Noxious inputs to supraoptic neurosecretory cells in the rat

Neuroscience Research, 2 (1984)49-61

49

Elsevier Scientific Publishers Ireland Ltd. NSR 00047

Noxious Inputs to Supraoptic Neurosecretory Cells in the Rat Mitsuko Hamamura, Katsuei Shibuki and Kinji Yagi Department of Physiology, Jichi Medical School, Minamikawachi-machi, Tochigi-ken 329-04 (Japan) (Received June 14th, 1984; Revised version received July 19th, 1984; Accepted July 26th, 1984)

Key words: neurosecretory cell - - nociceptor - - noxious heat stimulation - - rat - - supraoptic nucleus - tail pinching - - vasopressin

SUMMARY The effects of noxious stimuli were studied on discharge activity of the neurosecretory cells identified in the supraoptic nucleus by antidromic excitation after pituitary stimulation, in anaesthetized rats. Tail pinching excited 24 % and inhibited spontaneous discharge of 6% of the 91 cells tested. Noxious heat stimuli (44-63 °C) applied to the hindlimb paw produced a transient excitation in 26% of the 23 ceils tested. Electric stimulation of either the sciatic or cutaneous nerve with 20-Hz pulses for 1 s, at an intensity 5 times stronger than the threshold for evoking the changes in respiratory movements and blood pressure similar to those aider tail pinching or noxious heat stimuli, excited about 30% of the cells tested. The excitation produced by these noxious stimuli preceded, on some occasions, the respiratory movement and blood pressure decrease which occurred concomitantly. Peristimulus time histograms of spontaneous discharges constructed during stimulation of either nerve at 1 I-Iz, revealed the presence of excitatory synaptic inputs in about 35 % of the neurosecretory cells tested. These data indicate the existence of direct neural pathways which mediate excitatory synaptic inputs originating from nociceptors to supraoptic neurosecretory cells. Since 9 of the 22 cells which were excited by tail pinching exhibited a "phasic" pattern of spontaneous discharge which is known to characterize certain vasopressin-socreting neurones in rats, it is suggested that these excited cells were, at least in part, vasopressinergic.

INTRODUCTION

Noxious stimuli have been shown to increase plasma antidiuretic activity, in the early experiments with bioassay methods 2'1°,15'16. Based on recent experiments employing radioimmunoassay techniques, however, it has been claimed that electric foot shocks do not elevate the plasma vasopressin level6'9. On the other hand, our previous radioCorrespondence: M. Hamamura, Department of Physiology, Jichi Medical School, Minamikawachi-machi, Tochigi-ken 329-04, Japan. 0168-0102/84/$03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd.

50 immunoassay study has demonstrated a significant increase in the plasma vasopressin level after electric foot shocks TM. It is thus tempting to discover whether vasopressinsecreting cells in the rat supraoptic nucleus receive excitatory synaptic inputs after a variety of noxious stimuli. A decrease in arterial blood pressure is known to potentiate antidiuretic hormone secretion by the pituitary 13"19, and to excite neurosecretory cells in the supraoptic nucleus 7"8'18. Since noxious stimuli alter blood pressure, an increase in the rate of vasopressin secretion after noxious stimuli may be a secondary effect of blood pressure decrease. On the other hand, certain pontine neurones which were suggested to project to the supraoptic nucleus have been shown to be excited by tall pinching without appreciable change in blood pressure 8. This finding raises the possibility that the direct neural pathway originating from nociceptors exists and mediates neural signals to supraoptic neurosecretory cells after noxious stimuli. Thus, in the present experiments, we recorded unit discharges of supraoptic neurosecretory cells and arterial blood pressure at the same time, and attempted to fred whether excitatory synaptic inputs after noxious stimuli depend upon a blood pressure decrease or not. Preliminary accounts of the present data have been reported at the annual meeting of the Physiological Society of Japan 4.

