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J. Physiol. (1977), 271, pp. 253-271 With 8 text-figurem Printed in Great Britain
CHARACTERIZATION OF THE RESPONSES OF OXYTOCIN- AND VASOPRESSIN-SECRETING NEURONES IN THE SUPRAOPTIC NUCLEUS TO OSMOTIC STIMULATION BY M. J. BRIMBLE AND R. E. J. DYBALL From the A.R.C. Institute of Animal Physiology, Babraham, Cambridge CB2 4AT
(Received 21 February 1977) SUMMARY
1. Extracellular action potentials were recorded from forty antidromically identified single units in the supraoptic nucleus of lactating, urethaneanaesthetized female rats. The activity was monitored both during reflex milk ejection and during an increase of 10-15 m-osmole/kg in plasma osmotic pressure induced by intraperitoneal injection of 1 ml. of 1-5 MNaCl solution. 2. About half (eighteen) the cells showed a burst of activity before reflex milk ejection and were dubbed oxytocin cells. Oxytocin cells responded to a hypertonic injection with a smooth sustained threefold increase in firing rate. 3. The remainder (twenty-two) showed no burst of activity before reflex milk ejection and were dubbed vasopressin cells. Vasopressin cells doubled their firing rate as plasma osmotic pressure increased. Neither cell type increased its firing rate after injections of isotonic NaCi. 4. A phasic firing pattern was rarely seen in slow firing vasopressin cells (< 2 spikes/sec) but was seen in almost all vasopressin cells (twelve out of fourteen) firing between 3 and 8 spikes/sec. Above 8 spikes/sec, some vasopressin cells fired continuously. Phasic firing was only once encountered in an oxytocin cell. 5. The firing rate of both oxytocin and vasopressin cells decreased when plasma osmotic pressure was reduced 10-15 m-osmole/kg by an intragastric water load of 10 ml. 6. Hypothalamic cells lying just outside the supraoptic nucleus did not show a consistent response to injection of hypertonic NaCl. 7. Clearly, both oxytocin and vasopressin cells are osmoresponsive, but phasic firing is characteristic of stimulated vasopressin cells. Thus, osmotic activation allows discrimination between oxytocin- and vasopressin-
secreting neurones.
254
M. J. BRIMBLE AND R. E. J. DYBALL INTRODUCTION
Intracarotid injections of hypertonic NaCl solution, which produce relatively small changes in plasma osmotic pressure in the carotid circulation, lead to secretion of the antidiuretic hormone (vasopressin; Verney, 1947). All the nervous elements necessary for this response are situated in the basal parts of the hypothalamus (Jewell & Verney, 1957). Accordingly, subsequent attempts to obtain an electrophysiological correlate of vasopressin release have been concentrated in the hypothalamus (for references see Cross, Dyball, Dyer, Jones, Lincoln, Morris & Pickering, 1975; Hayward, 1975). In the majority of these investigations, the activities of cells in the supraoptic nucleus were recorded during intracarotid injections of hypertonic NaCl solution. However, intracarotid injections do not constitute an ideal stimulus for use in single cell recording experiments. They are almost inevitably accompanied by transient and unpredictable changes in B.P. Such pressure changes may themselves influence nerve cell firing by altering local oxygen tension, by initiating pressor reflexes or by mechanical stimulation of the cell by the electrode. Any effect of plasma osmotic pressure change may thus be obscured. In order to overcome the problem, the more chronic stimulus of dehydration has been applied (Arnauld, Dufy & Vincent, 1975; Dyball & Pountney, 1973; Walters & Hatton, 1974). However, this alternative suffers from the disadvantage that single cells cannot be recorded continuously during the time of the plasma osmotic pressure change. Furthermore, in all previous studies no attempt was made to distinguish between the responses of oxytocin- and vasopressin-secreting neurones although it is known that both cell types occur in the supraoptic nucleus (Swaab, Nijveldt & Pool, 1975; Vandesande & Dierickx, 1975). We have attempted to extend the earlier investigations of the osmoresponsiveness of supraoptic neurones, but have altered the experimental design in two important respects. Before testing each cell, we established whether or not it showed a burst of activity just before reflex milk ejection (Wakerley & Lincoln, 1973). Such cells probably secrete oxytocin (Poulain, Wakerley & Dyball, 1977) and the remaining cells probably secrete vasopressin. We also used the i.P. route for the injection of hypertonic NaCl. A single i.P. injection of hypertonic NaCl increases plasma osmotic pressure slowly over approx. 15 min and leads to a release of vasopressin (Dunn, Brennan, Nelson & Robertson, 1973). Transient B.P. changes at the time of the injection subside within 5-10 min. Thus, any alterations of firing rate due to B.P. changes can be dissociated from those due to plasma osmotic pressure change. The time course is sufficiently short to enable continuous recording to be maintained from a single unit throughout the plasma
255 OXYTOCIN AND VASOPRESSIN NEURONES osmotic pressure change. Plasma osmotic pressure also remains elevated for some time after the hypertonic injection so that any changes in firing pattern can readily be discerned. Using these techniques, we hoped to establish whether both oxytocin and vasopressin cells in the supraoptic nucleus were osmoresponsive and, if they were, whether any differences could be detected between the responses of the two cell types. A preliminary report of this work has already been published (Brimble & Dyball, 1976). METHODS
Animals Female Wistar rats, 250-350 g in body wt., were used throughout the investigation. They were maintained in a constant environment at 21 TC with 10 hr dark and 14 hr light periods and given food (Breeders Diet, Oxoid Ltd) and water ad lib. Becaurreflex milk ejection occurs readily when the mammary tissue is engorged with milk, the rats were used between days 8 and 12 of lactation and each mother was separated from all but one of its litter on the night before the experiment. Only mothers with a litter of ten or more pups were used, and the procedures were similar to those described by Poulain et al. (1977). Surgery On the day of the experiment, the mother rats were anaesthetized by a single i.P. injection of urethane (ethyl carbamate; 1-2 g/kg). If additional anaesthesia was required during surgery, small doses (5 mg) of methohexitane sodium (Brietal, Elanco) were given I.P. A flexible cannula (Silastic, Dow Corning i.d. 0-02 in., o.d. 0-037 in.) was then inserted into the right atrium through the right jugular vein to collect blood samples (0.6 ml.) for plasma osmotic pressure determination (carried out by the freezing point method using a Knauer, Semimicro Osmometer). A Polythene cannula (Portex PP25) was inserted into the right saphenous artery for B.P. measurement, and a slightly larger cannula (Portex PP50) into the most caudal teat duct on the right side for intramammary pressure recording. For those experiments during which it was proposed to reduce plasma osmotic pressure, a stomach tube was also inserted (Portex PP40).
