow were transferred to a fixation medium (4% formaldehyde with 0.1 mM phosphate buffer) and stored overnight at 4°C. The following day slices were cleared for 45-60 min with dimethylsulfoxide and viewed under epifluorescence.
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RESULTS |
Observations were derived from a total of 273 lateral horn neurons. Within this population were 68 cells that we classified as SPNs on the basis of their antidromic activation (23/60 tested) after electrical stimulation of their axons in the ventral root exit and/or typical morphology with dendrites extending toward the central canal (45/100 tested) (cf. Shen and Dun 1990
; Krupp and Feltz 1993
; Pickering et al. 1991
). SPNs had a resting membrane potential of
65.1 ± 0.8 mV and input conductance of 3.2 ± 0.2 nS, which differed little from that for all lateral horn neurons, where the mean resting membrane potential was
63.4 ± 0.5 mV (range
51 to
82 mV) and input conductance measured 3.6 ± 0.2 nS. In all cells, depolarizing current pulses elicited action potentials of 76 ± 1 mV (measured from threshold; width at half-amplitude of 1.6 ± 0.1 ms), and these were usually followed by fast and slow afterhyperpolarizations with amplitudes of 23.1 ± 0.7 mV (n = 258) and 13.8 ± 0.4 mV (n = 222), respectively. Whereas~60% of lateral horn neurons displayed a hyperpolarization-activated time-dependent inward current (Ih) of varying degrees, this feature was seldom noted among the SPN population.
AVP V1 receptor-mediated depolarization and inward current
A majority (90%) of 273 cells tested, including all 68 SPNs, responded to AVP. A typical response in current-clamp mode (n = 11) was a slowly rising (30-200 s) and prolonged (5-20 min) membrane depolarization (mean 14.5 ± 2.1 mV) that triggered a burst of action potentials on a plateau that lasted several minutes (Fig. 1A). Although an increase in baseline noise at the peak of the response suggested a possible presynaptic component, we focused on a postsynaptic site of action. This was confirmed in voltage clamp in ACSF containing 1 µM TTX where, at a holding potential of
65 mV, responsive cells featured a dose-dependent (0.01-1 µM) inward current, also with a prolonged time course (Fig. 1B). Applications of AVP at intervals <15 min could be seen to undergo a progressive reduction in amplitude, possibly reflecting receptor desensitization; therefore, when repetitive tests were utilized (e.g., Fig. 3C), intervals of 25-30 min were allowed between successive applications.

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| FIG. 1.
Arginine vasopressin (AVP) induces a prolonged depolarization and dose-dependent inward current in lateral horn cells. A: whole cell current-clamp recording from a sympathetic preganglionic neuron (SPN, resting membrane potential 64 mV) illustrates that a 30-s AVP application (horizontal open bar) is followed by a slowly rising, prolonged, and reversible membrane depolarization, with spike discharges at the peak of the response. B: traces obtained in voltage-clamp mode (Vh 65 mV) from another unidentified lateral horn neuron illustrate AVP-induced dose-dependent inward currents. Downward deflections are inward currents resulting from hyperpolarizing voltage pulses (10 mV, 400-ms duration). Right: histogram of data pooled from 134 neurons illustrate the dose-dependent increase of maximal inward current amplitude produced by AVP (0.01 µM, n = 5; 0.1 µM, n = 47; 1 µM, n = 82; for 20-60 s). Data are expressed as means ± SE in all figures.
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| FIG. 3.
Subsequent AVP responses are affected by G-protein modulation. A and B: sample illustrations of the 1st (left traces) and 2nd (right traces) AVP-induced current responses recorded with electrodes containing GTP- -S (A) or GDP- -S (B). In the GTP- -S-loaded cell, note that the current fails to return to baseline (indicated by the dotted line) and the response to the 2nd AVP application produces only a minimal effect. In the GDP- -S-loaded cell, there is recovery of the current to baseline after the initial AVP application, but the response to a 2nd application is significantly smaller than the 1st or in control GTP-loaded cells. C: histograms of pooled data from 20 cells tested with 2 applications of AVP. *P < 0.05, and ***P < 0.001, compared with values obtained during 1st AVP application.