MATERIALS AND METHODS Twenty-nine male rats of Wistar strain, weighing between 368 and 548 g, were anaesthetized with a mixture of ~-ehlorarose and urethane at an i.p. dose o f 70 and 700 mg/kg dissolved in 7 ml of distilled water, respectively. Additional anaesthetics of 0.3 ml were intravenously administered when the corneal reflex was detected upon touching, tested at 1-h intervals. The trachea was cannulated with polyethylene tubing. A polyethylene catheter was inserted into the femoral artery and connected to a blood pressure transducer. Another catheter was attached to the femoral vein for injection of additional anaesthetics and Locke's or glucose solution. The rat was placed in a stereotaxic apparatus. Respiratory movement was continuously monitored with a length transducer made of rubber tubing containing carbon powder, attached to the chest. The rectal temperature was monitored and maintained between 36 and 37 ° C throughout the experiment. For stimulating the posterior pituitary, a stainless steel insect pin (no. 0; Shiga) insulated with Insl-X E33 (Insl-X Products) except for the section about 0.3 mm from the tip, was inserted stereotaxically into the posterior pituitary. Coordinates of the electrode placement were 4.3 mm rostral to the inter-aural line and 1.5 mm left of the midline. The electrode was lowered at an angle of 10 ° to the right until the electrode tip reached the bottom of the skull and slight bending of the electrode was detected under the dissecting microscope. The dectrode tip was then lit~ed 0.7 mm from the bottom and fixed. A piece of silver wire (100 #m in diameter) was placed on the dura

51 mater as the reference electrode. A negative stimulating pulse of 0.5-ms duration supplied by a stimulus isolator (Tektronix 2620) was applied to the posterior pituitary. After each experiment, a positive current of 0.5-mA intensity and 50-s duration was applied to the electrode. The tip location was verified to be within the posterior pituitary from the lesion mark on the histological sections of 50-/~a thickness which were cut from the frozen brain and stained with hematoxylin and eosin. For activating somatic noxious afferents, the tail was pinched for 4 s or, in some cases, 10 s by a pair of forceps. As the control, the tail was touched gently for 4 s with the forceps. For applying noxious heat stimuli, a heat irradiator was constructed with a microscope light source (8 V) and an electrically controlled shutter (Copal EC-601) attached in front of the focusing lens. The palm skin of the left hind paw was painted black with India ink and irradiated for 10 s. The irradiated area was a 5-ram square. The skin temperature was monitored with a piece of hand-made thermocouple (diameter: 0.4 mm) made of copper and constantan wires. For stimulating the peripheral nerves, the trunks of the left sciatic nerve and the left cutaneous nerve to the thigh were isolated and the central parts of the cut nerves were placed on two pairs of chlorided silver wire. The surgical area was covered with a piece of translucent plastic film to protect the nerves from drying. Negative pulses of 1-ms duration repeated at 1 or 20 Hz were used for the stimulation. For recording single unit discharges, a tungsten microelectrode insulated with Insl-X was inserted vertically through the cortical surface and into the right supraoptic nucleus, while the posterior pituitary was stimulated with single negative pulses (intensity: 0.3 mA) repeated at 0.89 Hz. Coordinates of the electrode placement ranged between 8.0 and 9.2 mm rostral to the inter-aural line, and between 1.5 and 2.8 mm to the right of the midline. A piece of silver wire was placed in the right temporal muscle as the reference electrode for unit recording. Unit action potentials were displayed on a storage oscilloscope (Tektronix R5113N) and photographed. The units were identified antidromically as those of neurosecretory cells when the unit spike evoked by posterior pituitary stimulation showed constant latency and was cancelled by collision with a spontaneously occurring spike. Each unit spike was used to trigger a pulse generator to produce a square pulse, and the pulse was fed into a pen recorder and a minicomputer (Nova 4/S, Data General). A pulse was also generated at the onset of tail pinching, noxious heat stimuli or each stimulating pulse and was fed into the minicomputer to record the time of stimulus. Peristimulus time histograms (PSTHs) of spike discharges were constructed with 250-ms time bins during repeated tail pinchings, noxious heat stimuli or 20-Hz nerve stimulation of 1-s duration, and with 5-ms time bins during nerve stimulations at 1 Hz. The effects of tail pinching were appraised by comparing the number of spikes per bin between 0 and 5 s and between 5 and 10 s after the onset of pinching, with that during a prestimulus 5-s period. The effects of noxious heat stimuli were estimated by comparing the number of spikes per bin during a poststimulus period of between 7.5 and 12.5 s, with that during a prestimulus 5-s period. Synaptically mediated responses to nerve stimulations were judged by comparing the number of

