NOBUKUNI OGATA* 0-3 mM-EGTA. 3. Amplitude of the vasopressin

J. Physiol. (1983), 337, pp. 665-685 665With 14 text-figure8Printed in Great Britain

THE EFFECTS OF VASOPRESSIN ON ELECTRICAL ACTIVITY IN THEGUINEA-PIG SUPRAOPTIC NUCLEUS IN VITRO

BY HIDEHIKO ABE, MASUMI INOUE, TADASHI MATSUO ANDNOBUKUNI OGATA*

From the Department of Pharmacology, Faculty of Medicine, Kyushu University,Fukuoka 812, Japan

(Received 14 September 1982)

SUMMARY

1. Brain slices of the guinea-pig hypothalamus were used to determine the effectsof vasopressin on intracellular potentials in neurones of the supraoptic nucleus.

2. Vasopressin (0-05-1 i.u./ml.) depolarized the membrane without apparentchange in the input resistance and decreased the spontaneous firing rate. This actionof vasopressin was retained in the medium containing 0 mM-Ca2+, 12 mMMg2+ and0-3 mM-EGTA.

3. Amplitude of the vasopressin-induced depolarization was voltage-independent.Ion-substitution experiments showed that the changes in [K+]O, [Cl-10 and [Ca2+]0had little effect upon the amplitude of vasopressin-induced depolarization, whereasthe depletion of [Na+]. slightly reduced the amplitude.

4. The vasopressin-induced depolarization was blocked at a temperature of 15 0Cand by ouabain in a dose of 10-4 M.

5. Dibutyryl cyclic AMP (2 mM) produced electrophysiological effects similar tothose seen with vasopressin, and actions of both agents were potentiated by eitherpapaverine (10-4 M) or theophylline (10-2 M).

6. Contents of cyclic AMP in tissues incubated with vasopressin were significantlyhigher than in cases of incubation with normal Krebs solution.

7. We conclude that vasopressin directly modulates the activity of supraopticneurones, possibly through activation of adenylate cyclase.

INTRODUCTION

The hypothalamic magnocellular system which secretes vasopressin or oxytocinwas the first peptidergic system detected in the mammalian brain (Bargmann &Scharrer, 1951), and is the best understood of such systems in the mammalian brain.Electrophysiological properties of the magnocellular neurosecretory cells have longbeen investigated, mainly using extracellular unit recordings, in particular the firingpatterns in relation to secretion of the neurohormones (for review, see Poulain &Wakerley, 1982).

Micro-ionophoretic application of vasopressin inhibits the firing of supraoptic

* To whom correspondence should be addressed.

H. ABE, M. INOUE, T. MATSUO AND N. OGATA

neurones (Nicoll & Barker, 1971). The cells in the supraoptic nucleus appear to beextensively interconnected, and about two thirds of the synaptic boutons impingingon neurosecretory cells seem to be of intranuclear origin (Leranth, Zaiborszky, Marton& Palkovits, 1975). These observations suggest that vasopressin itselfmay play a rolein the modulation of neuronal excitability in the magnocellular neurosecretorysystem.

Vasopressin may also act as a neurotransmitter or neuromodulator, outside ofthe hypothalamo-neurohypophysial system. Immunohistochemical and biochemicalstudies indicate that this peptide is present in a wide variety of regions in the centralnervous system (e.g. Swanson, 1977; Dogterom, Snijdewint & Buijs, 1978; Sofroniew& Weindl, 1978). Vasopressin has a number of central effects: e.g. brain-stemvasopressin may be involved in altering cardiovascular reflex activity (M6hring,Schoun, Kintz & McNeill, 1980); behavioural studies suggest a role for vasopressinin learning and memory (De Wied & Gispen, 1977); this peptide might activatestress-induced mesocortical dopamine synthesis (Delanoy, Kramarcy & Dunn, 1982);and vasopressin may mediate febrile convulsions (Kasting, Veale, Cooper & Lederis,1981). Furthermore, there is considerable evidence to suggest that this peptide canmodify neuronal excitability of mammalian central neurones (Miihlethaler, Dreifuss& Gihwiler, 1982) and of invertebrate neurones (Barker & Gainer, 1974). Despitethese numerous central actions, little is known of the cellular mechanism of actionsof vasopressin on neurones in the central nervous system.We now report the action of vasopressin on the electrophysiological property of

cells in the supraoptic nucleus of guinea-pig hypothalamic slices. The mechanismsof vasopressin action, particularly in relation to activation of adenylate cyclase, arediscussed, since some actions of this peptide in the non-neural target organs arethought to be mediated via cyclic AMP-generating systems (e.g. Orloff & Handler,1967).

METHODS

Adult guinea-pigs of either sex were decapitated, and coronal slices of the hypothalamus(400-600 ,tm thick) were prepared using a stainless-steel slicer.

Incubation and recordingThe procedures for incubation and recording were as described previously (Abe & Ogata, 1982).

The brain slice was placed on a small agar block, and the temperature-controlled perfusate floweddown directly onto the slice (about 2 ml./min) from a polyethylene tube (inner diameter, 1 mm)the open end of which was positioned just above the slice. The volume of the medium pool on theagar block was about 0-1 ml. The standard medium was of the following composition (mM): NaCl,124; NaHCO3, 13; KCl, 5; CaCl2, 2-6; KH2PO4, 1-24; MgSO4, 1-3; glucose, 10. The slices were super-fused with the standard medium equilibrated with 97 %02 and 3 % C02, and the pH of the mediumwas kept at 7-3. Unless otherwise specified, the temperature of the medium was kept at 32 'C.