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As anticipated for V1-type AVP receptors, similar membrane depolarizations and/or inward currents (
25.4 ± 1.4 pA) were induced by application of a V1 receptor agonist, [(Phe2,Orn8)-vasotocin], 1 µM, n = 12/14 cells), and reversibly blocked after pretreatment with a V1 receptor antagonist (Manning compound; 0.1-1 µM; 14/14 cells). Only 2/9 cells responded to application of DDAVP, a V2 receptor agonist, but with markedly reduced currents (
6.5 ± 1 pA) and at concentrations of 10-25 µM (not illustrated).
Response to oxytocin
Because oxytocin-immunoreactive fibers and oxytocin binding was observed in rat spinal cord (Rousselot et al. 1990
; Tribollet et al. 1997
), AVP-sensitive neurons were also tested for their response to oxytocin. Although cells were found to be responsive, oxytocin (1-2 µM for 60 s) induced significantly smaller membrane depolarizations when compared with 1 µM AVP (3 ± 0.8 mV vs. 15.4 ± 3.9 mV; n = 5; P < 0.05). Under voltage-clamp conditions, tests with a specific oxytocin agonist [Thr4,Gly7]-oxytocin (1-2 µM for 30-60 s) revealed small inward currents (
18 ± 3 pA) in three of seven tested cells. Thus neonatal lateral horn cells expressed more robust responses to AVP than to oxytocin (see also Sermasi and Coote 1994
; but cf. Desaulles et al. 1995
).
Role of G-proteins
Because AVP receptors are G-protein coupled, and to verify that these electrophysiological responses involved G-proteins, we first compared data where the recording pipette solutions lacked GTP. Although not achieving a level of significance, the mean AVP-induced inward current measured for 39 cells (
27.2 ± 2.3pA) in the absence of GTP was lower than for 40 cells recorded in the presence of GTP (
33.8 ± 3.4 pA). We next loaded 17 cells (including 7 SPNs) with GTP-
-S (200 µM), a nonhydrolyzable derivative of GTP that activates G-proteins in an irreversible manner (Gilman 1987
). In 9/17 cells (including all 7 SPNs) a gradual inward current of
17.8 ± 3.9 pA was measurable at the 5-min interval after establishment of whole cell voltage clamp. Moreover, the response to the first application of AVP failed to demonstrate recovery (Fig. 2, A, bottom trace, and B, and Fig. 3A), still showing a significant residual current after 10 min (Fig. 2, B and D) and only a marginal response to a second AVP application after 25 min of wash (Fig. 3, A and C). Additionally, the initial AVP application resulted in a maximum current of
52.7 ± 6.1 pA, significantly greater than the
33.8 ± 3.4 pA measured in control cells (Fig. 2C). Similar results were obtained in five cells dialyzed with another G-protein activator, GMP-PNP (Fig. 2, C and D).

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| FIG. 2.
G-proteins contribute to the AVP-induced inward currents in lateral horn neurons. A: in voltage-clamp mode (Vh 65 mV) and in the presence of tetrodotoxin, the top trace displays a control AVP-induced inward current that recovers after several minutes. The bottom trace from another cell dialyzed with GTP- -S (0.2 mM, for 10 min) illustrates a larger inward current that fails to recover. B: averaged data illustrate the time course of the inward current induced by 1 µM AVP, measured as percentage of the maximum current vs. time in cells recorded with guanosine-5'-triphosphate (GTP, open symbols, control cells, n = 20) or with GTP- -S (closed symbols, n = 10). Note the persisting current in GTP- -S loaded cells. C: summary histograms of data for the maximal inward current produced by 1 µM AVP in control cells (n = 40, 3.9 ± 0.7nS) and in cells pretreated either with GDP- -S (n = 9, 3.4 ± 0.5 nS), GMP-PNP (n = 5, 3.7 ± 0.9 nS), or GTP- -S (n = 17, 3.8 ± 0.4 nS). Note that there was not significant difference in input conductances. All analogues of GDP or GTP were applied intracellularly through the patch pipette solution for at least 10-min before the AVP test. D: summary data to illustrate the effect of pretreatment cells with GDP- -S, GMP-PNP, and GTP- -S on the residual current measured 10 min after the beginning of the response, expressed as a percentage of maximal current. Note the significant increase in cells pretreated with nonhydrolyzable analogues of GTP. In control cells the internal solution contained GTP. *P < 0.05 and **P < 0.01, compared with values obtained without pretreatment.
