Oxytocin predominantly excites putative oxytocin neurons in the rat supraoptic nucleus in vitro

BrainResearcl 410 1987) 3()4-368

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Hiroshi Y a m a s h i t a 1, Shigeru O k u y a 2, Kiyotoshi I n e n a g a 1, Masanori Kasai I , Sayuri U e s u g i 1, Hiroshi K a n n a n 1 and T o s h i o K a n e k o ~ 1Department of Physiology, Universityof Occupational and Environmental Health, School of Medicine, Kitakyushu (Japan) and 2Third Department oflnternal Medicine, Yamaguchi University, School of MediCine, Ube (Japan) (Accepted 28 April 1987)

Key words." Oxytocin; Oxytocin neuron; Supraoptic nucleus; Oxytocin analogue; Brain slice

To determine the oxytocin (OXT) sensitivity of neurons in the supraoptic nucleus (SON), extrace!lular recordings were made from the rat hypothalamic slice preparation. OXT added to the bathing medium (3 × 10-7 M) excited 13 (93%) of i4 cells which fired continuously (average 4.9 + 0.7 spikes/s) and 26 (81%) of 32 cells which fired slowly and irregularly (average 1.4 .+_0.4 spikes/s). By contrast, only 2 (8%) of 26 phasically firing neurons were excited and none of the SON cells tested were inhibited~ The excitation was reversibly antagonized by a synthetic OXT analogue, 1-deamino-[2-(O-methyltyrosine),4-valine, 8,D-arginine]vasopresSin. Th e results suggest that OXT exerts predominantly excitatory effects in the SON and that putative OXT cells are more likely to be affected than putative vasopressin cells.

Oxytocin ( O X T ) is well known for its p e r i p h e r a l action during lactation and parturition after its release into the b l o o d s t r e a m from the n e u r o h y p o p h y sis. H o w e v e r , O X T m a y also be released centrally to modify the activity of neurons in the central nervous system 4,11'14. including that of O X T neurons themselves. It has also been d e m o n s t r a t e d that exogenous O X T enhances O X T release from isolated magnocellular nuclei in vitro 12 and that O X T injected into the third ventricle modifies the activity of O X T cells in suckled lactating rats 7-9. It increases the basal firing rate of O X T neurons, and the frequency and amplitude of the bursts of spikes normally shown before each reflex milk ejection. F u r t h e r m o r e . magnocellular neurons can be excited by iontophoretically applied O X T t3 and an i m m u n o h i s t o c h e m i c a l study has revealed that O X T - i m m u n o r e a c t i v e terminals synapse on O X T neurons in the supraoptic nucleus ( S O N ) t9. The present electrophysiological study was undertaken to clarify the effects of O X T on SON neurons using the rat h y p o t h a l a m i c slice p r e p a r a t i o n .

Conventional h y p o t h a l a m i c slices 11"15'2°. 350 ~ m in thickness, were cut coronally from male Wistar rats weighing 150-300 g. Before recording, the slices were t r i m m e d carefully to leave a piece of tissue contaming the S O N with a total a r e a less than 1.8 × 1.8 mm. The small piece of tissue contained only the SON. a part of the perinuclear zone of the S O N and a part of the optic chiasm. A f t e r preincubation for at least 1 h in an incubation m e d i u m oxygenated with 95% 0 2 and 5% CO2, the t r i m m e d slice was transferred to a recording c h a m b e r , placed on a sylgard mat glued to its b o t t o m and h e l d in place with a nylon net and platinum weights. The recording c h a m b e r was perfused with perfusing medium at a rate of 1 - 2 ml/min and m a i n t a i n e d at 35 _+ 0.5 °C. T h e perfusion system (see refs. 10. 11. 15) was gravity-fed and excess m e d i u m was r e m o v e d by suction. The incubation m e d i u m was a m o d i f i e d Y a m a m o to's solution, which contained the following concentrations (in mM): NaCI 124, KCI 5, KH2PO 4 1.24. MgSO4 1.3. CaC12 2.1. N a H C O 3 20. and glucose 10.