Intracellular recordings were made from the supraoptic nucleus through conventional glassmicro-electrodes filled with 3 M-KCl (d.c. resistance, 100-200 MQ). Electrical signals were ledthrough a high input impedance d.c. amplifier, displayed on an oscilloscope, photographed, andrecorded on a pen-recorder. Two types of pen-recordings were employed to facilitate illustrationof both d.c. potential change and spike component: one, a linear amplification but with a narrowhigh-frequency band width; the other, a relatively broader high-frequency band width but witha non-linear amplification. Unless otherwise stated, the recording was made by a pen-recorder witha linear amplification and a narrow high-frequency band width.Membrane input resistance was measured routinely by passing hyperpolarizing current pulses

(0 4 Hz, 0-5 sec pulse duration) of known intensity through the recording electrode by means of a

666

VASOPRESSIN AND SUPRAOPTIC NUCLEUS

conventional bridge circuit. Spontaneous firing rate was counted every 10 sec by a rate-meter andplotted on another channel of the pen-recorder.

Assay for cyclic AMP levelThe tissues were frozen with liquid nitrogen immediately after incubation, homogenized

(3000 rev/min) in 0-5 ml. cold 6 % (w/v) trichloracetic acid, and centrifuged at 3000 rev/min for15 min. The supernatant was treated three times with water-saturated ether and then bubbled with100% N2 gas to evaporate the residual ether. Total levels of cyclic AMP in the tissues weredetermined with a radioimmunoassay kit (Yamasa Shoyu Co. Ltd), and expressed as p-mole/mgwet weight.

DrugsThe following drugs were used: lysine-vasopressin (Sigma); oxytocin (Sandoz); dibutyryl-

adenosine 3',5'-cyclic monophosphate monosodium (dibutyryl cyclic AMP, P-L biochemicals);adenosine; tetrodotoxin, theophylline, papaverine, ouabain, y-aminobutyric acid (GABA); mono-sodium L-glutamate; tetraethylammonium chloride (TEA); ethylene glycol-bis (aminoethyl ether)N,N'-tetraacetic acid (EGTA); atropine sulphate.The experimental data are given as mean + S.E. of the mean, and n represents the number of cells

examined.

RESULTS

Some of the basic properties of neurones in the supraoptic nucleus have beendescribed elsewhere (Abe & Ogata, 1982). The stable intracellular recordings from thesupraoptic nucleus could be maintained for several hours (3-10 hr). The restingmembrane potential measured upon withdrawal of the electrode at the end of theexperiment was - 58-8 + 1 9 mV (n = 25). Membrane input resistance measured byinjecting hyperpolarizing current pulses was 157-2 + 15-2 MQ (n = 25). Among 171cells examined, 131 cells were spontaneously active whereas forty cells were silent.Spontaneous small depolarizing potentials of variable amplitude (2-10 mV) andduration (10-50 msec) (see insets in Fig. 5C-F where these potentials changed theiramplitude voltage-dependently) were observed in about three-quarters of the totalnumber of cells examined.

Effects of vasopressin on membrane potential and input resistanceVasopressin reversibly depolarized supraoptic cells, dose-dependently (Fig. 1A,

upper traces). Tachyphylaxis was not apparent with repeated application of vaso-pressin. The relationship between the concentration of vasopressin and the amplitudeof the depolarization is shown in Fig. 1B. Vasopressin in a concentration of0.05 i.u./ml. depolarized the membrane slightly (less than 1 mV) in four cells out ofthirteen cells tested. With 1 i.u. vasopressin/ml., fifty-six out of sixty-five supraopticcells depolarized by 6-8 + 0-8 mV, whereas the remaining nine cells were unaffected.When the concentration of vasopressin was increased to 5 i.u./ml., the depolarizationwas much the same as that produced by 1 i.u./ml. in all three vasopressin-responsivecells tested.During the vasopressin-induced depolarization, no change in the input resistance

was observed in any cell tested, as shown in Fig. 1 A.Oxytocin also depolarized the membrane, but this effect was about 100 times less

potent than that of vasopressin, i.e. a dose of 20 i.u. oxytocin/ml. was required toproduce the same amplitude of depolarization as that induced by vasopressin at0'2 i.u./ml. (Fig. 1B).

667


A

Vasopressin 1 i.u./ml.

dsac 30 sec 1 s4sec 40se 60Bsec

8

E5:

C0

N1

0.10'36V

E

8B

4

0i

-L0-05

f

0-1 0X2Vasopressin (i.u./ml.)

I

0.5

Fig. 1. Effects of vasopressin on the electrical activity of supraoptic neurones recordedwith an intracellular electrode. In A, two identical electrical phenomena are presented intwo types of pen-recordings (see Methods): the upper trace has a linear amplification butwith a narrow high-frequency band width, the lower trace has a relatively broaderhigh-frequency band width but a non-linear amplification. In this and subsequent Figures:downward and upward arrows represent the duration of superfusion of test solution;repetitive negative deflexions reflect the electrotonic potentials to inward currentinjections (0 4 Hz, 0-5 see pulse duration) of constant intensity for measurement of inputresistance; time shown under gaps in the trace indicates an omitted period; and upwarddeflexion respresents positive polarity. Spikes were almost entirely abolished in the uppertraces and only partially recorded in the lower traces, due to limited frequency bandwidth of pen-recordings. B, dose-response relationship for membrane depolarizationproduced by vasopressin. Each point represents the mean + S.E. of the mean from four tofifty-six cells which were responsive to vasopressin. The membrane potential changesinduced by oxytocin (20 i.u./ml.) is also illustrated.

