561
Journal of Physiology (1992), 458, pp. 561-577 With 9 figures Printed in Great Britain
EFFECTS OF VASOPRESSIN AND ANGIOTENSIN II ON NEURONES IN THE RAT DORSAL MOTOR NUCLEUS OF THE VAGUS, IN VITRO BY ZUN-LI MO, TOSHIHIKO KATAFUCHI, HIROSHI MURATANI AND TETSURO HORI From the Department of Physiology, Faculty of Medicine, Kyushu University 60, Fukuoka 812, Japan
(Received 5 March 1991) SUMMARY
1. Extracellular recordings were made from 297 spontaneously firing neurones in the dorsal motor nucleus of the vagus (DMV) in slice preparations of rat medulla oblongata. Some of the neurones recorded were identified to be vagal motoneurones by antidromic stimulation. The cells fired with a slow irregular pattern at an average rate of 1 1 + 041 spikes/s (mean + S.E.M.). 2. Arginine vasopressin (AVP) was applied by perfusion in 196 of the 297 cells. Most of the neurones (190/196, 97 %) were excited by 10-6 M AVP with an increase in firing rate from the basal level of 11 + 0-1 to a maximum of 2-5 + 0-2 spikes/s. There was a dose-dependent relation between the concentration of AVP and the increased firing rate in all DMV neurones tested (n = 38). The threshold concentration of the peptide to produce changes in firing rate was assumed to be about 10-10 M. The remaining six neurones were not affected by application of AVP. 3. Application of oxytocin (OXT, 10-6 M) increased the firing rate of all thirtyeight neurones tested. The effects of AVP and OXT on all neurones examined (n = 20 and 4, respectively) still persisted after blocking the synaptic transmission in a low-Ca2+ or Ca2+-free-high-Mg2+ solution, indicating the direct action of both AVP and OXT on the postsynaptic membranes. 4. The AVP-induced excitatory responses were completely but reversibly blocked by the Vl-type receptor antagonists, [1-(,f-mercapto-/,,i-cyclopentamethylenepropionic acid), 2-(O-methyl)tyrosine]-arginine vasopressin (d(CH2)jTyr(Me)AVP) (n = 5) and Phaa-D-Tyr(Et)Phe-Gln-Asn-Lys-Pro-Arg-NH2 (n = 6), whereas a selective and reversible OXT receptor antagonist, desGly-NH2d(CH2)jTyr(Me)2Thr4]ornithine vasotocin, which suppressed the OXT-induced excitation, did not block the responses to AVP (n = 11). 5. Application of angiotensin II (AII, 10-6 M) to 153 neurones increased the firing rates of 60 (39%) neurones. The firing rate was increased from the basal level of 1-0+041 to a maximum of 1-8+0-2 spikes/s (n = 60). The effect of All was completely abolished by an All receptor antagonist, [Sarl,lle8]angiotensin II (n = 6). There was a dose dependence of the excitatory response on All concentration in all of eleven neurones tested. The threshold concentration was assumed to be about MS 9206
562
Z -L. MO AND OTHERS
10-9 M. The activity of 5 (3 %) of 153 neurones was decreased, and the remaining 88 (58%) neurones were not affected by AII. 6. After blocking synaptic transmission with low-Ca2+-high-Mg2+ medium, ten of sixteen neurones that had been excited by application of All in normal medium still responded to All, and the effects were abolished in the remaining six cells. 7. Of fifty-nine cells tested with both AVP and AII applied at 10-6 M, responses of fifty-four (92 %) to AVP were excitation. Of these fifty-four, fifteen were also excited by application of All, one was inhibited, and All had no effect on the remaining thirty-eight (70 %). 8. The excitatory responses induced by AVP were completely or partially blocked by simultaneous perfusion of All in normal medium, which were restored by [Sarl,lle8]AII, whereas the application of All alone had little effect. The suppressive effect of All on the AVP-induced excitation was abolished during perfusion with Ca2+-free-high-Mg2+ solution. 9. The results suggest that both AVP and OXT modulate vagal output mainly by increasing the firing rates of DMV neurones through an action on receptors for each peptide. All also increased the activity of DMV neurones, but the number of cells excited and the increase in activity were less by All than by AVP. In addition, All suppresses AVP-induced excitation, an effect that may be mediated by synaptic transmission. INTRODUCTION
Recent morphological and physiological evidence has suggested that the neurohypophyseal hormones, arginine vasopressin (AVP) and oxytocin (OXT), may act as neurotransmitters and/or neuromodulators in the dorsal brainstem, including the dorsal motor nucleus of the vagus (DMV) and the nucleus of the solitary tract (NST) (Swanson, 1977; Charpak, Armstrong, Muhlethaler & Dreifuss, 1984; Sofroniew, 1985; Riphagen & Pittman, 1986; Dubois-Dauphin & Zakarian, 1987; Raggenbass, Tribollet, Dubois-Dauphin & Dreifuss, 1989). The actions of these peptides in the brainstem are considered to be related to some autonomic regulation, such as cardiovascular (Riphagen & Pittman, 1986) and gastric functions (McCann & Rogers, 1990). Although both AVP and angiotensin II (All) are vasoconstrictor agents and are simultaneously released into the blood stream during volume depletion resulting from, for instance, haemorrhage (Hall & Hodge, 1971; Szczepanska-Sadowska, 1973), it has been shown that they have opposing effects on the sensitivity of the cardiac baroreflex in dogs, sheep and rats. AVP facilitates efferent cardiac vagal activity, reflex bradyeardia and reflex inhibition of sympathetic nerve activity, whereas All attenuates all of these (Lumbers, McCloskey & Potter, 1979; Courtice, Kwong, Lumbers & Potter, 1984; Schmid, Guo & Abboud, 1985; Peuler, Edwards, Schmid & Johnson, 1990). Interaction between the effects of the two peptides has also been reported; All attenuates the cardio-inhibitory effects of AVP (Caine, Lumbers & Reid, 1985). Although it has been suggested that these effects may be mediated by their central actions, and partly by peripheral actions, the underlying mechanisms are not yet completely understood. It has been demonstrated that circulating and central AVP are released