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We also dialyzed nine cells with GDP-
-S (800 µM for 10 min), a stable analogue of GDP that competitively inhibits G-protein binding of and activation by GTP (Gilman 1987
). In this group which included three SPNs, response to the initial AVP application measured
18.7 ± 4.3 pA, a significant (P < 0.05) reduction of 55% from the control (Figs. 2C and 3C), whereas the response to a subsequent AVP application was markedly blunted (Fig. 3, B and C).
A number of investigations focused on signaling mediated through AVP receptors. The consensus appears to indicate that V1 receptors stimulate production of inositol phosphates and likely couple with cholera- or PTX-insensitive G-proteins of the Gq/11 family in vascular and hepatic membrane and in brain (Thibonnier 1992
). However, there is now evidence for involvement of additional G-proteins in different tissues (e.g., Thibonnier et al. 1997
). To examine the PTX sensitivity of G-proteins involved in AVP-induced depolarization, slices were incubated for 12-18 h either in ACSF or in ACSF containing PTX. The data from eight cells recorded from eight PTX-pretreated slices (Fig. 4) revealed a significantly smaller mean AVP-induced inward current when compared with those from nine cells in control slices (
23.6 ± 7.6 pA vs.
53.4 ± 10.8 pA; P < 0.05). The duration of response in PTX-exposed cells was increased only marginally over controls. In view of the apparent PTX sensitivity of the response, we also tested the effects of NEM, a sulfhydryl-alkylating agent that has been shown to block G-protein-effector interactions by alkylating the
-subunits of PTX-sensitive GTP-binding proteins (Shapiro et al. 1994
; Viana and Hille 1996
). After a 5-min bath application of NEM (50 µM), AVP-induced currents were reduced to 34% of control amplitudes (Fig. 4, n = 5, P < 0.01, includes 1 SPN), an effect that was partly reversible (to 80% of control) in three of five neurons tested. The duration of the AVP-induced response was also affected, with residual current at 10 min being 18.1 ± 13.2% of the maximum for control versus 6.4 ± 6.4% for responses with NEM (P > 0.05). These observations suggest that both PTX-sensitive and -insensitive G-proteins participate in the AVP-induced current in neonatal spinal lateral horn neurons.

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| FIG. 4.
AVP-induced current requires the activation of pertussis toxin (PTX)-sensitive G-proteins. Left histogram: normalized control AVP-induced current for the other columns. Middle histogram: overnight incubation in media containing PTX (4-5 µg/ml) results in a marked reduction in the maximal AVP-induced inward current (n = 8, 3.3 ± 1 nS). The control group of neurons was recorded from slices incubated under the same conditions but without PTX (n = 9, 3.8 ± 0.6 nS). Right histogram: data from AVP-induced currents in cells exposed to bath applied N-ethylmaleimide (50 µM for 5 min, n = 5, 3.2 ± 0.6 nS). *P < 0.05, compared with values obtained without pretreatment.
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AVP activates different membrane conductances
Despite the increase in mean peak currents with higher AVP concentrations (Fig. 1B), we noted a marked variation among neurons in membrane conductances associated with the AVP responses, with some cells showing an obvious inward current but only a marginal conductance change. For 57 cells, we therefore looked at the profile of the net AVP current (obtained by subtraction of the I-V relationships obtained under control conditions and at the peak of a response) induced by 1 µM AVP (Fig. 5, A-D). The net AVP current for all cells revealed an overall reduction but no reversal in membrane conductance in the hyperpolarizing direction and some voltage rectification above
40 mV (Fig. 5E). However, when we compared the data from individual cells, 37 cells demonstrated a decrease in conductance (from 3.9 ± 0.3 to 3.3 ± 0.3 nS; P < 0.001), 7 cells showed an increase (from 3.5 ± 0.5 to 3.9 ± 0.6 nS; P < 0.05), and the remainder showed no consistent change. We then separated cells into three groups according to their I-V relationships, recognizing that SPNs were represented within each group.