1 20Oxytocin(i.u./ml.)

I

668

LfI


Effects of vasopressin on the spikeSpontaneous spikes and spikes evoked by outward or inward (anode break) current

injections were suppressed dose-dependently by vasopressin in eighty-three out of 120spontaneously active cells (e.g. see Figs. 1A and 3A), whereas in six cells the firingrate was increased and in thirty-one cells the rate was unchanged, during applicationof various doses of vasopressin. The effect of vasopressin on the spike generation thusseems to be mainly inhibitory.However, the inhibitory effect ofvasopressin was often reversed to excitatory when

vasopressin was applied to a hyperpolarized membrane, in both spontaneously active(Fig. 2A) and silent (Fig. 2B) cells. The spontaneously active cell in Fig. 2A ceased

A Vasopressin 1 i.u./ml. t

40sec 1 min 40sec 2min 6 min

Original membrane potential p.,

2 1

UK'JL 5 min1 min 110 sec 50 sec 4 min 160 sec

B

Original membrane potential a

b ti10 mV

5min1mi

Fig. 2. Effects of vasopressin on the spike generation ofspontaneously active (A ) and silent(B) neurones in the supraoptic nucleus. In A, identical electrical phenomena are shownin two types of pen-recordings (see Methods). In B, patterns of spike discharges recordedby a pen-recorder with a broader high-frequency band width and a non-linear amplificationin periods indicated as a and b are shown in insets. Further explanation in the text.

669


firing when vasopressin was applied (A, 1). When the membrane of this cell washyperpolarized by application of inward d.c. current injections to a level wherespontaneous firing did not occur (see lower trace in A, 2), application of vasopressinled to a generation of the firing (A, 2). Such a phenomenon was observed in five outof nine cells tested. It should be noted that, among the above five cells, three showedburst discharges during application of vasopressin as shown in A, 2, whereas in theremaining two, tonic or irregular firings developed. The bursts had a duration ofabout15-40 sec and were separated by silent periods of similar duration. These bursts werestrikingly reminiscent of the phasic activity, which is a characteristic firing patternof vasopressin containing neurosecretory cells in vivo (e.g. Poulain, Wakerley &Dyball, 1977) and in vitro (e.g. Haller & Wakerley, 1980).

A

a~~~~~~~~~~

VUoprUin 0e5 i.u./ml \

2m in 2min

d

+ ttI' tl 1, ll lIt

mgm10 Inc

I II

a b cQ d-L

| L-glUmMat 10-3M t11 II " U# 1~

mmrn "1_min _I min 2

5sc 59sc

20 mV

4+02 nASC

Fig. 3. Responses of supraoptic neurones to direct depolarizing current pulses duringapplication of vasopressin or L-glutamate. In both A and B, a, b, c and d illustrate thepotential (upper trace) and the current (lower trace) recorded at points indicated by arrowsin the continuous pen-recordings. Lower traces in A andBwere recorded with a pen-recorderhaving a broader high-frequency band width and a non-linear amplification. Currentintensity: a, b and d, 0-2 nA; c, 05 nA. The spike in A, c was truncated.

The silent cell in Fig. 2B showed anode break excitation (see inset a), and here,vasopressin (1 i.u./ml.) suppressed the anode break spike (not illustrated). When themembrane was hyperpolarized, the cell began to fire spontaneously, and vasopressinapplied during the hyperpolarized state further increased the firing rate (inset b). Sucha phenomenon was observed in three out of seven cells tested, and in one of the threecells, burst discharges developed as was the case with findings in A.

a

0--

670

VASOPRESSIN AND SUPRAOPTIC NUCLEUS 671

The inhibition of spike generation by vasopressin occurred without any transientenhancement of the firing rate (Fig. 3 A). Vasopressin elevated the threshold for spikegeneration; e.g. as shown in Fig. 3A, the depolarizing current pulse evoked the spikein the control medium (a) but failed to activate the cell during application ofvasopressin (b), and the stimulus increased to 2-5 times that of the control couldevoke the spike (c). On the other hand, depolarization (12-23 mV) induced byL-glutamate (10-3 M) was always accompanied by a transient but marked increasein the firing rate before cessation of spike generation. Suppression of spikes inducedby L-glutamate was observed even under an augmented stimulus (B, c).

A Vasopressin 1 i.u./mi. t

a~M . '37sec 2min 2m050 -

25-ILa 0

B ~~~~~~~~~~~~~~~~~~~Vasopressin1 i.u./mI.Ca-free, Mg 12 mm, EGTA 0-3 mm

V

1 min ~~~10mV1 min 4 sec

50-25-0

Fig. 4. Effects of Ca2+-free, Mg2+-rich EGTA solution on the vasopressin-induceddepolarization. A, control vasopressin-induced depolarization obtained in the standardmedium. In B, the medium in which CaCl2 was totally depleted, MgCI2 was raised to 12 mm,and 0-3 mM-EGTA added was superfused for the period indicated by the bar.