Electrophy8iologioal techniques After insertion of the cannulae, the rat was fixed in the head-holder of a stereotaxic apparatus (SN2 Narishige Instrument Co) and a longitudinal trephine hole was drilled in the skull through which both stimulating and recording electrodes were lowered. The bipolar concentric stimulating electrode (SNE 100, Rhodes Medical Instruments) was lowered in the midline at an angle of 13° from the vertical with the lower end forward, so that its tip rested on the neural stalk. It was lowered slowly until a matched biphasic pulse train (0.4 mA peak to peak) at 50 HEz for 5 sec reliably increased intramammary pressure to an extent similar to the intra-atrial injection of 1 m-u. oxytocin. InslX (InslX Products Corp) coated steel microelectrodes (approx. tip diameter 2 ,um) were then lowered into the supraoptic nucleus and extracellular action potentials recorded using conventional techniques. When a unit was encountered which was antidromically activated by stimulation of the neural stalk, ten pups were allowed to suckle the mother to induce reflex milk 9
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M. J. BRIMBLE AND R. E. J. DYBALL
ejection. In this way, the antidromically identified units could be classified into two groups. The first showed a burst of activity before reflex milk ejection (oxytocin neurones). The second group contained the unresponsive cells (putative vasopressin neurones; see Fig. 1). At this stage, two blood samples of 0-6 ml. were taken through the atrial cannula for plasma osmotic pressure and haematocrit determinations. After each sample was taken, blood volume was restored by the immediate injection of 0-6 ml. isotonic NaCl solution, through the same cannula. The blood sampling technique itself had only a transient (< 2 min) effect on the firing rate of the cells tested. After at least 20 min of recording, the plasma osmotic pressure was altered, either by an I.P. injection of 1 ml. of a 1-5 M-NaCl solution or an intragastric infusion of 10 ml. of distilled water. Further (0.6 ml.) blood samples were taken 30 min and 1 hr after the injection of either distilled water or hypertonic NaCl. At the end of each experiment, a small direct current was passed down the electrode (10 #A for 10 see) to deposit iron from the electrode tip. The brain was dissected out and fixed in 10% formaldehyde saline containing a small quantity of potassium ferro- and ferricyanide. The resulting Prussian blue spot was then used to locate the position of the tip of the recording electrode during a subsequent histological reconstruction of the hypothalamus (60 ,um frozen sections). RESULTS
1. Firing charracteritstic of supraoptic neurones Neurones antidromically identified as being in the supraoptic nucleus
exhibited one of the following spontaneous firing patterns (see Arnauld et al. 1975 and Fig. 1): Continuous (n = 11); Bursts of high-frequency activity separated by silent periods (phasic firing, n = 10); Slow (< 3 spikes/sec) and irregular (n = 19). Most cells could be classified readily as showing one of these patterns before stimulation, but some were hard to classify. Any cell which was not clearly firing continuously or physically was included in the slow and irregular category. We encountered only one silent cell in the rat supraoptic nucleus, although almost silent (< 5 spikes/min) cells were quite frequently encountered in adjoining hypothalamic areas. When supraoptic neurones were recorded during reflex milk ejection, the continuously active neurones always (eleven out of eleven) showed a burst of activity a few seconds before the increase on intramammary pressure, and thus were classified as oxytocin-secreting cells (see Discussion). In contrast, only one of the ten phasically firing cells showed a burst of firing Fig. 1. Polygraph recordings showing examples of the firing patterns of cells encountered in the supraoptic nucleus. Slow irregular firing is seen in both oxytocin (A) and vasopressin (B) neurons. At frequencies of 3-8 spikes/sec, oxytocin cells usually fire continuously (C), but vasopressin cells fire phasically (D). In each panel, the upper trace is intramammary pressure (I.M.P.), the middle trace a ratemeter record (RATE) and, on the lower trace, each pen deflection represents 1 spike (UNIT).
OXYTOCIN AND VASOPRESSIN NEURONES
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Fig. 2. The mean change in firing rate of eleven oxytocin (open circles), eight vasopressin (closed squares) and eleven adjacent hypothalamic cells (filled triangles) induced by an i.P. injection of 1 ml. 1*5 M-NaCl solution (given at the arrow). The mean changes in plasma osmotic pressure (O.P.) and blood pressure (B.P.) are also shown. The closed circles in the plot of plasma O.P. represent blood samples taken during the recordings. The open circles represent values from a separate series of six similarly treated animals which were used to avoid the necessity of taking multiple blood samples from the animals used for recording. Bars indicate s.E. of mean.
259 OXYTOCIN AND VASOPRESSIN NEURONES In the unstimulated preparation, the median firing rate of oxytocin cells in the supraoptic nucleus was 1-5 spikes/sec (range 0.6-4.4), for vasopressin cells the median was 2-3 (range 0-9.6). The respective mean (± S.E. of mean) values were 1-8 + 0-2 and 2-7 + 0-6. Since the distribution of the values was, in no case, significantly different from normal (Kolmogorov Smirnov, one sample test) values are, for simplicity, presented as means + 8.E. of mean in the remainder of this paper.
2. The effects of hypertonic NaCi injections on the firing rates of supraoptic neurotes Intraperitoneal injections of 1 ml. of 1-5 M-NaCl increased plasma osmotic pressure progressively over a time course of 15-30 min (Fig. 2). Thirty min after the injection, plasma osmotic pressure was ca. 12 mosmole/kg (4 %) higher than during the control period. It then remained constant for at least 1 hr. In one experiment, plasma osmolality was monitored for 3 hr after the injection, but there was no sign of a return to the pre-injection value. Systemic blood pressure showed variable fluctuations after the injection of hypertonic NaCl solution. Frequently, there was a rapid decrease (20 mm Hg or more), which lasted 1-2 min, followed by a smaller (5 mm Hg) but longer (10 min) increase. By 15 min after an injection, B.P. was the same as that before injection (see Fig. 2). Haematocrit decreased as the experiments progressed because of the removal of red blood cells with successive blood samples (a mean decrease of 2 % for each 0-6 ml. blood sample). In a supplementary series of experiments, smaller (0-1 ml.) samples were taken from seven rats at 5 min intervals from 15 min before to 25 min after an injection of hypertonic saline. Here too, haematocrit decreased slightly (2.5 % for eight samples), but there was no evidence of any substantial change in blood volume following the hypertonic injection. The increase in plasma osmotic pressure elicited by i.P. injection of hypertonic saline was associated with a significant (P < 0-01, t or MannWhitney test) increase in the firing rate of each type of neurone in the supraoptic nucleus. The excitation depended neither on the initial firing pattern nor on whether the cell secreted oxytocin or vasopressin. The increase in firing rate occurred at the same time as the increase in osmotic pressure. Both firing rate and osmotic pressure reached a max. about 15 min after the injection. After attaining the max. mean firing rate 6-7 + 0-5 spikes/sec for oxytocin cells and 6-5 + 0 7 spikes/sec for vasopressin cells (Fig. 2) the firing rates of both types of neurone tended to decrease somewhat although plasma osmotic pressure remained constant. Despite some variation between cells, Fig. 2 shows that the firing rate of vasopressin cells tended, at first, to decrease more rapidly than that of oxytocin cells,
M. J. BRIMBLE AND R. E. J. DYBALL 260 but then remained stable. Oxytocin cells showed a slower, but more prolonged, decrease in rate which continued throughout the recording period. Intraperitoneal injections of 1 ml. of isotonic (0.15 M) NaCl solution had no effect on any of the three oxytocin and three vasopressin cells tested.