52 spikes per bin during a poststimulus period of between both 75 and 125 ms, and t50 and 300 ms, with that of a period of 200 ms during the prestimulus period. The difference in number of spikes per bin was tested by Mann-Whitney's U-test at the significance level of 1% to judge the presence of excitation or inhibition of spontaneous discharge. After each experiment, a positive current of 0.1-mA intensity and 20-s duration was applied to the recording microelvctrod¢ to produce a lesion mark. The recording sites in the supraoptic nucleus were examined by histological sections stained with cresyl violet. RESULTS

At the beginning of unit recording in each rat, systolic blood pressure was between 87 and 135 (106 + 13, mean + S.D., n = 18) mm Hg. 105 units were antidromicaUy identified as magnocellular neurosecretory cells in the supraoptic nucleus after stimulation of the posterior pituitary. Fig. 1 displays examples of histological data indicating

A

B

| I I i

! SON

OC

Fig. 1. A: a horizontal section of the pituitary. The arrow indicates electrolytic lesion. B: a frontal section of the hypothalamus showing a lesion in the supraoptic nucleus (arrow). Another lesion 1 mm dorsal to this lesion was made for size calibration. AL, the anterior lobe of the pituitary; IL, the intermediate lobe; NL, the neural lobe; OC, the optic chiasm; SON, the supraoptic nucleus.

53 tip location of the stimulating electrode within the posterior pituitary, and that of the recording microelectrode in the supraoptic nucleus. The threshold for evoking an antidromic spike in these supraoptic units ranged between 15 and 450 (165 _+ 104, mean + S.D., n = 104)/~A. Latency of the antidromic spike was between 8.6 and 24.5 (13.2 _+ 3.2, n = 105) ms.

Effects of tail pinching Tail pinching induced a transient and strong inspiratory movement and a change in arterial blood pressure (Fig. 2A), while tail touching did not. Thus, effects of tail pinching or other noxious stimuli on discharge activity were analyzed in the present study only when the stimuli evoked these respiratory and/or cardiovascular responses. Tall pinching produced a marked transient excitation in some of the supraoptic neurosecretory cells tested, and the excitatory response could be repeatedly evoked in the same single units (Fig. 2B). The change in blood pressure which also occurred after tall pinching was either pressor (Fig. 3A), depressor (Fig. 3B) or biphasic (Fig. 2A). The increase in discharge rate after tail pinching occurred even during the pressor response in some cells (Fig. 3A), and in some others preceded the blood pressure decrease (Fig. 3B). PSTHs of spontaneous discharge constructed during repeated tail pinchings showed two excitatory peaks in certain neurones (Fig. 3B). Of the 91 units tested for tail pinching, 24% were found to produce an excitation as shown in Fig. 3C, and the other 6% an inhibition of discharge activity after tail pinching (Table I). 9 of the 22 excited cells exhibited the "phasic" pattern of spontaneous discharge which was characterized by alternating a bursting period of intra-burst frequency higher than 3 spikes/s, and silence1'~2. Tail touching did not excite 5 tested cells that were responsive to tail pinching. A

~TP

B 15

d o

.2oo E E

0 TP

<

o

n l

I

I

l

I

l

I

I

I

I

l

I

I

I

I

]

I

I

I

I

I

2O0

o

Time (s)

lo s Fig. 2. An example of the supraoptic neurosecretory cells which showed an excitation after tail pinching (TP). A: a pen-writer record showing respiratory movement (Resp.), arterial blood pressure (ABP) and unit discharge (Unit). Downward deflection in the upper trace shows inspiratory movement. Note abrupt and strong inspiration accompanied by a blood pressure change. B: a rate-meter display of spontaneous discharge. Note a transient excitation after each of the tail pinchings. An asterisk indicates the time of recording of A.