Effect of vasopressin in the absence of external Ca2+Fig. 4 shows the effect of vasopressin in the medium containing 0 mM-Ca2+,

12 mM-Mg2+ and 0-3 mm EGTA. In this solution, the membrane was depolarized(2-5 mV) and spontaneous spikes were suppressed. The depolarizing action ofvasopressin (Fig. 4A) persisted in the above Ca2+-free medium (Fig. 4B) in all of fivecells tested.

Voltage independency of the vasopressin-induced depolarizationFig. 5 shows the vasopressin-induced depolarization at various membrane poten-

tials. The membrane potential was held at -28, -46, -59 (original restingmembrane potential), -93, - 129 and - 163 mV by depolarizing or hyperpolarizingd.c. currents, in the cell shown in Fig. 5. The amplitude of the vasopressin-induceddepolarization evoked at the original resting membrane potential was little affectedby the membrane polarization. The relationship between the amplitude of thevasopressin-induced depolarization and the membrane potential is summarized inFig. 6.

672 H. ABE, M. INOUE, T. MATSUO AND N. OGATA

A Vasopressin 1 i.u./ml.

10 min

Original resting membrane potential

B

3 minF

C

2 min

D

+

20mV

1 min

Fig. 5. Voltage dependency of the vasopressin-induced depolarization. Effects ofvasopressin were examined at various membrane potentials produced by inward andoutward d.c. current injections. Detailed electrical phenomena at portions indicated byarrows in C, D, E and F are shown as X-ray photographs (a.c. recording, time constant0-2 sec) in insets of respective traces. Depolarizing potentials shown in insets of E and Fwere reflected on the pen-recording as large positive deflexions due to their slow timecourse. The horizontal line in each trace represents the level of the original restingmembrane potential.

O (mV)E -13

0 00

a)-10

0

vaE

0 IVacnnmrocun i-i Im I-

A---A&GABA 10-4M v

-110 -100

A\o \

-50

Membrane-potential displacement (mV)

-5

--5

Fig. 6. Voltage dependency of the membrane potential change induced by vasopressin andGABA. The amplitude of the depolarization or hyperpolarization induced by the testsolution was plotted as a function of the membrane potential displacement produced byinward or outward d.c. current injection. The data for vasopressin are shown in triplicateand were obtained from three different cells.

E

0 0

NN"

NINIIN-II /.C

N

N, NIN-

NI

50

N,

NIA~

0-


To determine whether or not the voltage independency of the vasopressin-induceddepolarization is due to an inappropriate current clamping of the membrane, thevoltage dependency ofthe response toGABA was also studied. GABA, which is knownto increase membrane permeability to Cl-, reduced the input resistance to 37 % ofthe control and depolarized the membrane at the resting membrane potential level,

A

Vasopressin 1 i.u./ml. +1K+]0 Jo

15 sec Original rating membrane potential

t[K1lo

624mM Irl-l;-_wI - 30sec

1-24 mm _d Pr .10

235sac1

0mi

231 - Filled symbols: control20 - Open symbols: Vasopressin _ 130 -

E - 1 i.u/mlA

& soC ~~ ~~~~~~~~01P

010 _ 0 0 f1 200 5 0 + A

o 100A0C 0~~~~~~~~~~

F0. beCL

0

L4 70LI1 2 5 10 20 50 1 2 5 10 20 50

[KIC (mm) [lKfl0 (mm)

Fig. 7. Relationship between [K+]0 and the amplitude of vasopressin-induced depolari-zation. In A, vasopressin was applied during perfusions with media containing 25 mm-(upper trace), 6-24 mm- (middle trace) and 1P24 mm- (lower trace) K+. The concentrationof Cl- was kept constant by reducing equimolar amounts of NaCl. B, left: abscissa, K+concentration in the medium; ordinate, membrane potential shift from the original restinglevel (horizontal line) in the absence (filled symbol) or presence (open symbol) ofvasopressin. B, right, amplitude of vasopressin-induced depolarization expressed aspercentage of that obtained at control [K+]o (6-24 mM) was plotted as a function of [K+]o.In A and B, the same symbols represent the measurements from the same cell.

as shown in Fig. 6. Displacement of the membrane potential in the hyperpolarizingdirection increased the amplitude of the depolarization, whereas on displacement inthe depolarizing direction GABA produced hyperpolarization (reversal potential,about -55 mV). The measured reversal potential may have deviated to a moredepolarized level since the recordings were made with 3 M-KCl-containing micro-

22 PHY 337


pipettes, and repetitively applied inward currents might have shifted the Cl-equilibrium potential towards the positive value. Thus, the voltage-independentnature of the vasopressin-induced depolarization is probably not due to inappropriatecurrent clamping of the membrane.

Effects of vasopressin on synaptic potentialsThe spontaneous small depolarizing potentials (see inset in Fig. 5C) changed their

amplitude voltage-dependently, i.e. depolarizing d.c. currents successively diminishedthe amplitude of these depolarizing potentials, and the polarity was reversed to ahyperpolarization at membrane potentials above -30 mV, whereas hyperpolarizingd.c. currents successively augmented the amplitude of the depolarizing potential (seeinsets in Fig. 5D-F). The small depolarizing potential, which often had a spikesuperimposed, was abolished in the medium containing 0 mM-Ca2+, 12 mMMg2+ and0 3 mM-EGTA. These observations suggest that the small depolarizing potential wasan excitatory post-synaptic potential produced by a neurotransmitter other thanvasopressin.

Vasopressin exerted only a minimal effect on the small depolarizing potentials atany membrane potential level (e.g. see Fig. 5E and F, where the small depolarizingpotential could be seen on the pen-recording as a large positive deflexion).