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| FIG. 5.
Net AVP-induced current. A: voltage-clamp trace (dotted line depicts holding current at Vh = 65 mV) illustrates inward current in response to application of AVP. B and C: current responses to a series of voltage pulses (duration 600 ms, from 130 to 20 mV with 10-mV steps) applied before (control) and at the peak (AVP) of the AVP-induced response shown in A. D: I-V plots constructed from values taken at the end of the pulse (open and closed symbols, representing control and drug-induced states, respectively). Net AVP-induced current (triangles) was determined by subtraction of the I-V obtained at the peak of the AVP response (AVP) from that obtained before application of AVP (control). E: mean net AVP current for 57 cells indicates reduction in the hyperpolarizing direction but lacks an obvious reversal in this voltage range; note the rectification at potentials more positive than 40 mV.
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For one group of 21 cells (37%, 7 being identified as SPNs), I-V plots converged near
100 mV, with a net AVP-induced current that reversed at
102.8 ± 3.1 mV (Fig. 6A, solid symbols); in 17 of these cells, membrane conductance decreased from 3.8 ± 0.5 to 3.2 ± 0.4 nS (P < 0.01) and was unchanged in the rest. Near resting membrane potentials, the shape of these plots suggested that the net AVP current was not particularly voltage sensitive in that its amplitude was linearly related to the driving force on K+ (Vm
EK+). The linearity of this current assures that it would contribute substantially to the leak conductance of the membrane potential, even at rest.

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| FIG. 6.
Net AVP-induced inward current analysis indicates 3 patterns in I-V relationships. A, open symbols: data from 24 cells with no obvious reversal potentials. Solid symbols: data from 21 cells where there was inward current that reversed close to EK+. Shaded symbols: data from 12 cells demonstrating an increase in membrane conductance with a reversal close to 40 mV. B: amplitude histogram of the AVP-induced inward currents for the different conductance patterns. Note that the maximum amplitude for cells where no reversal was observed is significantly larger than that for cells with reversals around either 100 or 40 mV. *P < 0.05, compared with values between cells with no reversal vs. cells with other I-V patterns.
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Another group of 12 cells (21%, includes 4 identified as SPNs) displayed I-V plots that also indicated a reversal of the net AVP-induced current, but at a more depolarized level of
37.6 ± 2.8 mV (Fig. 6A, shaded symbols). For this group, measurements of membrane conductance revealed an increase in five cells (from 3.8 ± 0.5 to 4.4 ± 0.7 nS; P < 0.05), a decrease in four cells (from 2.7 ± 0.5 to 2.3 ± 0.5 nS; P < 0.05), and no change in the remainder. The I-V relationships in these cells displayed outward rectification at potentials more positive than
40 mV.
By contrast, the remaining group of 24 cells (42%, 3 identified as SPNs) had I-V relationships and net AVP-induced currents that indicated no reversal (Fig. 6A, open symbols). In this group, 16 cells showed an AVP-induced decrease in membrane conductance (from 4 ± 0.4 to 3.4 ± 0.4 nS; P < 0.01), and the remainder was without change. It is notable that the maximum inward current induced by AVP was significantly larger for cells with no reversal (Fig. 6B).
These data suggest that AVP can depolarize neonatal spinal lateral horn neurons, including SPNs, by at least two conductances. One is largely voltage independent, coupled with a decrease in membrane conductance and reverses close to the equilibrium potential for potassium (
98 mV in our conditions); the other has outward rectification, is coupled with a modest increase in membrane conductance, and reverses near
40 mV.