A

B

TP

£-,oo r ~ ~

TP

:i!2'

E E Q. =B

C

0

2s 16r

w

i'

18

j

"K

i off

i..

i

~

"

uuui

-5

0

0.5

u TP

5

°°uvw~n ~ 10

O

15

1

O-O.5 ~ 5 tt" -5

I 0

TP

, 5

~ 10

:~_ 1,~

TP

Time after stimulus (s)

Fig. 3. Excitation evoked by tail pinching. A: an example of the units in which excitation occurred during the pressor response to tail pinching (TP). The upper panel illustrates the result of one trial of TP, and the lower panel shows the PSTH with 250-ms time bins obtained in the same unit during 7 repeated TPs. A dotted curve indicates the moving averages calculated from data in consecutive 12 bins in this and following figures. B: the upper panel represents an example of the excitatory responses in which an increase in discharge rate preceded the depressor response to TP. The lower panel is the PSTH obtained similar to that in A but for the unit in the upper panel of B. C: superposed display of moving average curves derived from the 22 excited cells. The ordinate is the relative discharge activity in each unit as expressed by the relative moving average value at a 250-ms interval when the mean discharge rate during prestimulus 5-s period and the peak rate during poststimulus period were taken as 0 and 1.0, respectively. TABLE I RESPONSES OF SUPRAOPTIC NEUROSECRETORY CELLS TO A VARIETY OF NOXIOUS STIMULI Stimulation

Number of cells which exhibited: excitation

inhibition

no response

Tail pinching

22 (24%)

5 (6%)

64 (70%)

Noxious heat

6 (26%)

0 (0%)

17 (74%)

Sciatic nerve 20 Hz 1 Hz

5 (31~o) 15 (36%)

2 (13~o) 4 (10~o)

9 (56%) 23 (55~o)

Cutaneous nerve 20 Hz 1 Hz

5 (26~o) 13 (35%)

1 (5%) 1 (3%)

13 (68%) 23 (62%)

55

Effects of noxious heat stimuli Before irradiation, the skin temperature of the paw of the left hindlimb was between 20 and 24 ° C in the 11 tested rats. Changes in respiratory movements and arterial blood pressure similar to those observed after tail pinching occurred abruptly as the skin temperature reached between 44 and 63 °C during irradiation for 10 s. These noxious heat stimuli evoked reproducibly a transient increase in discharge rate, while the control heat stimulation at the peak temperature of between 35 and 42 °C did not (Fig. 4). The P S T H constructed during repeated stimulations demonstrated the response (Fig. 4A). Of the 23 supraoptic neurosecretory cells that were tested two times or more for noxious heat stimuli, 2 6 ~ were excited by the stimuli and the remaining cells were unresponsive (Table I). Fig. 4C illustrates the excitatory response in all 6 responsive cells. 10 of the 23 units were further tested for control heat stimulation and 9 of them were unresponsive, while one was excited. 11 of the 23 cells were tested for both noxious heat stimuli and tail pinching, and three of them were excited by both stimuli.

Effects of peripheral nerve stimulation Stimulation of the sciatic or cutaneous nerve (pulse width: 1 ms; duration of 20-Hz pulses: 1 s) evoked the similar changes in respiratory movements and blood pressure to those induced by tail pinching and noxious heat stimuli (Fig. 5A). The mean of the A

B H8

CH$

C

,

•

2a

i -5

0

ln,

....

5 HS

10

15 0 - 5 Time

r after

n

5 10 OHS stimulus (s)

z.

0. 15 Do-O" -' 5

,

0

5 HS

' 10

15

Fig. 4. Excitation evoked by noxious heat stimuli. A: the upper panel illustrates an example of a change in respiratory movement and an excitation evoked by noxious heat stimulation (HS) with a peak skin temperature (Temp.) of 52 °C. The PSTH in the lower panel was derived from the same unit during 4 repeated HSs. Bin width is 250 ms. B: no change in respiratory movement and discharge rate after control heat stimulation(CH S) with a peak temperature of42 ° C in the same unit as that of A. C: superposeddisplay of moving average curves derived from the 6 excited cells after noxious heat stimulation.

56

A

NS

d

o @ O

-IE E

200

13. ~D

0

los

B

C 12

16 12 8

"5.