563 EFFECTS OF A VP AND AII ON DMV NEURONES simultaneously by osmotic and hypovolaemic stimuli (Pittman, Veale & Lederis, 1982; Riphagen & Pittman, 1986). All, as well as AVP, has also been suggested to exert some cardiovascular functions acting as a neurotransmitter/neuromodulator in the DMV and NST (Diz, Barnes & Ferrario, 1987). These findings raise the possibility that AVP and All may be involved in the reflex regulation of blood pressure when they are released from nerve terminals. Regulation of baroreflex sensitivity by these two peptides may also occur, at least in part, at the level of the DMV, a major site in the efferent pathway of the vagus. In the present study, the activity of rat DMV neurones was recorded extracellularly in vitro to compare the effects of AVP and OXT. Furthermore, the possibility of interactions between the effects of AVP and AII was also examined. METHODS
Tissue preparation Experiments were performed on brainstem transverse slices from 120-150 g male Wistar rats. A rat was stunned by a blow to the back and the whole brain, including the upper cervical spinal cord, was quickly removed and cut into blocks freehand with a razor blade. Coronal brain slices, 400 4um thick, containing the dorsal motor nucleus of the vagus (DMV) were prepared with a vibratome-type slicer and preincubated in Krebs-Ringer solution equilibrated with 95% 02 and 5 % CO2 at room temperature. Three slices of DMV tissue were usually obtained from each brain. After preincubation for at least 1 h, the slices were transferred to a 1-0 ml recording chamber with the Krebs-Ringer solution flowing through at a rate of 2-3 ml/min. The temperature of the recording chamber was kept at 32 + 0-5°C by heating the mantle. The composition of the Krebs-Ringer solution was (mM): NaCl, 124; KCI,50;M,g4,1-3; KH2P 4,1-23; CaCl2,24; NaHCO3, 26; and glucose, 10 (pH 7 4). Synaptic transmission was blocked by changing the normal solution to free- or low-(0-2 mM) Ca2+-high-Mg2+ (12 or 6 mM) solution. The pH of the perfusing medium was not affected by adding peptides at the concentrations used.
Extracellular recordings Extracellular single neurone activity was recorded from the DMV with a glass micropipette (tip diameter 2-3 ,um; DC resistance, 8-15 MQl) filled with 0 5 M sodium acetate containing saturated Pontamine Sky Blue. Action potentials were amplified, displayed on an oscilloscope and stored on magnetic tape for further analysis. After passing through a window discriminator, the number of impulses per 2 s were counted by a rate-meter and recorded on a chart recorder. In some experiments, in order to determine whether the recording DMV neurones were the vagal motoneurones, a single pulse stimulation (0 1-0{3 mA, 0 5 ms) was given antidromically to the axons of vagal motoneurones running ventrolaterally from the DMV in the slice. The stimulation was delivered through bipolar stimulation electrodes which were made of twisted tungsten wires and insulated except for their tips. Criteria for identifying an antidromically evoked action potential were: (1) constant latency of the evoked spike; (2) action potentials following stimuli at more than 200 Hz in a one-to-one relation; (3) occasional failure of evoking a somatodendritic component of an action potential, leaving an initial segment spike only; and (4) a collision of the evoked action potential with a spontaneously occurring spike. At the end of each experiment, the recording site was marked with Pontamine Sky Blue by applying a constant cathodal current through the micropipette (1-5 1sA for 3 min). The slices were fixed in 10 % formalin solution for 6 h and sectioned at 100 ,um thickness with a freezing microtome. They were stained with Neutral Red in order to localize the blue spots. Peptides The peptides used in the experiments were arginine vasopressin (AVP), oxytocin (OXT), angiotensin II (All), All antagonist, [Sar1,Ile8]AII and the AVP antagonist, [1-(/8-mercapto-,/,?cyclopentamethylene propionic acid), 2-(O-methyl)tyrosine]-arginine vasopressin (d(CH2)5Tyr (Me)AVP; Peptide Institute, Minoh, Japan). Furthermore, we used a more potent and selective
564
Z.-L. MO AND OTHERS
AVP V1 antagonist, Phaa-D-Tyr(Et)Phe-Gln-Asn-Lys-Pro-Arg-NH2 (where Phaa is the abbreviation for phenylacetic acid), and OXT antagonist, desGly-NH2d(CH2)5[Tyr(Me)2Thr4]OVT (where OVT represents ornithine vasotocin), which were kindly supplied by M. M. Manning (Department of Biochemistry, Medical College of Ohio, Toledo, OH, USA). All peptides were dissolved in the Krebs-Ringer solution. Peptides were applied for 1 min by switching the perfusate from the control solution to the appropriate peptide-containing solution. The dead space in the tube leading to the chamber was about 3 0 ml.