Ionic mechanisms
In cells whose I-V relationships demonstrated a reversal near
100 mV, changing [K+]out from 3.1 to 10 mM shifted the AVP current reversal from
106 ± 3.1 mV to
69 ± 2.6 mV, respectively (Fig. 7, A-C), as anticipated according to the Nernst equation. In ACSF containing 10 mM [K+], the net AVP-induced inward current (at a holding potential approximately
65 mV) was substantially reduced from
25.8 ± 8.2 pA (in normal ACSF) to
10.8 ± 4.2 pA (n = 4; P < 0.05).

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| FIG. 7.
AVP-induced inward current is associated with a decrease in a potassium conductance. A: in normal artificial cerebrospinal fluid (ACSF), ramp analysis (voltage increases linearly from 110 to 40 mV in 7 s) reveals that AVP decreases the membrane conductance with a reversal potential close to the potassium equilibrium potential. B: in ACSF containing 10 mM potassium, reversal potential shifts toward the predicted EK+ (from 98 to 68 mV according Nernst equation) C: histograms illustrate that the I-Vs during application of AVP (0.1-1 µM) in ACSF with [K+]o of 3.1 mM reverses at 106 ± 3.1 mV (n = 15) and the shift of this reversal to 69 ± 2.6 mV (n = 4) after increasing [K+]o to 10 mM.
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To characterize this potassium conductance(s) further, cells were tested for the effects of inhibitors of various K+ currents. After switching to ACSF containing 2 mM barium, nine cells (including 3 identified SPNs) displayed a persistent mean inward current of
18.3 ± 2.6 pA, similar to that induced by AVP, accompanied by a reduction in membrane input conductance, from 2.7 ± 0.2 to 1.9 ± 0.2 nS (P < 0.01). The I-V relationships indicated that this current decreased with hyperpolarization and reversed at a Vm of approximately
91 mV, close to the presumed EK+ (Fig. 8A), suggesting that the barium-activated inward current resulted from a decrease in one or more resting potassium conductances. Because of the similarity between the barium- and AVP-induced conductances (compare solid symbols in Fig. 6A with Fig. 8A), we hypothesized that AVP and barium might influence the same conductances. In fact, in five cells tested in ACSF that contained barium, we observed a significant reduction in the magnitude of the AVP-induced current (Fig. 8, B and C), whereas none of the cells tested with several other potassium channel blockers, including 4AP (5 mM), TEA (10 mM), and glibenclamide (20 µM), demonstrated a similar effect (Fig. 8C). The I-V relationship of the barium-sensitive component of inward current induced by AVP (obtained by subtracting the I-V plot obtained in normal media from that obtained in ACSF with barium, Fig. 8B) had characteristics similar to the net current produced by barium or AVP respectively, with a current that reversed at approximately
92 mV, close to the estimated EK+. On the other hand, the residual barium-insensitive component of the net AVP current had a quite different I-V relationship, with an opposite slope and a reduction to zero near
40 mV (Fig. 8B).

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| FIG. 8.
AVP-induced currents display both barium-sensitive and -insensitive components. A: data from 9 cells reveal a net barium-induced inward current (for calculation of net current see legend for Fig. 5). Note the resemblance between the barium and the AVP-induced current in Fig. 6A (solid symbols). B: data from 5 cells where AVP (1 µM for 20-50 s) were applied 1st in normal ACSF and (15-20 min later) after preincubation in ACSF containing barium (2 mM for 5-10 min). The I-V plots were obtained before and during each AVP application. Solid symbols represent the net AVP-induced current under control conditions. Net barium-insensitive AVP-induced current was calculated from I-Vs obtained during barium pretreatment and at the peak of the AVP response in ACSF containing barium (open squares); this represents the residual AVP-induced current in the presence of barium. The net barium-sensitive AVP-induced current (open diamond) was obtained by subtracting the barium-insensitive from the control AVP-induced current. Note that the barium-insensitive plot displays an opposite slope when compared with the control or the barium-sensitive plot, suggesting that some other conductance likely contributes to this effect. C: summary histograms illustrate the influence of different potassium channel blockers on the AVP-induced inward current. In each cell, AVP was 1st applied (control), and then cells were exposed to ACSF containing blockers for 5-15 min before testing with a 2nd AVP application. The amplitude of AVP-induced inward current was measured at the maximum of response. *P < 0.05, compared with values obtained without pretreatment. Blockers included glibenclamide (GLB, 20 µM), 4-aminopyridine (4AP, 5 mM), tetraethylammonium chloride (TEA, 10 mM), cesium (Cs, 1 mM), and barium (Ba, 2 mM).