3

4 0

n0 '-s

In o

NS

nnnn nn

On

J

s

Io

Is

-'5

nn0 o

NS

'

i'o

11s

Time a f t e r stimulus (s) Fig. 5. Excitation after sciatic and cutaneous nerve stimulation repeated at 20 Hz. A: characteristic responses in respiratory movement and blood pressure repeatedly evoked by the sciatic nerve stimulation for l s. Asterisks indicate that that part of the record includes stimulus artifacts as well as unit discharges. B: the PSTH derived from the same unit as that of A aRer sciatic nerve stimulations repeated 6 times. Stimulus artifacts were removed from the P S T H by the computer. C: the PSTH derived from the same cell as that of B after cutaneous nerve stimulation repeated 6 times. Note that the discharge rate reached the m a x i m u m within 500 ms aRer the onset of l-s stimulation, whereas the blood pressure change was evoked later than l s after stimulus initiation.

threshold for evoking such responses was 0.14 _+ 0.30 (S.D.) m A for sciatic and 0.12 _+ 0.21 m A for cutaneous nerve stimulation in 10 tested rats. The stimulus intensity used for testing the effect on discharge activity was 5 times the threshold in each rat. PSTHs of spontaneous discharge constructed during repeated 20-Hz stimulations showed an excitation during poststimulus period (Fig. 5). The peak latency was shorter than 500 ms (Fig. 5B and C), whereas the depressor response occurred later than 1 s from the onset of the stimulation. Of the 17 and 19 cells tested for sciatic and cutaneous nerve stimulations at 20 Hz, respectively, about 30% were excited (Table I). Nerve stimulation with 1-Hz pulses for 100 s induced a blood pressure change which was either a sustained increase, decrease or an undulatory alteration. An increase in discharge rate was observed aflzr the stimulation along with these blood pressure changes. Fig. 6 shows an example of the supraoptic units that increased discharge rates

57

A NS

A

~d~[~]~"`~""ji~:Li~h~i~i~'~'`h"`i~""``;'J[i`~1.~..i"'~i""`~""~"`1i~"i"''i'L'iL~

O~ 2 0 0 -r-

I

E E 0. m <

B

o 10s

12 ¢o

~m

'Q.

U}

NS

•

i

i

i

i

I

I

I

I

I

I

I

0

I

I

I

I

I

i

i

!

I

2OO

Time

(s)

Filg. 6. An example of the excitation during the pressor response to repetitive sciatic nerve stimulation at 1 Hz. NS indicates the stimulus timing. Arterial blood pressure in A and the discharge rate in B were sir~ultaneously recorded and these stimuli could be repeatedly evoked on the same single neurosecretory cells.

dm'ing the simultaneously occurring pressor response. PSTHs constructed during 1-Hz stimulation of either the sciatic or cutaneous nerve revealed a marked excitation during the poststimulus period (Fig. 7A and C). As shown in Table I, about 35 ~o of the tested coils were excited by stimulation of these nerves (Fig. 7B and D). In 12 of the 15 cells e~cited by sciatic nerve stimulation, a transient excitation during the poststimulus period was found to occur in PSTHs even though both systolic and diastolic pressures were sustained at an elevated level during the whole stimulation period, and the mean blood pressure increase was more than 5 mm Hg. 2 of the 15 excited neurones exhibited a "phasic" pattern of spontaneous discharge. Two excitatory peaks were noted in some cells (Fig. 7C). Peak latency of excitation (the shortest peak) after sciatic nerve stimulation was 70-290 (165 + 83, mean + S.D.) ms, and that after cutaneous nerve stimulation was 80-345 (192 + 98)ms. 14 of the 15 cells that were excited by sciatic nerve stimulation were also tested for cutaneous nerve stimulation, and 79~o were excited after .both stimulations. Of the 17 cells that were excited by either or both nerve stimulations, 53 ~o were also excited by tail pinching.

58

B

A 44r

1.0

3a i

0.5

22 ~4,,*

•

,l 4,*

o

o q)

-200

0

200

400

600

800

k.

II J: o

q) "~

c0 @ -0.5 -200

C 2s

"o

200

400

600

800

200

400

600

800

D t.O

o 4-*

m @

"14

0.5

ni

o

7 0 -200

0

200

400

800

800

-0.5 -200

0

Time after stimulus (ms) Fig. 7. Peristimulus time histograms of spontaneous discharge after stimulation of the sciatic and cutaneous nerves repeated at 1 Hz. A: an example of the units that were excited by sciatic nerve stimulation. Bin width is 5 ms. The number of repetition was 213. This unit is the same as that of Fig. 6. B: superposed display of moving average curves obtained from the 15 excited cells after sciatic nerve stimulation. C: an example of the units that were excited by cutaneous nerve stimulation. The number of stimulus repetitions was 161. D: superposed display of moving average curves of the 13 excited cells after cutaneous nerve stimulation.