Analysis of data Each neuronal response to a peptide was studied at least twice to verify the results. If the mean firing rate during 1 min changed by at least 20 % from its basal firing level (mean firing rate during 1 min) in response to application of a peptide at less than 10-6 M, the neurone was considered to be sensitive to that drug. The changes in firing rates after application of AVP and AII, and differences in the changes induced by the peptides were compared by Dunnett's multiple-range test following the analysis of variance. A simple x2 test was used to analyse differences between the numbers of neurones responding to the peptides. A 95 % level of confidence was accepted as statistically significant in all analyses. RESULTS
The DMV could be readily distinguished from surrounding structures under a dissecting microscope as a translucent area with light transmitted through the slice. This area was verified with reference to the stereotaxic atlas of Paxinos & Watson (1986). Histological verification by the dye marking technique indicated that all of the blue deposits were located in the DMV. Figure 1 illustrates typical examples of the histological sections. The locations of the other recording sites were determined by referring to the distance from the staining points. Extracellular recordings were obtained from a total of 297 spontaneously firing cells, which were all located within the boundaries of the DMV. Neurones located outside the DMV were discarded from the results. Some neurones were confirmed to be vagal motoneurones by the presence of an antidromic spike induced by stimulation of the efferent fibres of the DMV neurones. The average spontaneous firing rate of the DMV neurones was 1 1+0-1 spikes/s (-.iean+s.E.M., n = 297). Effects of AVP, AII and OXT were examined in, respectively, 196, 153 and 38 of the 297 neurones. Of these, 90 neurones were studied for their responsiveness to the three peptides applied either separately or
simultaneously. Effects of A VP on DMV neurones Out of 196 neurones, 190 (97%) were excited by bath application of 10-6 M AVP. The average firing rate of these cells increased from 1-1 + 0-1 to 2-5 + 0-2 spikes/s (mean+s.E.M., n = 190) after AVP application. As shown in Fig. 2A, the neuronal activity started to increase within 3 min and reached a maximum about 5 min after application of the peptide. The firing rate recovered to the pre-infusion level 10-30 min after switching back to the control solution. The remaining six of the 196 cells did not respond to AVP. To investigate dose-response relationships, thirty-eight neurones which responded to 106 M AVP were tested at different AVP concentrations ranging from 10-10 to 10' M. Although twenty-eight of thirty-eight neurones were not examined, the
EFFECTS OF A VP AND AII ON DMV NEURONES
565
remaining ten cells were electrophysiologically identified to be vagal motoneurones. The responses of unidentified neurones were essentially the same as those of the vagal motoneurones. The increase in firing rates of the DMV neurones after application of
Fig. 1. Histological sections showing deposits made by ejecting Pontamine Sky Blue (arrows) in the dorsal motor nucleus of the vagus (DMV) at the level of area postrema (B) and more rostral part (A). The DMV on the left was surrounded by a dashed line. A and B from different animals. AP, area postrema; NST, nucleus of the solitary tract; XII, hypoglossal nucleus. Calibration, 500 ,um. Neutral Red stain. Magnification x 44.
AVP at each concentration was calculated from the rate-meter records and plotted against concentration (Fig. 2B). The threshold AVP concentration to evoke responses of the DMV neurones was about 10-10 M and the responses seemed to reach maximum at 10- M.
Z.-L. MO AND OTHERS
566
Effects of A VP on DMV neurones in Ca2+-free-high-Mg2+ solution About 15 min after changing the control perfusing medium to a Ca2+-free-highMg2+ medium, AVP was applied to ten neurones. Perfusion with this solution has been reported to completely abolish both spontaneous and evoked postsynaptic A A
[I I''''l1 2sj
1 mV
B
AP
AVP,MI
8
8
O
10-7M
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10 8M 1 09M N 0 E(33")"'S - ""-'I-''S'l'l''IIL
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10-M
(3A1) , 211
(4
r
(27) m
10O,6m 10-9210-810-
10-6 10 10-5 Concentration (M) Fig. 2. Responses of DMV neurones to AVP. A, rate-meter records show dose-dependent increase in firing rate after 10-' to 10-6 M AVP. Continuous records from upper to lower trace. Oscilloscope traces were obtained at the times indicated by the arrows. B, dose-response curve of increase in firing rate with perfusion of AVP. The data were obtained from thirty-eight DMV neurones. Each point represents mean + S.E.M. of three to six different doses. 5 min
potentials in DMV neurones within 10 min (Fukuda, Minami, Nabekura & Oomura, 1987). No neurone that had been excited in response to 10-6 M AVP in normal medium lost its responsiveness in the Ca2+-free medium (n = 10). Furthermore, we investigated the effects of AVP on ten other neurones which were identified to be motoneurones in the DMV by the presence of antidromic action potentials, and obtained the same results. Figure 3 shows an example in which the response of a vagal motoneurone to AVP is preserved in the Ca2+-free-high-Mg2+ solution.
Effects of OXT on DMV neurones Application of OXT at a concentration of 10-6 M excited all of DMV neurones tested (n = 38). During OXT perfusion the firing rate increased with a similar time course and intensity to those of AVP-induced excitation. The effects of OXT on all four neurones examined still persisted after blocking the synaptic transmission in a low-Ca 2+-high-Mg2+ solution (data are not shown).
Effects of A VP and OXT antagonists The effects of d(CH2)5Tyr(Me)AVP, an AVP antagonist which is specific to the V1 receptor (Kruszynski, Lammek, Manning, Seto, Haldar & Sawyer, 1980), on AVPinduced excitation of the DMV neurones were investigated. As shown in Fig. 4, the
EFFECTS OF A UP AND All OiN DMNllV 567 AVEURONKT5-E6S incrbease in firing rate following application of 10'6 M AVP (Fig. 4, top trace) was completely blocked by 10-6 M of the V -receptor antagonist. which by itself did not change the spontaneous discharge of the neurone (Fig. 4. middle trace). In all of five I)MV cells tested, d(CH2)5Tyr(Me)AVP completely or partially blocked the excitatory effects of AVP. 1 2~
~
AVP, 10
~
~
0
*
J1500 mV
M
6_L
12
Ca 2+-free-high- Mg2+
12
AVP,10o-
M
5 mi
Fig. 3. Effects of synaptic blockade on the response to AVP. The DMV motoneurone which had been excited by 10-6 M AVP in the normal solution (upper trace) was also
excited by lo-6 Mi AVP in Ca2+-free-high-Mg2+ medium (middle trace). In this case, the
response wvas enhanced. After wash-out, the neurone was excited again by AVP (bottom trace). Inset: left, fivTe superimposed sweeps showing constant latency of antidromic
spikes (0) evoked by stimulation19 (0 3 mA, 0 5 ins) of the vTagal efferent fibres (-); PHT right,45 cancellation of antidromic action potential in one sweep due to collision with a spontaneous spike ( *).