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As indicated in Fig. 6A (shaded symbols), a population of neurons displayed another AVP-induced conductance that was outwardly rectifying and reversed near
40 mV (Fig. 6A, shaded symbols), a profile that is suggestive of a nonselective cationic conductance (c.f. Swandulla and Partridge 1990
). One possibility for this conductance is Ih, a cesium-sensitive and nonselective cationic conductance that is permeable to both potassium and sodium ions (Pape 1996
). As mentioned earlier, ~60% of cells (excludes SPNs) display a time-dependent, hyperpolarization-activated inward current of varying degrees (e.g., Fig. 9, A and C). To explore the contribution of this conductance to the AVP-induced current, we examined cells that showed no I-V reversals and/or reversals at approximately
40 mV. Anticipating a difference between their instantaneous and steady-state I-V relationships, we found that the magnitude of the net AVP-induced currents in 22 cells was independent of where they were measured and that the net AVP-induced inward current for cells with reversal around
40 mV was still active at potentials of
50 to
60 mV, where Ih was already inactive (Fig. 6A, gray symbols, and Fig. 9, B and D). Additionally, the application of cesium (1 mM) blocked any Ih conductance (n = 8) but was without effect on the AVP-induced current (n = 4). Furthermore, an Ih conductance was also present in cells where the net AVP-induced current reversed close to EK+ (n = 12). Therefore these data suggest that the conductance underlying Ih has little role in the proposed cationic conductance observed to contribute to the AVP-induced currents in certain spinal lateral neurons.

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| FIG. 9.
Hyperpolarization-activated inward current (Ih) does not contribute to AVP-induced inward current. A and C: current traces in response to 10-mV voltage steps (600-ms duration; Vh 65 mV) recorded from a cell where the net AVP-induced current reversed around 40 mV. B: I-V plots before AVP application, where the open symbols represent instantaneous current (Iins) measured at the end of the capacitive transient and closed symbols represent steady-state current (Iss) measured at the end of voltage steps. The difference between Iins and Iss (diamond symbol) represents the time- and voltage-dependent inward current Ih. D: I-V plots during the peak of an AVP-induced response, where the open symbols again represent instantaneous current (Iins) and the closed symbols represent steady-state current (Iss). The net currents (square symbols), representing the difference between I-V plots taken during and before the AVP application, are virtually identical for measurements of instantaneous and steady-state currents and suggest minimal, if any, contribution of Ih to the AVP response.
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A possible explanation for a parallel shift in the I-V relationships is that AVP activates ionic flow via an electrogenic Na+-K+ pump or exchange mechanism, thereby affecting membrane potential without changing membrane resistance. Indeed, vasopressin's depolarizing actions in guinea pig supraoptic nucleus neurons was reported to be blockable with ouabain, an irreversible Na+-K+ ATPase inhibitor (Abe et al. 1983
). To test for a possible role for Na+-K+ ATPase in the AVP-induced currents, we first applied strophanthidin (50 µM), a reversible inhibitor of Na+-K+ ATPase. This resulted in an inward current of
93 ± 32.9 pA in four cells, and two cells showed no obvious reversal in their I-V relationships. When AVP was applied during the strophanthidin effect, an additional inward current of
69.3 ± 23.5 pA, similar to that in control neurons, was recorded in four cells, with two cells showing a parallel shift in their I-V relationships. These data argue against a role for a Na+-K+ ATPase in the AVP-induced inward current.