DISCUSSION

In the present experiments, the stimulating electrode for antidromic activation was stereotaxically located in the posterior pituitary, and the dcctrolytic lesion of the electrode was in the posterior pituitary. However, it is possible that the observed antidromic spike might have be.¢n due to spreading of stimulating current to the neighbouring hypothalamus. But the pituitary was connected to the basal hypothalamus only with the narrow stalk, and covered with the dura mater. In addition, histological examinations demonstrated that recording sites were within the supraoptic nucleus where cell bodies of the magnoc¢llular ncurosccrctory cells resided. So, it is reasonable to conclude that the antidromic spike observed in the present experiments indicate the result of antidromic activation of the magnocc, ular ncurosecrctory axons in the posterior pituitary, rather than the cons¢qucnc¢ of cunvat spreading to the ncighbouring hypothalamus. In the present experiments, excitation was produced in a considerable number of

59 supraoptic magnocellular neurosecretory cells after tail pinching and noxious heat stimuli but not after tail touching or control heat stimuli. These data suggest that the supraoptic neurosecretory cells receive excitatory synaptic inputs originating from nociceptors. However, the noxious stimuli were also shown to induce a fall in blood pressure. A decrease in blood pressure has been demonstrated to elevate antidiuretic activity in the plasma 13'19 and to excite supraoptic neurosecretory cells 7's'ls. Thus, the possibility arises that a fall in blood pressure evoked by noxious stimuli might be the cause of the excitation in the supraoptic neurosecretory cells. In the present experiments, however, the early period of the excitation after noxious stimuli preceded a blood pressure decrease, and in some instances the excitation even occurred during a pressor response to noxious stimuli. Furthermore, nerve stimulation repeated at 20 Hz evoked excitation with a much shorter latency than that of the depressor response produced after the same stimuli. In addition, nerve stimulation repeated at 1 Hz produced a transient excitation on PSTHs, although blood pressure elevated during the stimulation. These data indicate the existence of direct neural pathways which mediate excitatory synaptic inputs originating from nociceptors to neurosecretory cells in the rat supraoptic nucleus. The present experiments demonstrated that certain supraoptic neurosecretory cells excited after both tail pinching and the application of noxious heat stimuli to the hindlimb paw. Furthermore, more than half of the neurones that were excited by stimulation of the sciatic nerve and the cutaneous nerve innervating the thigh were also shown to excite in response to tail pinching. These data suggest that supraoptic neurosecretory cells receive synaptic inputs originating from widely distributed nociceptors. Earlier bioassay studies have demonstrated that noxious stimuli such as electric foot shocks and tail pinching elevate the plasma antidiuretic activity2"1°'15,16. On the other hand, it was claimed in later radioimmunoassay studies that noxious stimuli do not increase the plasma vasopressin level6"9. Our previous study with radioimmunoassay techniques, however, has shown that electric foot shocks for 30 s significantly increase the plasma vasopressin level, while the plasma level of the hormone was low after intermittently applied foot shocks for 10 min TM. In the present experiments, a considerable number of supraoptic neurosecretory cells were shown to receive excitatory synaptic inputs after a variety of noxious stimuli, and a part of these responsive cells were found to exhibit a "phasic" pattern of spontaneous discharge. Magnocellular neurosecretory cells which show the "phasic" pattern of spontaneous discharge have been identified as the vasopressin-secreting cells in the rat La'SA2,17. Thus, the present study fh'st provides electrophysiological evidence to support the hypothesis that noxious stimuli potentiate vasopressin secretion by the posterior pituitary. It is known that during surgical operation plasma antidiuretic activity is elevated in patients under general anaesthesia 11. The present data demonstrate that supraoptic vasopressin-secreting neurones can be activated by a variety of noxious stimuli in the deeply anaesthetized rat, and that the excitation is mediated, at least in part, by direct

60 neural pathways originating from nociceptors. Thus, it appears that these neural pathways are involved in the increased rate of vasopressin secretion during surgical operation under anaesthesia. ACKNOWLEDGEMENTS