Ionic basis for the vasopressin-induced depolarizationThe amplitude of the vasopressin-induced depolarization was little affected by a

change of K+ concentration in the medium; i.e. as shown in Fig. 7 A, the amplitudeof the vasopressin-induced depolarization was constant when measured duringperfusion with media containing 25 mm-, 6-24 mm- (control concentration) or1-24 mM-K+. Fig. 7B summarizes the relationship between the amplitude ofvasopressin-induced depolarization and K+ concentration in the medium(1-24-45 mM). Fig. 8 shows the effects of TEA, which is known to block some formof K+-permeability, on the vasopressin-induced depolarization. TEA (10 mM)depolarized the membrane by 8-12 mV (B), and prolonged the duration of the spike.The amplitude of the vasopressin-induced depolarization generated during perfusionwith TEA (C) was similar to that obtained in the standard medium (A).The effects of vasopressin in Na+-deficient solution, in which the concentration of

Na+ was reduced from 137 to 13 mm by replacing total NaCl with an equimolaramount of choline Cl, were observed (Fig. 9A). In this solution, the membrane wasslightly depolarized (about 1 mV) and spontaneous spike discharges were totallysuppressed (lower trace). When vasopressin was applied during a perfusion of theNa+-deficient medium, depolarization occurred (lower trace), but the amplitude wasreduced to about 70% of the control depolarization (upper trace). The resultsobtained from seven cells are shown in Fig. 9 B. The vasopressin-induced depolarizationin the Na+-deficient medium (52 + 0-8 mV) was slightly but significantly (paired t test,P < 0 05) smaller than in the standard medium (7-2 + I 1 mV).

Application of 3 x 10-7 M-tetrodotoxin had no effect on the vasopressin-induceddepolarization in any of the three cells tested, whereas spike generation wascompletely blocked.

Neither reduction of Cl- from 132'4 to 10-2 mm nor depletion of Ca2+ from 2-6 mmto 0 mm affected the amplitude of the vasopressin-induced depolarization (Fig. 9B).

674


AVasopressin 05 i.u./ml.

it Original resting membrane potential

I minB

TEA 10 mM

c 2 sec

,I

Niggjij~gdjR 10 m)mmm

i min

Fig. 8. Effects of TEA on the vasopressin-induced depolarization. A, control response inthe standard medium. At the period indicated by a bar in B and C, the medium containing10 mM-TEA was superfused. 10 min elapsed between B and C. Insets in B and C wererecorded with a pen-recorder having a broader high-frequency band width and a non-linearamplification to illustrate the firing pattern of recordings indicated by arrows. Thepen-recorded spikes in insets ofB became progressively larger due to widening of the spikeduration.

Metabolic aspects of vasopressin actionThe response of a cell to vasopressin was that of depolarization of about 7 mV at

32 TC (Fig. 10A). In the same cell, decrease in the temperature to 15 TC led to aconsiderable depolarization (absolute value of the depolarization could not bedetermined; see Figure legend) and cessation of spike discharge (B). Vasopressinapplied at 15 0C did not produce any membrane potential change. Such a temperaturedependency of the vasopressin-induced depolarization was observed in all of threecells tested, and the Q1o value for membrane depolarization measured in one cellwas 3-7.

Fig. 11 shows the effects of ouabain on the vasopressin-induced depolarization.Ouabain (10-4 M) gradually depolarized the membrane and spike generation waseventually blocked (B). Vasopressin applied during the perfusion with ouabain didnot produce any membrane potential change (C), whereas vasopressin applied priorto the superfusion of ouabain caused a depolarization of about 6 mV (A). There wasno effect when the membrane potential was restored to the original resting level byinward d.c. current (D).

Effects of phosphodiesterase inhibitorsSeveral peptides have been shown to modify cyclic nucleotide metabolism in a

variety of tissues including brain (Borgeat, Chavancy, Dupont, Labrie, Arimura &Schally, 1972; Gagnon & Heisler, 1974; Duffy, Wong & Powell, 1975; Jard &Bockaert, 1975; Minneman & Iversen, 1976). If the actions of vasopressin are indeed

22-2

zz

675

iV


mediated via the cyclic AMP system, then the effects of vasopressin should bepotentiated by concurrent application of the phosphodiesterase inhibitors.

Papaverine, a known phosphodiesterase inhibitor, at a concentration of 10-4 Mdepolarized the membrane (4-2 + 0 5 mV), and induced total suppression of the spikein all of the ten cells tested. Pre-treatment with papaverine potentiated thedepolarization induced by vasopressin in both amplitude and duration in six of eightcells tested. For example, as shown in Fig. 12, the effect of vasopressin in the standardmedium (A) was about 2-fold potentiated in amplitude when applied during perfusionwith papaverine (B).