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The responses of both types of neurone to an increased plasma osmotic pressure are similar when expressed as mean firing rate/sec during consecutive 5 min periods. A second increase in plasma osmotic pressure elicited a second increase in firing rate of similar magnitude to the first. This is illustrated for individual oxytocin and vasopressin neurones in Fig. 3. For oxytocin cells, the average increase was 4 7 + 0 3 spikes/sec (n = 11) following the first injection, and 5X5 + 0 5 spikes/sec (n = 9) following the second. For vasopressin cells, the increases were 3-4 ± 0-6 (n = 8) and 2-6 ± 0 5 (n = 7) spikes/sec, respectively. The firing rates of a population
261 OXYTOCIN AND VASOPRESSIN NEURONES of eleven spontaneously active neurones located outside but close to the supraoptic nucleus were also recorded. These neurones showed little change in their firing rate in response to the injection of 1 ml. of 1-5 M-NaCl (see Fig. 2).
3. Firing pattern differences in the responses of oxytocin and vasopressin neurones After stimulation, important pattern differences between the cell types emerged. The oxytocin cells responded with a smooth sustained increase in firing rate, regardless of their initial firing rate (Fig. 4). None of the oxytocin cells tested fired phasically after a hypertonic injection. The response of the vasopressin cells varied according to whether they were firing irregularly or phasically before the test. Irregularly firing cells, like the other cell types encountered, typically responded to an i.P. hypertonic injection with an increase in firing rate. At first, the response was very similar to that seen in oxytocin cells, but after 10-15 min the firing became interrupted by silent periods and a phasic firing pattern was established. The irregularly firing vasopressin cells thus adopted a pattern of firing indistinguishable from that of the phasic vasopressin cells seen in untreated animals (see Fig. 5). The vasopressin cells that were already firing phasically in the control period responded somewhat differently from those firing irregularly (see Fig. 5). They did not display a period of continuous firing following the i.P. injection. Typically, they increased their firing rates by increasing the duration of their active periods at the expense of their silent periods. The cells which had changed from irregular to phasic firing in response to one hypertonic injection responded to a second injection in the same way as cells which were initially firing phasically, i.e. with increased burst duration and firing rate within the bursts. The mean response of all the phasic cells tested is shown in Fig. 6, together with the increase in firing rate within the bursts and the increase in the values for Q, the activity quotient (Wakerley, Poulain, Dyball & Cross, 1975). The value of Q was calculated by dividing the total duration of the bursts by the total time, and the results are plotted as increments so that the variation in initial firing rate did not obscure the responses. Clearly injections of hypertonic NaCl solution significantly (P < 0-01, paired t test/or Wilcoxon matched-pairs signed-ranks test) increased the firing rate of the cells. There was an increase both in the firing rate within the bursts and Q. Some 15 min after the injection, Q tended to diminish although firing rate within the bursts remained elevated. In three animals in which plasma osmotic pressure had been raised above 315 m-osmole/kg, phasic cells with a very long burst duration were encountered. These cells responded to a further increase in
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M. J. BRIMBLE AND R. E. J. DYBALL 264 plasma osmotic pressure by firing continuously (Fig. 5). However, not all phasic cells could be induced to fire continuously, even when plasma osmotic pressure was raised above 330 m-osmole/kg. Some appeared to reach a max. firing rate and activity quotient beyond which they would not respond. 3 U 0
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4. The relationship between firing rate and plasma osmotic pressure During the course of the experiments, the firing rate of each cell recorded was related to the plasma osmotic pressure of samples taken at the
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M. J. BRIMBLE AND R. E. J. DYBALL 266 same time. A number of cells were recorded at several values of plasma osmotic pressure and a total of 108 values of firing rate and plasma osmotic pressure are plotted in Fig. 7. There was a significant correlation (P < 0.01) between firing rate and plasma osmotic pressure for both oxytocin and vasopressin cells, although the scatter of values was rather greater for the vasopressin than for the oxytocin cells. The scattergrams also show that, although only one phasic oxytocin cell was seen, almost all the vasopresan cells firing between 3 and 8 spikes/sec were phasic. 5. Effects of water loading on the firing rate of supraoptic neurones In a small series of experiments (five), plasma osmotic pressure was reduced by administration of an intragastric water load of 10 ml. of distilled water by stomach tube. This reduced plasma osmotic pressure by 10-15 mosmole/kg over a time course of about 30 min. The firing rates of both oxytocin- and vasopressin-releasing cells were reduced by water loading. The oxytocin cell illustrated in Fig. 8 almost stopped firing. The vasopressm cell illustrated was initially firing considerably faster, due to a higher plasma osmotic pressure (the animal had already had one i.P. injection of hypertonic saline) and was firing in a phasic pattern. After an intragastric water load, the cell slowed down and the phasic firing pattern gave way to an irregular pattern. Increasing the plasma osmotic pressure again by a subsequent i.P. injection of hypertonic saline increased the firing rate of both cells almost to their original values and re-established the phasic firing pattern in the vasopressin cell. DISCUSSION
Recordings from single neurones in the supraoptic nucleus have shown that approx. 50 % of the units encountered display a characteristic burst of firing just before reflex oxytocin secretion (Lincoln & Wakerley, 1974). The proportion of cells in the nucleus which are involved in reflex milk ejection does not depend on the intensity of the stimulus. Any cell which shows a burst of activity always does so whenever there is a reflex milk ejection. Reducing the number of suckling pups diminished the magnitude of the oxytocin release (Lincoln & Wakerely, 1975) and there was a corresponding reduction in the spike discharge, but no change in the number of responsive cells. Vasopressin is not released along with oxytocin during milk ejection in the rat (Wakerley, Dyball & Lincoln, 1973), so it is probable that the neurones which are activated are involved solely with oxytocin secretion. They probably represent that population of neurones (also constituting approx. half the cells in the nucleus) which can be stained immunologically