This study was supported by the Grants-in-Aid for Scientific Research from the Japanese Ministry of Education, Science and Culture (Nos. 56440079, 58106007 and 58770076). REFERENCES 1 Brimble, M.J. and DybaU, R.EJ., Characterization of the responses of oxytocin- and vasopressmsecreting neurones in the supraoptic nucleus to osmotic stimulation, J. Physiol. (Lend.), 271 (1977) 253-271. 2 De Wied, D. and Mirsky, I.A., The action of ALhydrocortisone on the antidiuretic and adrenocorticotropic responses to noxious stimuli, Endocrinology, 64 (1959) 955-966. 3 Dreifuss, J.J., Hams, M.C. and Tribollet, E., Excitation of phasically fn'ing hypothaiamic supraoptic neurones by carotid occlusion in rats, J. Physiol. (Lend.), 257 (1976) 337-354. 4 Hamamura, M., Shibuki, K. and Yagi, K., ADH-secreting cell: excitation after noxious stimuli in rats, J. Physiol. Soc. Jap., 45 0983) 574. 5 Harris, M.C., Dreifuss, JJ. and Legros, J.J., Excitation of phasically firing supraoptic neurones during vasopressin release, Nature (Lend.), 258 (1975) 80-82. 6 Husain, M.K., Manger, W.M., Rock, T.W., Weiss, R.J. and Frantz, A.G., Vasopressin release due to manual restraint in the rat: role of body compression and comparison with other stressful stimuli, Endocrinology, 104 (1979) 641-644. 7 Kannan, H. and Yagi, K., Supraoptic neurosecretory neurons: evidence for the existence of converging inputs both from carotid baroreceptors and osmoreceptors, Brain Res., 145 (1978) 385-390. 8 Kannan, H., Yagi, K. and Sawaki, Y., Pontine neurones: electrophysiologicat evidence of mediating carotid baroreceptor inputs to supraoptic neurones in rats, Exp. Brain Res., 42 (1981 ) 362-370. 9 Knepel, W., Nutto, D. and Hertting, G., Evidence for inhibition by fl-endorphin of vasopressin release during foot shock-induced stress in the rat, Neuroendocrinology, 34 (1982) 353-356. 10 Mirsky, I.A., Stein, M. and Pauliseh, G., The secretion of an antidiuretic substance into the circulation of rats exposed to noxious stimuli, Endocrinology, 54 (1954) 491-505. l 1 Moran, W.H. Jr., Milt~'nberger, F.W. Shuayb, W.A. and Zimmerman, B., The relationship of antidiuretic hormone secretion to surgical stress, Surgery, 56 (1964) 99-108. 12 Poulain, D.A., Wakerley, J.B. and Dyball, R.E., Electrophysiological differentiation of oxytocin- and vasopressin-secreting neurones, Prec. roy. Soc. B, 196 (1977) 367-384. 13 Share, L. and Levy, M.N., Carotid sinus pulse pressure, a determinant of plasma antidiuretic hormone concentration, Amer. J. Physiol., 211 (1966) 721-724. 14 Shibuki, K., Ishikawa, S., Hamamura, M., Saito, T., Yoshida, S. and Yagi, K., Synergistic action between footshocks and water deprivation on the plasma AVP level in the rat, Neurosci. Lett., Suppl. 17 (1984) S96. 15 Tata, P.S. and Buzalkov, R., Vasopressin studies in the rat. III. Inability of ethanol anesthesia to prevent ADH secretion due to pain and hemorrhage, P r i e r s Arch. ges. Physiol., 290 (1966) 294-297: 16 Thompson, E.A. and De Wied, D., The relationship between the antidiuretic activity of rat eye plexus blood and passive avoidance behavior, Physiol. Behav., 11 (1973) 377-380.

61 17 Wakerley, J.B., Poulain, D.A., Dyball, R.E. and Cross, B.A., Activity of phasic neurosecretory cells during haemorrhage, Nature (Lond.), 258 (1975) 82-84. 18 Yamashita, H., Effect of baro- and chemoreceptor activation on supraoptic nuclei neurons in the hypothalamus, Brain Res., 126 (1977) 551-556. 19 Zucker, I.H., Gorman, A.J., Cornish, K.G., Huffman, L.J. and Gilmore, J.P., Influence of left ventricular receptor stimulation on plasma vasopressin in conscious dogs, Amer. J. Physiol., 245 (1983) R792-R799.