A

Vasopresmn 1 i.u./ml. tIhr Original resting membrane potential

TOT 2 rmi n

(Nall. 13mM t

mmflfk19011n-U11~_- Ius rD W_'U 11 20 sec _

9 min I min 125 minB~~~~~5sec Imin

B> 10i_

13 137 102 134-2 0 2-6[Na+l0 [C0+]O [m)[Ca2lJo

Fig. 9. Effects of vasopressin on the membrane potential in various ionic environments.A, upper trace: control vasopressin-induced depolarization. A, lower trace: effects ofdepletion of [Na+]0 on the vasopressin-induced depolarization. During the period indicatedby bar, NaCl in the medium was totally replaced by an equimolar amount of choline Cl(10-6 g atropine sulphate/ml. was added). B illustrates the relationship between theamplitude of vasopressin-induced depolarization (ordinate) and the concentration of Na+,Cl-, or Ca2+ in the medium (abscissa). Cl1-deficient medium was prepared by replacingtotal NaCl with an equimolar amount of Na isethionate. Ca2+-free medium was preparedas in Fig. 4. Each point represents the mean + S.E. of the mean from seven (Na+) or five(Cl- and Ca2+) experiments.

Theophylline, also an inhibitor of phosphodiesterase, exerted much weaker actionon the supraoptic cell, i.e. theophylline (10 mM) depolarized the membrane slightly(1 5 + 0-3 mV) without significant change of the firing rate in six out of fifteen cellstested, whereas the remaining nine cells were unaffected by this drug. Pre-treatmentwith theophylline potentiated the vasopressin-induced depolarization in only one outof four cells tested (about 30% increase in amplitude).

676


Effects of dibutyryl cyclic AMPDibutyryl cyclic AMP (2 mM) depolarized the membrane (4 3 + 1.5 mV) and

reduced the spontaneous firing rate to 53-5 + 12-0% of the control in six out of ninevasopressin-responsive cells, whereas the remaining three cells were unaffected by thisdrug. The depolarization induced by dibutyryl cyclic AMP resembled the vasopressin-induced depolarization in many respects, i.e. the depolarization induced by dibutyrylcyclic AMP was accompanied by a minimal change in input resistance, was voltage-independent, and was unaffected by alteration of external K+ or by depletion ofexternal Ca2+ in both cells tested.

A Vasopressin 0 5 i.u./ml.

35

25

15

Cooling off

Cooling on ~~p~~wmwuumm~~~~~dL.... ~~10mV4min 30 #c 3 min 1 min

Artifactual d.c. potential shift5 sec 1 mih

35 -

43 30-

L 20.

10(0C)

Fig. 10. Effects of cooling on the vasopressin-induced depolarization. The temperature ofthe medium was monitored continuously by an electrically insulated thermistor (diameter,1-5 mm; length, 3 mm) immersed in the medium pool on the agar block, as close as possibleto the slice. The output of the thermistor was connected to another channel of thepen-recorder. The temperature of the medium was controlled by passing it through awarming-cooling chamber which could vary the temperature rapidly between 10 and45 'C. A, control vasopressin-induced depolarization obtained at 32 'C. In B, the changein the medium temperature caused artifactual d.c. potential shift (continuous line).Accordingly, the trace of membrane potential during low temperature in B represents thealgebraic sum ofthe actual membrane potential change and artifactual d.c. potential shift.

Pre-treatment with papaverine (10-4 M) potentiated the response to dibutyrylcyclic AMP in all of three cells tested. Even in a cell on which dibutyryl cyclic AMPhad no effect in the standard medium (Fig. 12 A), dibutyryl cyclicAMP applied duringperfusion with papaverine depolarized the membrane by about 10 mV (Fig. 12B).

Theophylline (10 mM) potentiated the action of dibutyryl cyclic AMP in one outoftwo cells tested. In Fig. 13, dibutyryl cyclicAMP induced a transient depolarization

677


A

Vasopressin 0*t i.u./ml.Original resting membrane potential

30sic8

=_

x-~~-Ouaboin 104 M _pow00 2min

2 min

2min 2 minC

OIe11c IC30 sec

t-it -,ow

5 sec

D

t

r

l~VI 'I

-F--- 4 6 a -. 0| S

1 min

Fig. 11. Effects of ouabain on the vasopressin-induced depolarization. A, controlvasopressin-induced depolarization. At the period indicated by bars in B, C and D, themedium containing ouabain (10-4 M) was superfused. 20 min elapsed between B and C.At the end of C, the membrane which had been depolarized by ouabain was repolarized tothe original resting level by inward d.c. current injection.

ADibutyryl c-AMP 2 mM Vasopressin 05 u/mI Papaverine 10-i M

a- 28 min 5! min 5 minco--

_ ~____

_~~~~min 2 min2mn5 min 3 min

1 _v

tai1omV4miUI1 min

Fig. 12. Effects of dibutyryl cyclic AMP and vasopressin in the absence or presence ofpapaverine. Trace of firing rate seen in A was omitted in B, as the spike remainedsuppressed during recordings in B.

-

lw 11

Id' 11111 PikWYAL 11".31

I

W6..

678

.AI

' ,1 l_w ."-


with a relatively long latency in the standard medium (A). Dibutyryl cyclic AMPapplied during perfusion with theophylline produced a depolarization of a longerduration and with a shorter latency (C). The action of vasopressin in the same cellis shown for reference in D.Adenosine (1 mM) also depolarized the membrane by 2-5 mV and reduced the firing

rate to 35-70% of the control in all of four cells tested (not illustrated).

A Dibutyryl c-AMP 2 mM

402.F j20.c

LB Theophylline 10 mM

C

)Itk WAA

D Vasopressin 0O5 i.u/ml.Id- la mV

=>j~~~~illSW~~ ~ 5sec

Fig. 13. Effects ofdibutyryl cyclic AMP on the electrical activity in the absence or presenceof theophylline. Effects of vasopressin on the same cell as in A-C are shown in D. Insetsin C and D were recorded with a pen-recorder having a broader high-frequency band widthand a non-linear amplification to illustrate the firing pattern of portions indicated byarrows.