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M. J. BRIMBLE AND R. E. J. DYBALL 268 for oxytocin or its neurophysin (Swaab et at. 1975; Vandesande & Dierickx, 1975; Sokol, Zimmerman, Sawyer & Robinson, 1976). Thus, any cell which showed the characteristic burst of firing may, with confidence, be described as an oxytocin-secreting cell (Lincoln & Wakerley, 1974). The remaining 50 % of the cells within the nucleus can be stained for vasopressin or its neurophysin (Swaab et al. 1975; Vandesande & Dierickx, 1975; Sokol et at. 1976). Thus, cells recorded from within the nucleus which can be antidromically activated by stimulation of the neural lobe, but which do not secrete oxytocin, may with some justification be described as vasopressin-secreting cells. In the remainder of the Discussion section,this system of classification has been used to discriminate between the two major cell types in the supraoptic nucleus. Such a system of classification is essential if a successful correlation is to be made between the application of vasopressin-releasing stimuli and the activity of vasopressin-secreting neurones. Dunn et al. (1973) showed that, in the rat, i.P. injections of hypertonic NaCl solution increased plasma osmotic pressure. The increase was greatest approx. 15 min after the injection, and there was an increase in plasma vasopressin concentration which was linearly related to the rise in plasma osmotic pressure. Since there was no detectable change in plasma volume, the increase in vasopressin secretion was attributed to osmotic stimulation. In our hands, similar injections had little effect on plasma volume (as estimated by the haematocrit) but they increased plasma osmotic pressure to an extent which was similar to that reported by Dunn et al. (1973). Such injections produced quite dramatic but transient changes in B.P. immediately after they had been given. However, 15 min afterwards (at the time when the plasma osmotic pressure was greatest) B.P. had always returned to a value almost identical with that before injection. Vasopressin neurones frequently showed a transient excitation if B.P. fell at any time during an experiment (e.g. after the animal had been handled), but the excitation subsided as B.P. returned to normal. Furthermore, the magnitude of the increase in firing rate after an i.P. injection of hypertonic NaCl was not related to the magnitude of the B.P. change which accompanied it. Thus, any change in cell firing which persisted for 15 min or more after the hypertonic injections was probably due to a raised plasma osmotic pressure. It is clear, from the experiments reported here, that the firing rate of all the neurosecretory cells in the supraoptic nucleus increased as plasma osmotic pressure increased approx. 4 % following an i.P. injection of hypertonic NaCl. Both oxytocin and vasopressin cells responded to the stimulus but, if recording was continued for long enough, the firing of both cell types tended to diminish somewhat although plasma osmotic pressure
269 OXYTOCIN AND VASOPRESSIN NEURONES remained high. Neurones encountered outside the nucleus failed to respond, and injections of isotonic solution had no effect on either oxytocin or vasopressin cells. The responses of the neurosecretory neurones to hypertonic injections were repeatable so that a second or third injection increased the firing rates still further. They were also reversible in that a decreased firing rate was associated with the reduced plasma osmotic pressure which followed an intragastric water load. Vasopressin is known to be released by a raised plasma osmotic pressure (Verney, 1947), so that excitation of vasopressin cells by increased plasma osmotic pressure was not surprising. Excitation of oxytocin cells was more surprising, but not totally unexpected since depletion of the oxytocin content of the neural lobe occurs during dehydration (Jones & Pickering, 1969) and plasma oxytocin concentration also rises (Dogterom, van Rheenen-Verberg, van Wimersma Greidanus & Swaab, 1977). Furthermore, Haskins, Jennings & Rogers (1975) showed that all cells in the supraoptic nucleus were osmoresponsive. Cells in the supraoptic nucleus are osmoresponsive since they fire at a rate which is directly related to plasma osmotic pressure. Furthermore, since the firing rates of the immediately adjacent hypothalamic cells were not related to osmotic pressure, the supraoptic neurones may themselves be osmosensitive. At present, however, we cannot exclude the possibility that the neurones of the supraoptic nucleus are driven by osmosensitive neurones some distance away (Andersson, Leksell & Lishajko, 1975). Similarly, we cannot exclude the possibility that the change in firing rate is due to changes in plasma [Na+] rather than to changes in the osmotic pressure. This difference, however, may not be important in determining the response of the animal since Na+ ions are important in the maintenance of plasma osmotic pressure and, in most cases, when plasma [Na+] increases, so will plasma osmotic pressure. Phasic firing is plainly a much more characteristic feature of vasopressin cells than of oxytocin cells, a suggestion that has been made as a result of earlier studies (Dreifuss, Harris & Tribollet, 1976; Harris, Dreifuss & Legros, 1975; Wakerley et al. 1975; Poulain et al. 1977). Furthermore, it is clear that cells which fire phasically represent one aspect of the activity of cells which may at other times fire continuously or in a less structured pattern. The significance of the phasic pattern, whether it is intrinsic or reflects a cyclical pattern of input to the cells, and whether it is important in stimulus secretion coupling in vasopressin cells, is much less clear. Certainly, it allows the existence of short interspike intervals (which facilitate hormone release, Wakerley & Lincoln, 1973) with relatively slow average cell firing rates. The probable importance of short interspike intervals is emphasized by the observation that, when firing rate declines
M. J. BRIMBLE AND R. E. J. DYBALL 270 some time after a stimulus, it is the value of the activity quotient which is reduced, and not the firing rate within the bursts (see Fig. 6). It may be concluded that both oxytocin and vasopressin cells in the supraoptic nucleus are osmoresponsive and that their firing rate increases linearly as plasma osmotic pressure increases. In addition, phasic firing is a characteristic of vasopressin neurones when plasma osmotic pressure is raised and may thus be used as a means of discriminating between oxytocin
and vasopressin neurones. REFERENCES ANwDEnsso, B., LERSELL, L. G. & LisHjxo, F. (1975). Perturbations in fluid balance induced by medially placed forebrain lesions. Brain Re8. 99, 261-275. ARNAuLD, E., DuIFY, B. & VINCENT, J. D. (1975). Hypothalamic supraoptic neurones: rates and patterns of action potential firing during water deprivation in the unanaesthetised monkey. Brain Re8. 100, 315-325. BRIMBLE, M. J. & DYBALL, R. E. J. (1976). Contrasting pattern changes in the firing of vasopressin and oxytocin secreting neurones during osmotic stimulation. J. Phyeiol. 263, 189-190P. Cuoss, B. A., DYBATL, R. E. J., DYER, R. G., JoNss, C. W., LiNcoLN, D. W., MomRN, J. F. & PICKERING, B. T. (1975). Endocrine neurons. Recent Prog. Horm. Rem. 31, 243-286. DOGTEROm, J., vie RHEENEN-VERBERG, C. M. F., vAN WnRERs1 GREImAwus, Tj. B. & SwAAB, D. F. (1977). Vasopressin and oxytocin in cerebrospinal fluid of rats. J. Endocr. 72, 74-75P. DREnruss, J. J., HTRxIS, M. C. & TRIBOLLET, E. (1976). Excitation of phasically firing hypothalamic supraoptic neurones by carotid occlusion in rats. J. Phuysiol. 257, 337-354. DuAN, F. L., BRENNAN, T. J., NELSON, A. E. & ROBERTSON, G. L. (1973). The role of blood osmolality and volume in regulating vasopressin secretion in the rat. J. clin. Invest. 52, 3212-3219. DYBAIL, R. E. J. & PouwTNEY, P. S. (1973). Discharge patterns of supraoptic and paraventricular neurones in rats given a 2 % NaCl solution instead of drinkingwater. J. Endocr. 56, 91-98. HARRIS, M. D., DREIFUSS, J. J. & LEGROS, J. J. (1975). Excitation of phasically firing supraoptic neurones during vasopressin release. Nature, Lond. 258, 80-82. HASKINS, J. T., JENNINGS, J. P. & ROGERS, J. M. (1975). Response of neuroendocrine cell firing pattern types to measured changes in plasma osmolality. Phy8iologist, Lond. 18, 240. HAYWARD, J. N. (1975). Neural control of the posterior pituitary. A. Rev. Physiol. 37, 191-210. JEWELL, P. A. & VERNEY, E. B. (1957). An experimental attempt to determine the site of action of the neurohypophysial osmoreceptors of the dog. Phil. Trans. B. Soc. B 240, 197-324. JoNEs, C. W. & PICKERING, B. T. (1969). Comparison of the effects of water deprivation and sodium chloride inhibition on the hormone content of the neurohypophysis of the rat. J. Phyeiol. 203, 449-458. LINcoLN, D. W. & WAKERLEY, J. B. (1974). Electrophysiological evidence for the activation of supraoptic neurones during the release of oxytocin. J. Phy8iol. 242, 533-554.