We cannot exclude that closing of potassium conductances and an insufficient space clamp might cause a parallel shift in the AVP-induced I-V relationships. However, our data show that cells with a parallel shift actually demonstrate significantly larger amplitudes (Fig. 6B) in AVP-induced currents when compared with other cells, supporting a role for two conductances.
 |
DISCUSSION |
These observations support a neuronal distribution for the AVP binding that was noted in neonatal spinal tissue (Tribollet et al. 1991
). These data from the neonatal period indicate that a majority (90%) of lateral horn neurons express functional AVP receptors of the V1 subtype, responding to a transient exogenous application of AVP with an inward current that results in membrane depolarization. As anticipated, our findings are consistent with a role for G-proteins in signaling at these neuronal AVP receptors. In addition, responses appear to involve both PTX-sensitive and -insensitive components, suggesting involvement of multiple G-proteins. Extending earlier reports of a depolarizing action of AVP on neonatal spinal neurons, our analyses of I-V relationships and net AVP-induced currents in individual cells led us to conclude that AVP receptors may induce membrane depolarization by altering one or both of two conductances. In one population of cells where the response to AVP resulted in a decrease in membrane conductance at resting levels, we conclude that the dominant component of the AVP-induced inward current is due to reduction in a barium-sensitive resting or leak potassium conductance that decreases at hyperpolarizing potentials and reverses at a potential close to the estimated EK+. In another population of cells we noted that the AVP-induced inward current was associated with an increase in membrane conductance and involved an increase in a barium-insensitive conductance that reversed near
40 mV. We propose that the latter reflect a nonselective cationic conductance that is separate from Ih, with a charge carrier that remains to be defined. When individual cells expressed a predominance of one or other conductance, the profile of the net AVP currents permitted easy distinction. However, it appears that inward currents in some neurons can be mediated through both conductances, and their competition could explain both the parallel shifts observed in their I-V relationships and the uncertainty about net AVP-induced conductances. In fact, there are now several situations where postsynaptic actions of peptides or other transmitters increase a voltage-independent cation conductance and decrease an inward potassium conductance, for example, in hippocampus (Benson et al. 1988
), brain stem (Bayliss et al. 1992
; Dong et al. 1996
; Shen and North 1992a
,b
), and spinal cord lateral horn (Kolaj et al. 1997
).
It remains a matter of speculation as to why AVP receptors would couple to two different conductances. Because a leak potassium current contributes to the resting membrane potential in these neurons, one role for these AVP receptors might be to facilitate their initial responsiveness to excitatory neurotransmitters through membrane depolarization and an increase in input resistance. Indeed, one feature of neonatal SPNs is the presence of fast excitatory postsynaptic events mediated via non-N-methyl-D-aspartate (NMDA) and NMDA glutamate receptors (Krupp and Feltz 1995
; Mo and Dun 1987
; Spanswick and Logan 1990
; Spanswick et al. 1998
). A second nonselective cationic conductance could also serve to induce membrane depolarization at a stage when blockade of the resting potassium conductance would be an inefficient means of increasing cellular activity as, for example, during sustained hyperpolarization. Another possibility is that the latter is a dendritic conductance, as suggested for similar types of current changes during cholinergic stimulation in guinea pig hippocampus (Benson et al. 1988
). AVP might then serve to strengthen a dendrite-soma differential because it could promote increased synaptic efficiency in a somatic rather than a dendritic compartment by decreasing, rather than increasing, membrane conductance.
These observations contrast with results of investigations of AVP receptors in the brain stem of newborn rat, where depolarizing responses in hypoglossal and facial motoneurons were interpreted to arise through a TTX-resistant inward current and opening of persistent voltage-dependent sodium channels (Palouzier-Paulignan et al. 1994
; Raggenbass et al. 1991
). In the study on facial nucleus (Raggenbass et al. 1991
), the authors found no evidence of blockade of a potassium conductance, whereas the inclusion of cesium in the recording pipettes in the study on hypoglossal neurons (Palouzier-Paulignan et al. 1994
) likely precluded recognition of an altered potassium conductance. Reasons for these differences are unclear and may in part reflect development or neuronal specificity with receptors coupling through G-protein(s) that influence different conductances.