To further investigate the receptor specificity, we examined the effects of a more potent and selective AVP V1 antagonist (Phaa-D-Tyr(Et)Phe-Gln-Asn-Lys-Pro-ArgNH2) (Mfanning, Stoev, Kolodziejczyk, Klis, Kruszynski, Misicka & Olma, 1990) and a selective OXT antagonist (desGly-NH2d(CH2)5[Tyr(Me)2Thr4]OVT) (Manning, Kruszynski, Bankowski, Olma, Lammek, Cheng, Klis, Seto, Haldar & Sawyer, 1989) on the AVP- and OXT-induced excitation of the DMV neurones. As shown in Fig. 5, the increase in firing rate following application of OXT (06 M) (Fig. 5A-) was completely blocked by the OXT receptor antagonist (10-6 M) which by itself did not alter the spontaneous discharge of the neurone,-while the excitatory response to AVP 19
PHY 458
Z.-L. M1O AND OTHERS
568
(1o-6 M) neurones
not suppressed in the same neurone (n = 11). Five of such eleven were identified as vagal motoneurones which responded to AVP in a Ca2+_
was
deficient solution. In another series of experiments, we found that the selective V1 antagonist (10-I6 M) completely abolished the responses of six DMV neurones to AVP AVP, 10-6
IE
M
n _ d(CH2)5Tyr(Me)AVP, 1O'6 M AVP, 106'M
4 U,
00 1
111HUMMOMMLli
. .1
AVP, 10-6 M
40 5 min
Fig. 4. Effects of a V1-type receptor antagonist, d(CH )5Tyr(Me)AVP, on the DMV neurone response induced by AVP. An excitatory response to 1o-6 M AVP (upper trace) was completely blocked by the simultaneous perfusion 10-6 M of the V1 receptor antagonist without affecting the spontaneous firing rate of the neurone (middle trace). After washing out. AVP excited the neurone again (bottom trace).
without affecting those to OXT (Fig. 5B). Thus, the response of DMV neurones to AVP and OXT are mediated, respectively, through activation of each specific receptor.
Effects of All on DMUV neurones Sixty (39 %) of 153 neurones were excited by application of All at a concentration of 106 M with a time course similar to that of the AVP-induced responses (Fig. 6). The average firing rate of these cells increased from 1 0 + 0 I to 1 8 + 0-2 spikes/s after All application. All had no effect on eighty-eight (58 %) neurones, although in some neurones the firing rate changed by less than 10% from its basal level. Five (3 %) were inhibited by 10' M All. The dose dependence of the excitatory response to All was observed in eleven neurones tested. The threshold concentration of All was assumed to be around 10' M. In addition, the excitation with All was completely blocked by All anitagonist. [Sarl,lle8]AII in all of six neurones tested (data are not shown).
569 EFFECTS OF A VP ANTD All ON DM1-T NElURONES Effects of synaptic suppression on the All-induced excitation were examined in sixteen neurones. Of the sixteen cells, ten still responded to All in the low-Ca2+-highMg2+ medium (Fig. 6A). However, the excitatory effects were abolished in six neurones (Fig. 6B). Although the spontaneous activity was reduced in the Ca21A
B
OXT, 1 0 6 M
12
n
6 OXT antagonist *1 0. M OXT10-M 12 -I
6
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-
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106 M
6
** desGly-N H2d(CH 2)5slyr(Me)2"Fhi 43VT ** Phaa-oD -Tyrt(Et)Phe- Gin-Asn L.ys Pro -Arg-NH2 5 min
Fig. 5. Effects of selective OXT receptor antagonist, desGly-NH2d(CH2)5jTyr (Me)2Thr4]OVT, and AVP V1-type receptor antagonist, Phaa-D-Tyr(Et)Phe-Gln-AsnLys-Pro-Arg-NH2, on the excitatory responses of DMV neurones induced by OXT and AVP. In A, the excitatory response to OXT (10-6 M) was blocked by the simultaneous perfusion of the OXT receptor antagonist (10-6 M) without affecting the spontaneous firing rate of the neurone, whereas the excitatory response to AVP (10-6 M) was not blocked. In B, the excitatory response to AVP (10-6 M) was completely blocked by the perfusion of the VI antagonist (10-6 M), but the response to OXT (10-6 M) still remained.
deficient solution, the abolition of the responses to All was not due to a non-specific suppression of neuronal activity, since the application of AVP induced an excitation of activity in this condition (Fig. 6B, lower trace).