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LnrcoLw, D. W. & WAKxRimY, J. B. (1975). Factors governing the periodic activation of supraoptic and paraventricular neurosecretory cells during suckling in the rat. J. Phy8iol. 250, 443-461. POuLAiW, D. A., WAKERLEY, J. B. & DYBALL, R. E. J. (1977). Electrophysiological differentiation of oxytocin- and vasopressin-secreting neurones. Proc. R. Soc. 13. 196, 367-384. SOKOL, H. W., ZnIMERmAN, E. A., SAWYER, W. H. & ROBINSON, A. G. (1976). The hypothalamic neurohypophysial system of the rat: localization and quantitation of neurophysin by light microscopic immunocytochemistry in normal rats and in Brattleboro rats deficient in vasopressin and a neurophysin. Endocrinology, 98, 1176-1188. SwAAB, D. F., NUVELDT, F. & POOL, C. W. (1975). Distribution of oxytocin and vasopressin in the rat supraoptic and paraventricular nucleus. J. Enlocr. 67, 461-462. VANDESANDE, F. & DIERIcEX, K. (1975). Identification of the vasopressin producing and of the oxytocin producing neurons in the hypothalamic magnocellular neurosecretory system of the rat. Cell & Ti8oue Re8. 164, 153-162. VERNEY, E. B. (1947). The antidiuretic hormone and the factors which determine its release. Proc. R. Soc. B 135, 25-106. WAKERLEY, J. B., DYBATL, R. E. J. & LINCOL, D. W. (1973). Milk ejection in the rat: the result of a selective release of oxytocin. J. Endocr. 57, 557-558. WAKERLEY, J. B. & LINCOLN, D. W. (1973). The milk ejection reflex of the rat: a 20. to 40-fold acceleration in the firing of paraventricular neurones during oxytocin release. J. Endocr. 57, 477-493. WAKERLEY, J. B., PouIIN, D. A., DY8ALL, R. 13. J. & CRoss, B. A. (1975). Activity of phasic neurosecretory cells during hemorrhage. Nature, Lond. 258, 82-84. WALTERS, J. K. & HAT1rON, G. I. (1974). Supraoptic neuronal activity in rats during 5 days of water deprivation. Physiol. & Behov. 13, 661-667.
J. Physiol. (1977), 271, pp. 253-271 With 8 text-figurem Printed in Great Britain
CHARACTERIZATION OF THE RESPONSES OF OXYTOCIN- AND VASOPRESSIN-SECRETING NEURONES IN THE SUPRAOPTIC NUCLEUS TO OSMOTIC STIMULATION BY M. J. BRIMBLE AND R. E. J. DYBALL From the A.R.C. Institute of Animal Physiology, Babraham, Cambridge CB2 4AT
(Received 21 February 1977) SUMMARY
1. Extracellular action potentials were recorded from forty antidromically identified single units in the supraoptic nucleus of lactating, urethaneanaesthetized female rats. The activity was monitored both during reflex milk ejection and during an increase of 10-15 m-osmole/kg in plasma osmotic pressure induced by intraperitoneal injection of 1 ml. of 1-5 MNaCl solution. 2. About half (eighteen) the cells showed a burst of activity before reflex milk ejection and were dubbed oxytocin cells. Oxytocin cells responded to a hypertonic injection with a smooth sustained threefold increase in firing rate. 3. The remainder (twenty-two) showed no burst of activity before reflex milk ejection and were dubbed vasopressin cells. Vasopressin cells doubled their firing rate as plasma osmotic pressure increased. Neither cell type increased its firing rate after injections of isotonic NaCi. 4. A phasic firing pattern was rarely seen in slow firing vasopressin cells (< 2 spikes/sec) but was seen in almost all vasopressin cells (twelve out of fourteen) firing between 3 and 8 spikes/sec. Above 8 spikes/sec, some vasopressin cells fired continuously. Phasic firing was only once encountered in an oxytocin cell. 5. The firing rate of both oxytocin and vasopressin cells decreased when plasma osmotic pressure was reduced 10-15 m-osmole/kg by an intragastric water load of 10 ml. 6. Hypothalamic cells lying just outside the supraoptic nucleus did not show a consistent response to injection of hypertonic NaCl. 7. Clearly, both oxytocin and vasopressin cells are osmoresponsive, but phasic firing is characteristic of stimulated vasopressin cells. Thus, osmotic activation allows discrimination between oxytocin- and vasopressin-
secreting neurones.
254
M. J. BRIMBLE AND R. E. J. DYBALL INTRODUCTION
Intracarotid injections of hypertonic NaCl solution, which produce relatively small changes in plasma osmotic pressure in the carotid circulation, lead to secretion of the antidiuretic hormone (vasopressin; Verney, 1947). All the nervous elements necessary for this response are situated in the basal parts of the hypothalamus (Jewell & Verney, 1957). Accordingly, subsequent attempts to obtain an electrophysiological correlate of vasopressin release have been concentrated in the hypothalamus (for references see Cross, Dyball, Dyer, Jones, Lincoln, Morris & Pickering, 1975; Hayward, 1975). In the majority of these investigations, the activities of cells in the supraoptic nucleus were recorded during intracarotid injections of hypertonic NaCl solution. However, intracarotid injections do not constitute an ideal stimulus for use in single cell recording experiments. They are almost inevitably accompanied by transient and unpredictable changes in B.P. Such pressure changes may themselves influence nerve cell firing by altering local oxygen tension, by initiating pressor reflexes or by mechanical stimulation of the cell by the electrode. Any effect of plasma osmotic pressure change may thus be obscured. In order to overcome the problem, the more chronic stimulus of dehydration has been applied (Arnauld, Dufy & Vincent, 1975; Dyball & Pountney, 1973; Walters & Hatton, 1974). However, this alternative suffers from the disadvantage that single cells cannot be recorded continuously during the time of the plasma osmotic pressure change. Furthermore, in all previous studies no attempt was made to distinguish between the responses of oxytocin- and vasopressin-secreting neurones although it is known that both cell types occur in the supraoptic nucleus (Swaab, Nijveldt & Pool, 1975; Vandesande & Dierickx, 1975). We have attempted to extend the earlier investigations of the osmoresponsiveness of supraoptic neurones, but have altered the experimental design in two important respects. Before testing each cell, we established whether or not it showed a burst of activity just before reflex milk ejection (Wakerley & Lincoln, 1973). Such cells probably secrete oxytocin (Poulain, Wakerley & Dyball, 1977) and the remaining cells probably secrete vasopressin. We also used the i.P. route for the injection of hypertonic NaCl. A single i.P. injection of hypertonic NaCl increases plasma osmotic pressure slowly over approx. 15 min and leads to a release of vasopressin (Dunn, Brennan, Nelson & Robertson, 1973). Transient B.P. changes at the time of the injection subside within 5-10 min. Thus, any alterations of firing rate due to B.P. changes can be dissociated from those due to plasma osmotic pressure change. The time course is sufficiently short to enable continuous recording to be maintained from a single unit throughout the plasma
255 OXYTOCIN AND VASOPRESSIN NEURONES osmotic pressure change. Plasma osmotic pressure also remains elevated for some time after the hypertonic injection so that any changes in firing pattern can readily be discerned. Using these techniques, we hoped to establish whether both oxytocin and vasopressin cells in the supraoptic nucleus were osmoresponsive and, if they were, whether any differences could be detected between the responses of the two cell types. A preliminary report of this work has already been published (Brimble & Dyball, 1976). METHODS
Animals Female Wistar rats, 250-350 g in body wt., were used throughout the investigation. They were maintained in a constant environment at 21 TC with 10 hr dark and 14 hr light periods and given food (Breeders Diet, Oxoid Ltd) and water ad lib. Becaurreflex milk ejection occurs readily when the mammary tissue is engorged with milk, the rats were used between days 8 and 12 of lactation and each mother was separated from all but one of its litter on the night before the experiment. Only mothers with a litter of ten or more pups were used, and the procedures were similar to those described by Poulain et al. (1977). Surgery On the day of the experiment, the mother rats were anaesthetized by a single i.P. injection of urethane (ethyl carbamate; 1-2 g/kg). If additional anaesthesia was required during surgery, small doses (5 mg) of methohexitane sodium (Brietal, Elanco) were given I.P. A flexible cannula (Silastic, Dow Corning i.d. 0-02 in., o.d. 0-037 in.) was then inserted into the right atrium through the right jugular vein to collect blood samples (0.6 ml.) for plasma osmotic pressure determination (carried out by the freezing point method using a Knauer, Semimicro Osmometer). A Polythene cannula (Portex PP25) was inserted into the right saphenous artery for B.P. measurement, and a slightly larger cannula (Portex PP50) into the most caudal teat duct on the right side for intramammary pressure recording. For those experiments during which it was proposed to reduce plasma osmotic pressure, a stomach tube was also inserted (Portex PP40).