Assay for cyclic AMP levelAfter determining the electrical viability of the hypothalamic slice, the tissue was

microdissected undera binocular microscope. The symmetrical areaofeach hemispherewhich included the supraoptic nucleus and surrounding tissues was punched out forcontrol (one hemispheric tissue) or test (the other hemispheric tissue) assay. The wetweight of these tissues was 3-8 mg (51+ 04 mg, n = 28). If a pair of tissues showeda difference of more than 0f5 mg wet weight, they were discarded.


After an incubation of 1 hr, each hemispheric tissue was subjected to an additionalincubation for 5 min in the absence (control assay) or presence (test assay) of1 i.u. vasopressin/ml. Fig. 14 shows the contents of cyclic AMP obtained fromfourteen pairs of assays. The level of cyclic AMP in the test assay (I146 +0-24 p-mole/mg wet weight) was significantly greater than that in the control assay(0-85 ± 006 p-mole/mg wet weight) (paired t test, P < 0-05).

2 23 42

o-. o' U_ U i UUHffi _a0 Control

Ef Vasopressin

Q~~~~~~~ DII

Fig. 14. Effects of vasopressin on the cyclic AMP level in the basal hypothalamus mainlyconsisting of the supraoptic nucleus. Each pair of open and filled columns represent cyclicAMP contents in the control (open) and vasopressin-treated (filled) hemispheric basalhypothalamus obtained from the same slice.

DISCUSSION

Vasopressin produces membrane depolarization unaccompanied by apparentchange in input resistance in cells of the supraoptic nucleus, with minimal andmaximal effective concentrations of 0-05 and 1 i.u./ml., respectively. The doses usedin our study were somewhat high compared with those used in non-neural targetorgans for vasopressin. For example: renin secretion from rat renal cortical slices wasinhibited by vasopressin in doses of0-005-01 i.u./ml. (Churchill, 1981); the membraneof guinea-pig jejunal mesenteric artery was depolarized by vasopressin in doses of0-001-0.1 i.u./ml. (Karashima, 1981). Arelativelyhighdoseofvasopressin (0-2 i.u./ml.)was used to stimulate active sodium transport across the toad bladder (Walton,Delorenzo, Curran & Greengard, 1975).The possibility that the effects of vasopressin observed in the present study were

due to a non-specific action of the drug can be excluded. The sigmoid nature of thedose-response relationship, and the finding that the other neurohypophysial peptide,oxytocin (which differs from vasopressin only in amino acid positions 3 and 8 of themolecule), was much less effective, are consistent with a receptor-mediated event.Furthermore, the finding that vasopressin exerted only a minimal effect on thesynaptic potentials (see Fig. 5) indicates the existence of a group of cells, eitherintranuclear or peri-nuclear, which are not affected by vasopressin.Our finding that the depolarization induced by vasopressin persisted even in the

medium containing 0 mM-Ca2+, 12 mM-Mg2+ and 0-3 mm EGTA, the highly specific

680


Ca2+-chelating agent (Fig. 4) confirms that vasopressin can affect directly theneuronal activity of supraoptic cells.The hypothesis that vasopressin may be released from recurrent collaterals of

supraoptic cells at synapses back onto supraoptic cells, thus acting as the transmitterresponsible for antidromic inhibition (Nicoll & Barker, 1971) has not been confirmed(Dreifuss, Nordmann & Vincent, 1974). Our result that vasopressin depolarized themembrane of supraoptic cells also does not support this hypothesis.The effects of vasopressin on spike electrogenesis were complicated (see Figs. 2 and

3), and further experiments are required. Nevertheless, the finding that cells beganto fire phasically during application of vasopressin and under a hyperpolarized state(see Fig. 2A, 2) may be of great interest, in view of the suggestion that vasopressinmay induce phasic activity in vasopressin-producing neurones (Leng, 1981; Poulain& Wakerley, 1982).

The nature of vasopressin-induced depolarizationThe vasopressin-induced depolarization was not associated with a change in

membrane conductance and was voltage-independent. Vasopressin may activatereceptors on the fine dendritic tree remote from the soma and alter the membraneconductance at these areas. If such is the case, conductance change due to activationof these receptors may not be detectable by the electrode placed in the soma, andthe current-clamping of the remote dendrite by d.c. current injection may beinadequate. However, the following observations strongly suggest that the depolar-ization induced by vasopressin does not result from passive ion movement across themembrane linked to a change in membrane ionic permeability. That is, the amplitudeof vasopressin-induced depolarization was virtually the same in various [K+]O, andwas unaffected by TEA. Likewise, alteration of external concentrations of Cl- andCa2+ had no effect on the amplitude of vasopressin-induced depolarization. Only thereduction of [Na+]. significantly modified the amplitude of vasopressin-induceddepolarization, yet even in this case the change in amplitude was too small toattribute the depolarization induced by vasopressin entirely to a change in Na+-permeability of the membrane.The identified neurones in some invertebrates exhibit a characteristic 'bursting'

pattern of electrical activity, and this endogenous rhythm can be greatly modifiedby vasopressin (Barker & Gainer, 1974). The action of vasopressin which shifted themembrane potential but did not change the membrane conductance would modifyneuronal activity in a manner different from actions of conventional transmitterswhich in most cases increase or decrease the membrane conductance. The action ofvasopressin at post-synaptic cells may be more prominent on the voltage-dependentprocess (responsible for action potential generation) than on the voltage-independentprocess (responsible for synaptic potentials). Therefore, vasopressin may modulatethe endogenous firing pattern also in vertebrate neurones.