Comparison of the effects. of A VP and AII on DMV neurones There was a difference between responses of DMV neurones to AVP and All at concentrations of 10-6 M. The proportion of the cells excited by AVP was significantly higher than that excited by All (190/196 (97%) vs. 60/153 (39%), x2 = 138-, P < 0-01). In addition, AVP had a greater stimulating effect than All at the same concentration (10-6 M). Although the resting firing rates of neurones before application of AVP and All were not different (1 +01, n = 190 and 10+04 spikes/s, n = 60, respectively, P > 005), the maximum discharge rate after application of AVP (2-5 + 0-2 spikes/s) was significantly greater than that induced by All (1-8+0-2 spikes/s) (Dunnett's multiple-range test, P < 001). 19-2
Z.-L. MO AND OTHERS
570
Relationship between the responses to A VP and All Fifty-nine cells Mwere tested for the responsiveness to both AVP and All at concentrations of 10-6 Ai. Fifty-four (920%) of the fifty-nine cells were excited by AVP: fifteen (250%) of these were also excited by application of All, and one was A
B
Before _ 6
6
_______
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Journal of Physiology (1992), 458, pp. 561-577 With 9 figures Printed in Great Britain
EFFECTS OF VASOPRESSIN AND ANGIOTENSIN II ON NEURONES IN THE RAT DORSAL MOTOR NUCLEUS OF THE VAGUS, IN VITRO BY ZUN-LI MO, TOSHIHIKO KATAFUCHI, HIROSHI MURATANI AND TETSURO HORI From the Department of Physiology, Faculty of Medicine, Kyushu University 60, Fukuoka 812, Japan
(Received 5 March 1991) SUMMARY
1. Extracellular recordings were made from 297 spontaneously firing neurones in the dorsal motor nucleus of the vagus (DMV) in slice preparations of rat medulla oblongata. Some of the neurones recorded were identified to be vagal motoneurones by antidromic stimulation. The cells fired with a slow irregular pattern at an average rate of 1 1 + 041 spikes/s (mean + S.E.M.). 2. Arginine vasopressin (AVP) was applied by perfusion in 196 of the 297 cells. Most of the neurones (190/196, 97 %) were excited by 10-6 M AVP with an increase in firing rate from the basal level of 11 + 0-1 to a maximum of 2-5 + 0-2 spikes/s. There was a dose-dependent relation between the concentration of AVP and the increased firing rate in all DMV neurones tested (n = 38). The threshold concentration of the peptide to produce changes in firing rate was assumed to be about 10-10 M. The remaining six neurones were not affected by application of AVP. 3. Application of oxytocin (OXT, 10-6 M) increased the firing rate of all thirtyeight neurones tested. The effects of AVP and OXT on all neurones examined (n = 20 and 4, respectively) still persisted after blocking the synaptic transmission in a low-Ca2+ or Ca2+-free-high-Mg2+ solution, indicating the direct action of both AVP and OXT on the postsynaptic membranes. 4. The AVP-induced excitatory responses were completely but reversibly blocked by the Vl-type receptor antagonists, [1-(,f-mercapto-/,,i-cyclopentamethylenepropionic acid), 2-(O-methyl)tyrosine]-arginine vasopressin (d(CH2)jTyr(Me)AVP) (n = 5) and Phaa-D-Tyr(Et)Phe-Gln-Asn-Lys-Pro-Arg-NH2 (n = 6), whereas a selective and reversible OXT receptor antagonist, desGly-NH2d(CH2)jTyr(Me)2Thr4]ornithine vasotocin, which suppressed the OXT-induced excitation, did not block the responses to AVP (n = 11). 5. Application of angiotensin II (AII, 10-6 M) to 153 neurones increased the firing rates of 60 (39%) neurones. The firing rate was increased from the basal level of 1-0+041 to a maximum of 1-8+0-2 spikes/s (n = 60). The effect of All was completely abolished by an All receptor antagonist, [Sarl,lle8]angiotensin II (n = 6). There was a dose dependence of the excitatory response on All concentration in all of eleven neurones tested. The threshold concentration was assumed to be about MS 9206
562
Z -L. MO AND OTHERS
10-9 M. The activity of 5 (3 %) of 153 neurones was decreased, and the remaining 88 (58%) neurones were not affected by AII. 6. After blocking synaptic transmission with low-Ca2+-high-Mg2+ medium, ten of sixteen neurones that had been excited by application of All in normal medium still responded to All, and the effects were abolished in the remaining six cells. 7. Of fifty-nine cells tested with both AVP and AII applied at 10-6 M, responses of fifty-four (92 %) to AVP were excitation. Of these fifty-four, fifteen were also excited by application of All, one was inhibited, and All had no effect on the remaining thirty-eight (70 %). 8. The excitatory responses induced by AVP were completely or partially blocked by simultaneous perfusion of All in normal medium, which were restored by [Sarl,lle8]AII, whereas the application of All alone had little effect. The suppressive effect of All on the AVP-induced excitation was abolished during perfusion with Ca2+-free-high-Mg2+ solution. 9. The results suggest that both AVP and OXT modulate vagal output mainly by increasing the firing rates of DMV neurones through an action on receptors for each peptide. All also increased the activity of DMV neurones, but the number of cells excited and the increase in activity were less by All than by AVP. In addition, All suppresses AVP-induced excitation, an effect that may be mediated by synaptic transmission. INTRODUCTION