Electrophy8iologioal techniques After insertion of the cannulae, the rat was fixed in the head-holder of a stereotaxic apparatus (SN2 Narishige Instrument Co) and a longitudinal trephine hole was drilled in the skull through which both stimulating and recording electrodes were lowered. The bipolar concentric stimulating electrode (SNE 100, Rhodes Medical Instruments) was lowered in the midline at an angle of 13° from the vertical with the lower end forward, so that its tip rested on the neural stalk. It was lowered slowly until a matched biphasic pulse train (0.4 mA peak to peak) at 50 HEz for 5 sec reliably increased intramammary pressure to an extent similar to the intra-atrial injection of 1 m-u. oxytocin. InslX (InslX Products Corp) coated steel microelectrodes (approx. tip diameter 2 ,um) were then lowered into the supraoptic nucleus and extracellular action potentials recorded using conventional techniques. When a unit was encountered which was antidromically activated by stimulation of the neural stalk, ten pups were allowed to suckle the mother to induce reflex milk 9
PHY 271
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M. J. BRIMBLE AND R. E. J. DYBALL
ejection. In this way, the antidromically identified units could be classified into two groups. The first showed a burst of activity before reflex milk ejection (oxytocin neurones). The second group contained the unresponsive cells (putative vasopressin neurones; see Fig. 1). At this stage, two blood samples of 0-6 ml. were taken through the atrial cannula for plasma osmotic pressure and haematocrit determinations. After each sample was taken, blood volume was restored by the immediate injection of 0-6 ml. isotonic NaCl solution, through the same cannula. The blood sampling technique itself had only a transient (< 2 min) effect on the firing rate of the cells tested. After at least 20 min of recording, the plasma osmotic pressure was altered, either by an I.P. injection of 1 ml. of a 1-5 M-NaCl solution or an intragastric infusion of 10 ml. of distilled water. Further (0.6 ml.) blood samples were taken 30 min and 1 hr after the injection of either distilled water or hypertonic NaCl. At the end of each experiment, a small direct current was passed down the electrode (10 #A for 10 see) to deposit iron from the electrode tip. The brain was dissected out and fixed in 10% formaldehyde saline containing a small quantity of potassium ferro- and ferricyanide. The resulting Prussian blue spot was then used to locate the position of the tip of the recording electrode during a subsequent histological reconstruction of the hypothalamus (60 ,um frozen sections). RESULTS
1. Firing charracteritstic of supraoptic neurones Neurones antidromically identified as being in the supraoptic nucleus
exhibited one of the following spontaneous firing patterns (see Arnauld et al. 1975 and Fig. 1): Continuous (n = 11); Bursts of high-frequency activity separated by silent periods (phasic firing, n = 10); Slow (< 3 spikes/sec) and irregular (n = 19). Most cells could be classified readily as showing one of these patterns before stimulation, but some were hard to classify. Any cell which was not clearly firing continuously or physically was included in the slow and irregular category. We encountered only one silent cell in the rat supraoptic nucleus, although almost silent (< 5 spikes/min) cells were quite frequently encountered in adjoining hypothalamic areas. When supraoptic neurones were recorded during reflex milk ejection, the continuously active neurones always (eleven out of eleven) showed a burst of activity a few seconds before the increase on intramammary pressure, and thus were classified as oxytocin-secreting cells (see Discussion). In contrast, only one of the ten phasically firing cells showed a burst of firing Fig. 1. Polygraph recordings showing examples of the firing patterns of cells encountered in the supraoptic nucleus. Slow irregular firing is seen in both oxytocin (A) and vasopressin (B) neurons. At frequencies of 3-8 spikes/sec, oxytocin cells usually fire continuously (C), but vasopressin cells fire phasically (D). In each panel, the upper trace is intramammary pressure (I.M.P.), the middle trace a ratemeter record (RATE) and, on the lower trace, each pen deflection represents 1 spike (UNIT).
OXYTOCIN AND VASOPRESSIN NEURONES
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258 M. J. BRIMBLE AND R. E. J. DYBALL related to milk-ejection. Hence, with the one exception we classified all these cells as putative vasopressin-secreting cells. Slowly and irregularly firing cells fell into either category since some (six) showed milk-ejectionrelated bursts of firing, and some (thirteen) did not (Fig. 1). 8 rOxytocin cells 61C)0
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259 OXYTOCIN AND VASOPRESSIN NEURONES In the unstimulated preparation, the median firing rate of oxytocin cells in the supraoptic nucleus was 1-5 spikes/sec (range 0.6-4.4), for vasopressin cells the median was 2-3 (range 0-9.6). The respective mean (± S.E. of mean) values were 1-8 + 0-2 and 2-7 + 0-6. Since the distribution of the values was, in no case, significantly different from normal (Kolmogorov Smirnov, one sample test) values are, for simplicity, presented as means + 8.E. of mean in the remainder of this paper.