Correspondence." H. Yamashita. Department of Physiology, University of Occupational and Environmental Health. School of Medicine, Yahatanishi-ku, Kitakyushu. 807, Japan. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. IBiomedical Division I

365 while the calcium concentration was lowered to 0.75 mM in the perfusing medium in order to increase the spontaneous activity of SON neurons L~A6. When required, a low Ca 2+ (0.5 mM) and high Mg 2+ (9 mM) solution was used to block synaptic transmission mAklS. Extracellular recordings were made from neurons within the SON (which could be distinguished microscopically) using glass micropipettes filled with 0.5 M Na acetate containing 2% Pontamine sky blue (electrode resistance: 20-35 M~2). At the end of recording, a current of 5 ¢~A was passed through the electrode for 3-5 rain (tip negative) to deposit a blue spot from which the recorded sites could be precisely determined histologically. Recorded spikes were displayed using a conventional recording system and stored on magnetic tape for further analysis. The firing rates were expressed as spikes/s, although the reset time was sometimes 2 s or 5 s. The peptides used in this experiment were oxyto-

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cin (OXT; Peptide Institute, Minoh, Japan), arginine-vasopressin (AVP; Peptide Institute, Minoh, Japan) and 1-deamino-[2-(O-methyltyrosine), 4-valine, 8-D-arginine]vasopressin (D[Tyr(Me)2] VDAVP). The analogue, D[Tyr(Me):]VDAVP, was kindly supplied by Prof. M. Manning, Medical College of Ohio, Toledo. Seventy-two spontaneously active SON neurons were recorded extracellularly. On the basis of their firing pattern, 14 (19%) neurons were classified as fast continuous, 32 (44%) as slow irregular and 26 (36%) as phasic firing. The mean firing rates (mean _+ S.E.M.) of each type of neuron were 4.9 + 0.7 spikes/s, 1.4 + 0.4 spikes/s and 2.4 _+ 0.5 spikes/s respectively. The duration of the burst and silent periods of the phasic firing neurons fell within the range of 5-350 s and the mean intraburst firing rate ranged from 3 to 16 spikes/s. The amplitude of spikes recorded in this study ranged from (1.6 to 5.0 mV. After making a stable recording from a single cell

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Fig. 1. Ratemeter records of responses of SON neurons to application of peptides. A: a fast continuous firing neuron on which O X T exerted a clear excitatory effect: by contrast, A V P exerted a much weaker stimulatory effect at the same concentration. Application of an O X T analogue, d [ T y r ( M e ) 2 ] V D A V P 10 6 M, which acts as an oxytocic receptor antagonist, almost completely suppressed the excitation induced by O X T 3 x 10 7 M. B: a phasic firing neuron which failed to respond to OXT. C: another phasic firing neuron which was excited by OXT.

366 for at least 10 min, O X T was applied to the slice at a concentration of 3 x 10 -7 M. The responses were assessed by observing the change in firing rate before and during application of O X T . The change of the firing rate from the resting level (mean firing rate during 10 min) to the p e a k level (mean firing rate during 1 min) following a p p l i c a t i o n of O X T was calculated from the r a t e m e t e r records. Cells were classified as having r e s p o n d e d if their firing rates after application of O X T had changed by m o r e than 20%. The responses of 72 S O N cells to application of O X T were tested. Thirteen (93%) of 14 fast continuous and 26 (81%) of 32 slow irregular firing neurons were excited b y O X T (Figs. 1A and 2A). In contrast to the profound effects in these types of neurons, only 2 (8%) of 26 phasic firing neurons were excited by O X T (Fig. 1C). The remaining cells were unaffected (Fig. 1B). W h e n the actions of A V P were c o m p a r e d with those of O X T , all of 17 neurons tested, which had shown clear excitatory responses to O X T , showed a much w e a k e r excitatory responses to A V P

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continuous ratemeter record of successive responses of a slow irregular firing neuron to increasing concentrations of OXT (10-9-10 -6 M), B: dose-response curves obtained from 8 individual SON cells for OXT (O) and 4 for AVP ((3). The firing rate of each neuron increased with increased concentration of OXT and AVP.