Recent morphological and physiological evidence has suggested that the neurohypophyseal hormones, arginine vasopressin (AVP) and oxytocin (OXT), may act as neurotransmitters and/or neuromodulators in the dorsal brainstem, including the dorsal motor nucleus of the vagus (DMV) and the nucleus of the solitary tract (NST) (Swanson, 1977; Charpak, Armstrong, Muhlethaler & Dreifuss, 1984; Sofroniew, 1985; Riphagen & Pittman, 1986; Dubois-Dauphin & Zakarian, 1987; Raggenbass, Tribollet, Dubois-Dauphin & Dreifuss, 1989). The actions of these peptides in the brainstem are considered to be related to some autonomic regulation, such as cardiovascular (Riphagen & Pittman, 1986) and gastric functions (McCann & Rogers, 1990). Although both AVP and angiotensin II (All) are vasoconstrictor agents and are simultaneously released into the blood stream during volume depletion resulting from, for instance, haemorrhage (Hall & Hodge, 1971; Szczepanska-Sadowska, 1973), it has been shown that they have opposing effects on the sensitivity of the cardiac baroreflex in dogs, sheep and rats. AVP facilitates efferent cardiac vagal activity, reflex bradyeardia and reflex inhibition of sympathetic nerve activity, whereas All attenuates all of these (Lumbers, McCloskey & Potter, 1979; Courtice, Kwong, Lumbers & Potter, 1984; Schmid, Guo & Abboud, 1985; Peuler, Edwards, Schmid & Johnson, 1990). Interaction between the effects of the two peptides has also been reported; All attenuates the cardio-inhibitory effects of AVP (Caine, Lumbers & Reid, 1985). Although it has been suggested that these effects may be mediated by their central actions, and partly by peripheral actions, the underlying mechanisms are not yet completely understood. It has been demonstrated that circulating and central AVP are released
563 EFFECTS OF A VP AND AII ON DMV NEURONES simultaneously by osmotic and hypovolaemic stimuli (Pittman, Veale & Lederis, 1982; Riphagen & Pittman, 1986). All, as well as AVP, has also been suggested to exert some cardiovascular functions acting as a neurotransmitter/neuromodulator in the DMV and NST (Diz, Barnes & Ferrario, 1987). These findings raise the possibility that AVP and All may be involved in the reflex regulation of blood pressure when they are released from nerve terminals. Regulation of baroreflex sensitivity by these two peptides may also occur, at least in part, at the level of the DMV, a major site in the efferent pathway of the vagus. In the present study, the activity of rat DMV neurones was recorded extracellularly in vitro to compare the effects of AVP and OXT. Furthermore, the possibility of interactions between the effects of AVP and AII was also examined. METHODS
Tissue preparation Experiments were performed on brainstem transverse slices from 120-150 g male Wistar rats. A rat was stunned by a blow to the back and the whole brain, including the upper cervical spinal cord, was quickly removed and cut into blocks freehand with a razor blade. Coronal brain slices, 400 4um thick, containing the dorsal motor nucleus of the vagus (DMV) were prepared with a vibratome-type slicer and preincubated in Krebs-Ringer solution equilibrated with 95% 02 and 5 % CO2 at room temperature. Three slices of DMV tissue were usually obtained from each brain. After preincubation for at least 1 h, the slices were transferred to a 1-0 ml recording chamber with the Krebs-Ringer solution flowing through at a rate of 2-3 ml/min. The temperature of the recording chamber was kept at 32 + 0-5°C by heating the mantle. The composition of the Krebs-Ringer solution was (mM): NaCl, 124; KCI,50;M,g4,1-3; KH2P 4,1-23; CaCl2,24; NaHCO3, 26; and glucose, 10 (pH 7 4). Synaptic transmission was blocked by changing the normal solution to free- or low-(0-2 mM) Ca2+-high-Mg2+ (12 or 6 mM) solution. The pH of the perfusing medium was not affected by adding peptides at the concentrations used.
Extracellular recordings Extracellular single neurone activity was recorded from the DMV with a glass micropipette (tip diameter 2-3 ,um; DC resistance, 8-15 MQl) filled with 0 5 M sodium acetate containing saturated Pontamine Sky Blue. Action potentials were amplified, displayed on an oscilloscope and stored on magnetic tape for further analysis. After passing through a window discriminator, the number of impulses per 2 s were counted by a rate-meter and recorded on a chart recorder. In some experiments, in order to determine whether the recording DMV neurones were the vagal motoneurones, a single pulse stimulation (0 1-0{3 mA, 0 5 ms) was given antidromically to the axons of vagal motoneurones running ventrolaterally from the DMV in the slice. The stimulation was delivered through bipolar stimulation electrodes which were made of twisted tungsten wires and insulated except for their tips. Criteria for identifying an antidromically evoked action potential were: (1) constant latency of the evoked spike; (2) action potentials following stimuli at more than 200 Hz in a one-to-one relation; (3) occasional failure of evoking a somatodendritic component of an action potential, leaving an initial segment spike only; and (4) a collision of the evoked action potential with a spontaneously occurring spike. At the end of each experiment, the recording site was marked with Pontamine Sky Blue by applying a constant cathodal current through the micropipette (1-5 1sA for 3 min). The slices were fixed in 10 % formalin solution for 6 h and sectioned at 100 ,um thickness with a freezing microtome. They were stained with Neutral Red in order to localize the blue spots. Peptides The peptides used in the experiments were arginine vasopressin (AVP), oxytocin (OXT), angiotensin II (All), All antagonist, [Sar1,Ile8]AII and the AVP antagonist, [1-(/8-mercapto-,/,?cyclopentamethylene propionic acid), 2-(O-methyl)tyrosine]-arginine vasopressin (d(CH2)5Tyr (Me)AVP; Peptide Institute, Minoh, Japan). Furthermore, we used a more potent and selective
564
Z.-L. MO AND OTHERS
AVP V1 antagonist, Phaa-D-Tyr(Et)Phe-Gln-Asn-Lys-Pro-Arg-NH2 (where Phaa is the abbreviation for phenylacetic acid), and OXT antagonist, desGly-NH2d(CH2)5[Tyr(Me)2Thr4]OVT (where OVT represents ornithine vasotocin), which were kindly supplied by M. M. Manning (Department of Biochemistry, Medical College of Ohio, Toledo, OH, USA). All peptides were dissolved in the Krebs-Ringer solution. Peptides were applied for 1 min by switching the perfusate from the control solution to the appropriate peptide-containing solution. The dead space in the tube leading to the chamber was about 3 0 ml.