2. The effects of hypertonic NaCi injections on the firing rates of supraoptic neurotes Intraperitoneal injections of 1 ml. of 1-5 M-NaCl increased plasma osmotic pressure progressively over a time course of 15-30 min (Fig. 2). Thirty min after the injection, plasma osmotic pressure was ca. 12 mosmole/kg (4 %) higher than during the control period. It then remained constant for at least 1 hr. In one experiment, plasma osmolality was monitored for 3 hr after the injection, but there was no sign of a return to the pre-injection value. Systemic blood pressure showed variable fluctuations after the injection of hypertonic NaCl solution. Frequently, there was a rapid decrease (20 mm Hg or more), which lasted 1-2 min, followed by a smaller (5 mm Hg) but longer (10 min) increase. By 15 min after an injection, B.P. was the same as that before injection (see Fig. 2). Haematocrit decreased as the experiments progressed because of the removal of red blood cells with successive blood samples (a mean decrease of 2 % for each 0-6 ml. blood sample). In a supplementary series of experiments, smaller (0-1 ml.) samples were taken from seven rats at 5 min intervals from 15 min before to 25 min after an injection of hypertonic saline. Here too, haematocrit decreased slightly (2.5 % for eight samples), but there was no evidence of any substantial change in blood volume following the hypertonic injection. The increase in plasma osmotic pressure elicited by i.P. injection of hypertonic saline was associated with a significant (P < 0-01, t or MannWhitney test) increase in the firing rate of each type of neurone in the supraoptic nucleus. The excitation depended neither on the initial firing pattern nor on whether the cell secreted oxytocin or vasopressin. The increase in firing rate occurred at the same time as the increase in osmotic pressure. Both firing rate and osmotic pressure reached a max. about 15 min after the injection. After attaining the max. mean firing rate 6-7 + 0-5 spikes/sec for oxytocin cells and 6-5 + 0 7 spikes/sec for vasopressin cells (Fig. 2) the firing rates of both types of neurone tended to decrease somewhat although plasma osmotic pressure remained constant. Despite some variation between cells, Fig. 2 shows that the firing rate of vasopressin cells tended, at first, to decrease more rapidly than that of oxytocin cells,
M. J. BRIMBLE AND R. E. J. DYBALL 260 but then remained stable. Oxytocin cells showed a slower, but more prolonged, decrease in rate which continued throughout the recording period. Intraperitoneal injections of 1 ml. of isotonic (0.15 M) NaCl solution had no effect on any of the three oxytocin and three vasopressin cells tested.
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The responses of both types of neurone to an increased plasma osmotic pressure are similar when expressed as mean firing rate/sec during consecutive 5 min periods. A second increase in plasma osmotic pressure elicited a second increase in firing rate of similar magnitude to the first. This is illustrated for individual oxytocin and vasopressin neurones in Fig. 3. For oxytocin cells, the average increase was 4 7 + 0 3 spikes/sec (n = 11) following the first injection, and 5X5 + 0 5 spikes/sec (n = 9) following the second. For vasopressin cells, the increases were 3-4 ± 0-6 (n = 8) and 2-6 ± 0 5 (n = 7) spikes/sec, respectively. The firing rates of a population
261 OXYTOCIN AND VASOPRESSIN NEURONES of eleven spontaneously active neurones located outside but close to the supraoptic nucleus were also recorded. These neurones showed little change in their firing rate in response to the injection of 1 ml. of 1-5 M-NaCl (see Fig. 2).
3. Firing pattern differences in the responses of oxytocin and vasopressin neurones After stimulation, important pattern differences between the cell types emerged. The oxytocin cells responded with a smooth sustained increase in firing rate, regardless of their initial firing rate (Fig. 4). None of the oxytocin cells tested fired phasically after a hypertonic injection. The response of the vasopressin cells varied according to whether they were firing irregularly or phasically before the test. Irregularly firing cells, like the other cell types encountered, typically responded to an i.P. hypertonic injection with an increase in firing rate. At first, the response was very similar to that seen in oxytocin cells, but after 10-15 min the firing became interrupted by silent periods and a phasic firing pattern was established. The irregularly firing vasopressin cells thus adopted a pattern of firing indistinguishable from that of the phasic vasopressin cells seen in untreated animals (see Fig. 5). The vasopressin cells that were already firing phasically in the control period responded somewhat differently from those firing irregularly (see Fig. 5). They did not display a period of continuous firing following the i.P. injection. Typically, they increased their firing rates by increasing the duration of their active periods at the expense of their silent periods. The cells which had changed from irregular to phasic firing in response to one hypertonic injection responded to a second injection in the same way as cells which were initially firing phasically, i.e. with increased burst duration and firing rate within the bursts. The mean response of all the phasic cells tested is shown in Fig. 6, together with the increase in firing rate within the bursts and the increase in the values for Q, the activity quotient (Wakerley, Poulain, Dyball & Cross, 1975). The value of Q was calculated by dividing the total duration of the bursts by the total time, and the results are plotted as increments so that the variation in initial firing rate did not obscure the responses. Clearly injections of hypertonic NaCl solution significantly (P < 0-01, paired t test/or Wilcoxon matched-pairs signed-ranks test) increased the firing rate of the cells. There was an increase both in the firing rate within the bursts and Q. Some 15 min after the injection, Q tended to diminish although firing rate within the bursts remained elevated. In three animals in which plasma osmotic pressure had been raised above 315 m-osmole/kg, phasic cells with a very long burst duration were encountered. These cells responded to a further increase in
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M. J. BRIMBLE AND R. E. J. DYBALL 264 plasma osmotic pressure by firing continuously (Fig. 5). However, not all phasic cells could be induced to fire continuously, even when plasma osmotic pressure was raised above 330 m-osmole/kg. Some appeared to reach a max. firing rate and activity quotient beyond which they would not respond. 3 U 0
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4. The relationship between firing rate and plasma osmotic pressure During the course of the experiments, the firing rate of each cell recorded was related to the plasma osmotic pressure of samples taken at the
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Fig. 7. The relationship between firing rate and plasma osmotic pressure for both oxytocin cells (above) and vasopressin cells (below). Linear regression lines are fitted to the points by the least squares method. A clear relationship exists between firing rate and osmotic pressure in both types of neurone (r is the correlation coefficient). Cells firing irregularly are shown as open stars; those firing phasically as circled stars and those firing continuously as closed circles. Phasic firing occurs much more frequently in vasopressin than oxytocin cells.