The source of vasopressinIf vasopressin does act on the supraoptic cells under physiological conditions, the

source has to be clarified. At least three possibilities might be assumed, i.e. (1)vasopressin as a circulating or locally secreted neurohormone, (2) release from axon

681


collaterals of the neurosecretory cells, and (3) release from axon terminals ofextra-nuclear origin.

In support of the first possibility is the work by Kawakami & Saito (1969) whoshowed that intravenous injection ofoxytocin influences the activity ofhypothalamicneurones, whereas the paraventricular unit activity was not affected by massiveintravenous injections of this drug (Dyball & Dyer, 1971). There is evidence tosupport the second possibility that the firing pattern of the supraoptic cell whichshows phasic activity (i.e. probable vasopressin-producing cell; see Poulain et al. 1977)is influenced by the activity of its neighbouring cells (Leng, 1981).

Regarding the third possibility, one plausible site of origin is the suprachiasmaticnucleus. The parvocellular neurones of the suprachiasmatic nucleus contain vaso-pressin (Vandesande, Dierickx & De Mey, 1975; Sofroniew, 1980), and project ontothe supraoptic nucleus (Buijs, 1978) without projecting onto the pituitary (Fisher,Price, Burford & Lederis, 1979). In addition, latero-caudal and dorsal isolation of thesuprachiasmatic pathways abolishes drinking rhythms (Nunez & Stephan, 1977),suggesting a role of the suprachiasmatic nucleus in hypothalamo-neurohypophysialfunction.

Involvement of cyclic AMPPossible association of vasopressin action with the cyclic AMP generating system

in the supraoptic nucleus is suggested from the following results: (1) vasopressin anddibutyryl cyclic AMP provoked superficially similar membrane effects, (2) actions ofthese drugs were in most cases potentiated by papaverine, a potent inhibitor ofphosphodiesterase, and in some cases also by theophylline, another phosphodiesteraseinhibitor, (3) adenosine which stimulates cyclic AMP accumulation in brain slicesfrom virtually all species and regions studied (Skolnick & Daly, 1977) also producedelectrophysiological effects resembling those of vasopressin, (4) cyclic AMP contentin tissues incubated with vasopressin was significantly higher than that in tissuesincubated with normal Krebs solution, although whether or not the origin of theincrease is neural remains to be determined.The above postulation is supported by a number of studies on peripheral tissues

in which it is now widely accepted that some actions of vasopressin are mediated viaan increase in cyclic AMP formation by target cells, e.g. the effect of vasopressin onsodium transport in mucosal cells of the bladder (Orloff & Handler, 1967) and theantidiuretic action of vasopressin in the mammalian kidney (Brown, Clarke, Roux& Sherman, 1963).The possible involvement of cyclic AMP in the vasopressin action is further

supported by findings in invertebrate neurones. The electrical activity in molluscanneurones can be modified by perfusion with vasopressin (Barker & Gainer, 1974), andagents which alter cyclic nucleotide metabolism produce similar effects (Treistman& Levitan, 1976). Furthermore, treatment of molluscan ganglia with vasopressinleads to a large increase in cyclic AMP content in the tissue, apparently throughactivation of a membrane-bound adenylate cyclase (Levitan & Treistman, 1977).One possible mechanism by which the increase of cyclic AMP brings about the

ultimate physiological response of the neurone might be the stimulation of sodium

682


transport across the membrane as in non-neural tissues such as skin, bladder andkidney where vasopressin stimulates sodium transport in the epithelial tissues of theseorgans (e.g. Orloff& Handler, 1967). In the ion-substitution experiments in our study,only the alteration of [Na+] significantly modified the action of vasopressin. Specificmembrane protein phosphorylated by cyclic AMP may be associated with carrier-mediated and possibly active entry of sodium into the intracellular compartment ofthe neurone, although further experiments are required to support this idea. Thefinding that the action of vasopressin was abolished during application of ouabainmay be linked to the above postulation.

Miihlethaler et al. (1982) showed that unit firing in the rat hippocampus wasconsistently enhanced by vasopressin and this effect of the peptide did not appearto be linked to adenylate cyclase, since an antagonist for V,-receptor (which is notcoupled to adenylate cyclase and is present on smooth muscle cells) abolished theeffect of vasopressin, whereas an agonist for V2-receptor (which is linked to adenylatecyclase and is present on epithelial structures) had only a slight action on hippocampalneuronal firings. These observations indicate that there are subtypes in vasopressinreceptors in the central nervous system as in the peripheral tissues (Rajerison,Marchetti, Roy, Bockaert & Jard, 1974; Kirk, Michell & Hems, 1981), and that thereceptor distributed on the supraoptic neurone can probably be classified into theV2-subtype.

The authors are indebted to Professor H. Kuriyama for pertinent advice and criticism, to DrsM. Hirata and T. Kishikawa for instruction on the cyclic AMP radioimmunoassay and pertinentdiscussion, and to Dr Kate Creed and M. Ohara for critical reading of the manuscript. This studywas supported in part by the Japanese Ministry of Education (Scientific Research 577110,56770126).

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Documents

NOBUKUNI OGATA* 0-3 mM-EGTA. 3. Amplitude of the vasopressin