Analysis of data Each neuronal response to a peptide was studied at least twice to verify the results. If the mean firing rate during 1 min changed by at least 20 % from its basal firing level (mean firing rate during 1 min) in response to application of a peptide at less than 10-6 M, the neurone was considered to be sensitive to that drug. The changes in firing rates after application of AVP and AII, and differences in the changes induced by the peptides were compared by Dunnett's multiple-range test following the analysis of variance. A simple x2 test was used to analyse differences between the numbers of neurones responding to the peptides. A 95 % level of confidence was accepted as statistically significant in all analyses. RESULTS
The DMV could be readily distinguished from surrounding structures under a dissecting microscope as a translucent area with light transmitted through the slice. This area was verified with reference to the stereotaxic atlas of Paxinos & Watson (1986). Histological verification by the dye marking technique indicated that all of the blue deposits were located in the DMV. Figure 1 illustrates typical examples of the histological sections. The locations of the other recording sites were determined by referring to the distance from the staining points. Extracellular recordings were obtained from a total of 297 spontaneously firing cells, which were all located within the boundaries of the DMV. Neurones located outside the DMV were discarded from the results. Some neurones were confirmed to be vagal motoneurones by the presence of an antidromic spike induced by stimulation of the efferent fibres of the DMV neurones. The average spontaneous firing rate of the DMV neurones was 1 1+0-1 spikes/s (-.iean+s.E.M., n = 297). Effects of AVP, AII and OXT were examined in, respectively, 196, 153 and 38 of the 297 neurones. Of these, 90 neurones were studied for their responsiveness to the three peptides applied either separately or
simultaneously. Effects of A VP on DMV neurones Out of 196 neurones, 190 (97%) were excited by bath application of 10-6 M AVP. The average firing rate of these cells increased from 1-1 + 0-1 to 2-5 + 0-2 spikes/s (mean+s.E.M., n = 190) after AVP application. As shown in Fig. 2A, the neuronal activity started to increase within 3 min and reached a maximum about 5 min after application of the peptide. The firing rate recovered to the pre-infusion level 10-30 min after switching back to the control solution. The remaining six of the 196 cells did not respond to AVP. To investigate dose-response relationships, thirty-eight neurones which responded to 106 M AVP were tested at different AVP concentrations ranging from 10-10 to 10' M. Although twenty-eight of thirty-eight neurones were not examined, the
EFFECTS OF A VP AND AII ON DMV NEURONES
565
remaining ten cells were electrophysiologically identified to be vagal motoneurones. The responses of unidentified neurones were essentially the same as those of the vagal motoneurones. The increase in firing rates of the DMV neurones after application of
Fig. 1. Histological sections showing deposits made by ejecting Pontamine Sky Blue (arrows) in the dorsal motor nucleus of the vagus (DMV) at the level of area postrema (B) and more rostral part (A). The DMV on the left was surrounded by a dashed line. A and B from different animals. AP, area postrema; NST, nucleus of the solitary tract; XII, hypoglossal nucleus. Calibration, 500 ,um. Neutral Red stain. Magnification x 44.
AVP at each concentration was calculated from the rate-meter records and plotted against concentration (Fig. 2B). The threshold AVP concentration to evoke responses of the DMV neurones was about 10-10 M and the responses seemed to reach maximum at 10- M.
Z.-L. MO AND OTHERS
566
Effects of A VP on DMV neurones in Ca2+-free-high-Mg2+ solution About 15 min after changing the control perfusing medium to a Ca2+-free-highMg2+ medium, AVP was applied to ten neurones. Perfusion with this solution has been reported to completely abolish both spontaneous and evoked postsynaptic A A
[I I''''l1 2sj
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10-6 10 10-5 Concentration (M) Fig. 2. Responses of DMV neurones to AVP. A, rate-meter records show dose-dependent increase in firing rate after 10-' to 10-6 M AVP. Continuous records from upper to lower trace. Oscilloscope traces were obtained at the times indicated by the arrows. B, dose-response curve of increase in firing rate with perfusion of AVP. The data were obtained from thirty-eight DMV neurones. Each point represents mean + S.E.M. of three to six different doses. 5 min
potentials in DMV neurones within 10 min (Fukuda, Minami, Nabekura & Oomura, 1987). No neurone that had been excited in response to 10-6 M AVP in normal medium lost its responsiveness in the Ca2+-free medium (n = 10). Furthermore, we investigated the effects of AVP on ten other neurones which were identified to be motoneurones in the DMV by the presence of antidromic action potentials, and obtained the same results. Figure 3 shows an example in which the response of a vagal motoneurone to AVP is preserved in the Ca2+-free-high-Mg2+ solution.
Effects of OXT on DMV neurones Application of OXT at a concentration of 10-6 M excited all of DMV neurones tested (n = 38). During OXT perfusion the firing rate increased with a similar time course and intensity to those of AVP-induced excitation. The effects of OXT on all four neurones examined still persisted after blocking the synaptic transmission in a low-Ca 2+-high-Mg2+ solution (data are not shown).
Effects of A VP and OXT antagonists The effects of d(CH2)5Tyr(Me)AVP, an AVP antagonist which is specific to the V1 receptor (Kruszynski, Lammek, Manning, Seto, Haldar & Sawyer, 1980), on AVPinduced excitation of the DMV neurones were investigated. As shown in Fig. 4, the
EFFECTS OF A UP AND All OiN DMNllV 567 AVEURONKT5-E6S incrbease in firing rate following application of 10'6 M AVP (Fig. 4, top trace) was completely blocked by 10-6 M of the V -receptor antagonist. which by itself did not change the spontaneous discharge of the neurone (Fig. 4. middle trace). In all of five I)MV cells tested, d(CH2)5Tyr(Me)AVP completely or partially blocked the excitatory effects of AVP. 1 2~
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~
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12
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Fig. 3. Effects of synaptic blockade on the response to AVP. The DMV motoneurone which had been excited by 10-6 M AVP in the normal solution (upper trace) was also
excited by lo-6 Mi AVP in Ca2+-free-high-Mg2+ medium (middle trace). In this case, the
response wvas enhanced. After wash-out, the neurone was excited again by AVP (bottom trace). Inset: left, fivTe superimposed sweeps showing constant latency of antidromic
spikes (0) evoked by stimulation19 (0 3 mA, 0 5 ins) of the vTagal efferent fibres (-); PHT right,45 cancellation of antidromic action potential in one sweep due to collision with a spontaneous spike ( *).