M. J. BRIMBLE AND R. E. J. DYBALL 266 same time. A number of cells were recorded at several values of plasma osmotic pressure and a total of 108 values of firing rate and plasma osmotic pressure are plotted in Fig. 7. There was a significant correlation (P < 0.01) between firing rate and plasma osmotic pressure for both oxytocin and vasopressin cells, although the scatter of values was rather greater for the vasopressin than for the oxytocin cells. The scattergrams also show that, although only one phasic oxytocin cell was seen, almost all the vasopresan cells firing between 3 and 8 spikes/sec were phasic. 5. Effects of water loading on the firing rate of supraoptic neurones In a small series of experiments (five), plasma osmotic pressure was reduced by administration of an intragastric water load of 10 ml. of distilled water by stomach tube. This reduced plasma osmotic pressure by 10-15 mosmole/kg over a time course of about 30 min. The firing rates of both oxytocin- and vasopressin-releasing cells were reduced by water loading. The oxytocin cell illustrated in Fig. 8 almost stopped firing. The vasopressm cell illustrated was initially firing considerably faster, due to a higher plasma osmotic pressure (the animal had already had one i.P. injection of hypertonic saline) and was firing in a phasic pattern. After an intragastric water load, the cell slowed down and the phasic firing pattern gave way to an irregular pattern. Increasing the plasma osmotic pressure again by a subsequent i.P. injection of hypertonic saline increased the firing rate of both cells almost to their original values and re-established the phasic firing pattern in the vasopressin cell. DISCUSSION
Recordings from single neurones in the supraoptic nucleus have shown that approx. 50 % of the units encountered display a characteristic burst of firing just before reflex oxytocin secretion (Lincoln & Wakerley, 1974). The proportion of cells in the nucleus which are involved in reflex milk ejection does not depend on the intensity of the stimulus. Any cell which shows a burst of activity always does so whenever there is a reflex milk ejection. Reducing the number of suckling pups diminished the magnitude of the oxytocin release (Lincoln & Wakerely, 1975) and there was a corresponding reduction in the spike discharge, but no change in the number of responsive cells. Vasopressin is not released along with oxytocin during milk ejection in the rat (Wakerley, Dyball & Lincoln, 1973), so it is probable that the neurones which are activated are involved solely with oxytocin secretion. They probably represent that population of neurones (also constituting approx. half the cells in the nucleus) which can be stained immunologically
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M. J. BRIMBLE AND R. E. J. DYBALL 268 for oxytocin or its neurophysin (Swaab et at. 1975; Vandesande & Dierickx, 1975; Sokol, Zimmerman, Sawyer & Robinson, 1976). Thus, any cell which showed the characteristic burst of firing may, with confidence, be described as an oxytocin-secreting cell (Lincoln & Wakerley, 1974). The remaining 50 % of the cells within the nucleus can be stained for vasopressin or its neurophysin (Swaab et al. 1975; Vandesande & Dierickx, 1975; Sokol et at. 1976). Thus, cells recorded from within the nucleus which can be antidromically activated by stimulation of the neural lobe, but which do not secrete oxytocin, may with some justification be described as vasopressin-secreting cells. In the remainder of the Discussion section,this system of classification has been used to discriminate between the two major cell types in the supraoptic nucleus. Such a system of classification is essential if a successful correlation is to be made between the application of vasopressin-releasing stimuli and the activity of vasopressin-secreting neurones. Dunn et al. (1973) showed that, in the rat, i.P. injections of hypertonic NaCl solution increased plasma osmotic pressure. The increase was greatest approx. 15 min after the injection, and there was an increase in plasma vasopressin concentration which was linearly related to the rise in plasma osmotic pressure. Since there was no detectable change in plasma volume, the increase in vasopressin secretion was attributed to osmotic stimulation. In our hands, similar injections had little effect on plasma volume (as estimated by the haematocrit) but they increased plasma osmotic pressure to an extent which was similar to that reported by Dunn et al. (1973). Such injections produced quite dramatic but transient changes in B.P. immediately after they had been given. However, 15 min afterwards (at the time when the plasma osmotic pressure was greatest) B.P. had always returned to a value almost identical with that before injection. Vasopressin neurones frequently showed a transient excitation if B.P. fell at any time during an experiment (e.g. after the animal had been handled), but the excitation subsided as B.P. returned to normal. Furthermore, the magnitude of the increase in firing rate after an i.P. injection of hypertonic NaCl was not related to the magnitude of the B.P. change which accompanied it. Thus, any change in cell firing which persisted for 15 min or more after the hypertonic injections was probably due to a raised plasma osmotic pressure. It is clear, from the experiments reported here, that the firing rate of all the neurosecretory cells in the supraoptic nucleus increased as plasma osmotic pressure increased approx. 4 % following an i.P. injection of hypertonic NaCl. Both oxytocin and vasopressin cells responded to the stimulus but, if recording was continued for long enough, the firing of both cell types tended to diminish somewhat although plasma osmotic pressure
269 OXYTOCIN AND VASOPRESSIN NEURONES remained high. Neurones encountered outside the nucleus failed to respond, and injections of isotonic solution had no effect on either oxytocin or vasopressin cells. The responses of the neurosecretory neurones to hypertonic injections were repeatable so that a second or third injection increased the firing rates still further. They were also reversible in that a decreased firing rate was associated with the reduced plasma osmotic pressure which followed an intragastric water load. Vasopressin is known to be released by a raised plasma osmotic pressure (Verney, 1947), so that excitation of vasopressin cells by increased plasma osmotic pressure was not surprising. Excitation of oxytocin cells was more surprising, but not totally unexpected since depletion of the oxytocin content of the neural lobe occurs during dehydration (Jones & Pickering, 1969) and plasma oxytocin concentration also rises (Dogterom, van Rheenen-Verberg, van Wimersma Greidanus & Swaab, 1977). Furthermore, Haskins, Jennings & Rogers (1975) showed that all cells in the supraoptic nucleus were osmoresponsive. Cells in the supraoptic nucleus are osmoresponsive since they fire at a rate which is directly related to plasma osmotic pressure. Furthermore, since the firing rates of the immediately adjacent hypothalamic cells were not related to osmotic pressure, the supraoptic neurones may themselves be osmosensitive. At present, however, we cannot exclude the possibility that the neurones of the supraoptic nucleus are driven by osmosensitive neurones some distance away (Andersson, Leksell & Lishajko, 1975). Similarly, we cannot exclude the possibility that the change in firing rate is due to changes in plasma [Na+] rather than to changes in the osmotic pressure. This difference, however, may not be important in determining the response of the animal since Na+ ions are important in the maintenance of plasma osmotic pressure and, in most cases, when plasma [Na+] increases, so will plasma osmotic pressure. Phasic firing is plainly a much more characteristic feature of vasopressin cells than of oxytocin cells, a suggestion that has been made as a result of earlier studies (Dreifuss, Harris & Tribollet, 1976; Harris, Dreifuss & Legros, 1975; Wakerley et al. 1975; Poulain et al. 1977). Furthermore, it is clear that cells which fire phasically represent one aspect of the activity of cells which may at other times fire continuously or in a less structured pattern. The significance of the phasic pattern, whether it is intrinsic or reflects a cyclical pattern of input to the cells, and whether it is important in stimulus secretion coupling in vasopressin cells, is much less clear. Certainly, it allows the existence of short interspike intervals (which facilitate hormone release, Wakerley & Lincoln, 1973) with relatively slow average cell firing rates. The probable importance of short interspike intervals is emphasized by the observation that, when firing rate declines
M. J. BRIMBLE AND R. E. J. DYBALL 270 some time after a stimulus, it is the value of the activity quotient which is reduced, and not the firing rate within the bursts (see Fig. 6). It may be concluded that both oxytocin and vasopressin cells in the supraoptic nucleus are osmoresponsive and that their firing rate increases linearly as plasma osmotic pressure increases. In addition, phasic firing is a characteristic of vasopressin neurones when plasma osmotic pressure is raised and may thus be used as a means of discriminating between oxytocin
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