It is appreciated that AVP V1 receptors may alter different conductances in different tissues. For instance, in hepatocytes AVP increases [Na+]in and activates cation-selective channels, which could account for their AVP-activated calcium influx (Lidofsky et al. 1993
). In guinea pig ventricular myocytes and in insulin-secreting cells, AVP can potentiate voltage-sensitive calcium (probably L-type) channels (Thorn and Petersen 1991
; Zhang et al. 1995
). Even more complex is the AVP action on A7r5 rat smooth muscle cells; here, V1-type receptor activation stimulates IP3 formation and mobilizes intracellular calcium stores with consequent activation of capacitative calcium entry (Thibonnier et al. 1991
). Furthermore, vasopressin activates bivalent cation entry and a calcium efflux pathway (Byron and Taylor 1995
) and inward current caused by the opening of nonselective cation channels (Iwasawa et al. 1997
; VanRenterghem et al. 1988
).
Possible functions of AVP receptors in spinal cord
In the adult spinal cord, both AVP and oxytocin are proposed to have a neurotransmitter and/or neuromodulatory role in central regulation of autonomic function. Both AVP- and oxytocin-immunoreactive fibers observed in the dorsal horn, central gray, and intermediolateral horn areas (e.g., Buijs 1978
; Rousellot et al. 1990) are deemed to originate largely from parvocellular peptidergic neurons in the hypothalamic paraventricular nucleus (Cechetto and Saper 1988
; Hosoya et al. 1991
; Sawchenko and Swanson 1982
; Swanson 1977
; Swanson and McKeller 1979). Moreover, stimulation localized to PVN can increase the levels of AVP and oxytocin in spinal cord perfusates (Pittman et al. 1984
) and produce a V1 receptor-mediated increase in renal sympathetic nerve activity in rat (Malpas and Coote 1994
; Riphagen and Pittman 1989a
,b
) and cardioaccelerator and pressor responses in cat (Ciriello and Calaresu 1980
). In spinal cord, most of the attention has focused on a role for AVP, in part because its intrathecal administration can be seen to alter cardiovascular and renal function (Riphagen and Pittman 1985a
,b
, 1989a
,b
), and its iontophoretic application in cat was shown to increase the excitability of SPNs (Backman and Henry 1984
). These features are consistent with an AVP-induced membrane depolarization in neonatal SPNs and lateral horn neurons (see also Ma and Dun 1985
; Sermasi and Coote 1994
). Collectively, the data support the notion that AVP receptors may have an excitatory neurotransmitter/modulator role in autonomic outflow pathways. Although spinal lateral column neurons and SPNs are also responsive to oxytocin (Backman and Henry 1984
; Desaulles et al. 1995
), the present observations did not indicate a comparatively prominent role for oxytocin receptors in these neonatal neurons. However, the question remains as to whether these spinal AVP (and oxytocin) receptors have yet been reached by a descending peptidergic innervation at this stage of development.
In view of the preceding comment and other observations, alternative roles for AVP receptors that are unrelated to neurotransmission are worthy of consideration. With cerebrospinal fluid AVP demonstrating a circadian rhythmicity in several species (Reppert et al. 1987
; Schwartz et al. 1983
) at resting levels of 10-30 pg/ml in adult rat spinal subarachnoid space (e.g., Pittman et al. 1984
) and
100 pg/ml in the preoptic recess in fetal lambs in utero (Stark and Daniel 1989
), it is possible that AVP receptors may have a role in neuronal development. For example, in explanted spinal cord cultures, AVP, but not oxytocin, is reported to have a neurotrophic action (Iwasaki et al. 1991
). Furthermore, the demonstration by Tribollet et al. (1994
, 1997)
that the expression of AVP binding in spinal cord can be regulated by sex steroids and by nerve injury (Tribollet et al. 1997
) suggests that AVP receptors can be up- or down-regulated and apparently reexpressed under certain conditions, for example, after neuronal injury (Tribollet et al. 1994
). It may be of interest in future studies to determine how these manipulations in AVP receptors correspond with the electrophysiological properties reported here.