To further investigate the receptor specificity, we examined the effects of a more potent and selective AVP V1 antagonist (Phaa-D-Tyr(Et)Phe-Gln-Asn-Lys-Pro-ArgNH2) (Mfanning, Stoev, Kolodziejczyk, Klis, Kruszynski, Misicka & Olma, 1990) and a selective OXT antagonist (desGly-NH2d(CH2)5[Tyr(Me)2Thr4]OVT) (Manning, Kruszynski, Bankowski, Olma, Lammek, Cheng, Klis, Seto, Haldar & Sawyer, 1989) on the AVP- and OXT-induced excitation of the DMV neurones. As shown in Fig. 5, the increase in firing rate following application of OXT (06 M) (Fig. 5A-) was completely blocked by the OXT receptor antagonist (10-6 M) which by itself did not alter the spontaneous discharge of the neurone,-while the excitatory response to AVP 19
PHY 458
Z.-L. M1O AND OTHERS
568
(1o-6 M) neurones
not suppressed in the same neurone (n = 11). Five of such eleven were identified as vagal motoneurones which responded to AVP in a Ca2+_
was
deficient solution. In another series of experiments, we found that the selective V1 antagonist (10-I6 M) completely abolished the responses of six DMV neurones to AVP AVP, 10-6
IE
M
n _ d(CH2)5Tyr(Me)AVP, 1O'6 M AVP, 106'M
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00 1
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Fig. 4. Effects of a V1-type receptor antagonist, d(CH )5Tyr(Me)AVP, on the DMV neurone response induced by AVP. An excitatory response to 1o-6 M AVP (upper trace) was completely blocked by the simultaneous perfusion 10-6 M of the V1 receptor antagonist without affecting the spontaneous firing rate of the neurone (middle trace). After washing out. AVP excited the neurone again (bottom trace).
without affecting those to OXT (Fig. 5B). Thus, the response of DMV neurones to AVP and OXT are mediated, respectively, through activation of each specific receptor.
Effects of All on DMUV neurones Sixty (39 %) of 153 neurones were excited by application of All at a concentration of 106 M with a time course similar to that of the AVP-induced responses (Fig. 6). The average firing rate of these cells increased from 1 0 + 0 I to 1 8 + 0-2 spikes/s after All application. All had no effect on eighty-eight (58 %) neurones, although in some neurones the firing rate changed by less than 10% from its basal level. Five (3 %) were inhibited by 10' M All. The dose dependence of the excitatory response to All was observed in eleven neurones tested. The threshold concentration of All was assumed to be around 10' M. In addition, the excitation with All was completely blocked by All anitagonist. [Sarl,lle8]AII in all of six neurones tested (data are not shown).
569 EFFECTS OF A VP ANTD All ON DM1-T NElURONES Effects of synaptic suppression on the All-induced excitation were examined in sixteen neurones. Of the sixteen cells, ten still responded to All in the low-Ca2+-highMg2+ medium (Fig. 6A). However, the excitatory effects were abolished in six neurones (Fig. 6B). Although the spontaneous activity was reduced in the Ca21A
B
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Fig. 5. Effects of selective OXT receptor antagonist, desGly-NH2d(CH2)5jTyr (Me)2Thr4]OVT, and AVP V1-type receptor antagonist, Phaa-D-Tyr(Et)Phe-Gln-AsnLys-Pro-Arg-NH2, on the excitatory responses of DMV neurones induced by OXT and AVP. In A, the excitatory response to OXT (10-6 M) was blocked by the simultaneous perfusion of the OXT receptor antagonist (10-6 M) without affecting the spontaneous firing rate of the neurone, whereas the excitatory response to AVP (10-6 M) was not blocked. In B, the excitatory response to AVP (10-6 M) was completely blocked by the perfusion of the VI antagonist (10-6 M), but the response to OXT (10-6 M) still remained.
deficient solution, the abolition of the responses to All was not due to a non-specific suppression of neuronal activity, since the application of AVP induced an excitation of activity in this condition (Fig. 6B, lower trace).
Comparison of the effects. of A VP and AII on DMV neurones There was a difference between responses of DMV neurones to AVP and All at concentrations of 10-6 M. The proportion of the cells excited by AVP was significantly higher than that excited by All (190/196 (97%) vs. 60/153 (39%), x2 = 138-, P < 0-01). In addition, AVP had a greater stimulating effect than All at the same concentration (10-6 M). Although the resting firing rates of neurones before application of AVP and All were not different (1 +01, n = 190 and 10+04 spikes/s, n = 60, respectively, P > 005), the maximum discharge rate after application of AVP (2-5 + 0-2 spikes/s) was significantly greater than that induced by All (1-8+0-2 spikes/s) (Dunnett's multiple-range test, P < 001). 19-2
Z.-L. MO AND OTHERS
570
Relationship between the responses to A VP and All Fifty-nine cells Mwere tested for the responsiveness to both AVP and All at concentrations of 10-6 Ai. Fifty-four (920%) of the fifty-nine cells were excited by AVP: fifteen (250%) of these were also excited by application of All, and one was A
B
Before _ 6
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_
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