or no response at all at the same concentration (Fig, IA). A d o s e - r e s p o n s e relationship at different concentrations ranging from 10 -m to 10 ~ M was obtained for 8 SON neurons excited by O X T and for 4 SON neurons excited by A V P (Fig. 2). The increase in firing rate following application of O X T or A V P at each concentration was calculated from the r a t e m e t e r records and, when plotted against the concentration, the firing rate of each neuron generally increased with increase of O X T or A V P concentration (Fig. 2B). The threshold concentrations which e v o k e d responses were a p p r o x i m a t e l y 10-" M for O X T and 10 .-7 for AVP. The much greater effectiveness ot O X T suggested that the responses resulted from an action on oxvtocic receptors, tn an a t t e m p t to confirm this. a svnthetic structural analogue, D[Tyr(Me)2]VDAVP. known to block both the oxytoclc and vasopressor effects of neurohypophysial peptides 4A~, was applied to 6 neurons. Fig. I A shows the effect of D[Tyr(Me)~] V D A V P on the O X T - i n d u c e d activity of a S O N neuron. The analogue (10 '~ M) ahnost completely blocked the effect of O X T (3 x t l ) - MI on the neuron. A f t e r washing out both peptides, application of O X T (3 × 10 -7 M) again excited the cell. The analogue (11)-~' M) substantially blocked the effects of O X T (3 × 10 7 M) on each of 6 neurons tested. While the experiments described above clearly d e m o n s t r a t e d responses of SON neurons to O X T . it was not clear whether the responses were due to direct or indirect effects on the neurons. To d e t e r m i n e which alternative was correct, a low (-a :+ and high Mg ~+ medium was used to block synapuc transmission 2, m.l!As. After changing the perfusing m e d i u m to the low Ca 2. and high Mg e+ medium_ the spontaneous activity of SON neurons was strongly depressed. Most of the synaptic drive lo the cells is eliminated or strongly depressed by such a procedure and the slices were t r i m m e d as far as possible to minimize synaptic drive from elsewhere in the slice. Four neurons which had shown excitatory responses to O X T in control perfusion m e d i u m were also excited after blockade of synaptic transmission (Fig. 3). Although the amplitude of responses were decreased, it is thus p r o b a b l e that at least some S O N neurons were themselves sensitive to OXT. The concentration of O X T used in this experiment

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ranged from 10 ~0 to 10 6 M and the threshold concentration was approximately 10 -9 M. The normal concentration of O X T in the cerebrospinal fluid (CSF) of rats is known to be 7 x 10 -11 M (ref. 6). If O X T was released locally in the SON and acted as a neurotransmitter or neuromodulator, the concentration of O X T near the cell bodies in the SON might be much higher than that in the CSF. It thus seems reasonable to suggest that the threshold concentration observed in this study, which was very similar to that found in previous experiments TM, lies within the physiological range. According to their firing pattern, the electrical activity of SON neurons can normally be assigned to one of 3 categories: slow irregular, fast continuous or phasic 17. Phasic activity is known to be a feature of vasopressin (VP) neurons and fast continuous activity is characteristic of O X T neurons 3'17'2°. The slow irregularly firing neurons probably constitute a mixed population of O X T and VP cells. In the present study, almost all the fast continuous firing neurons were excited by OXT, but the phasic firing neurons were relatively unaffected by OXT. It is thus probable that O X T excites predominantly putative O X T cells in the SON. There was the existence of a marked discrepancy between the results obtained in the rat SON and in the guinea pig SON relating to the type of receptors to neurohypophysial peptides. In the guinea pig SON, lysine-vasopressin (LVP) decreased the number of action potentials, though it caused depolarization of the membrane 1. The amplitude of its depolarization for LVP was much bigger than that for OXT. However, the present study showed that A V P

exerted a much weaker stimulatory effect or no effect at all on the neurons which had shown clear excitatory responses to O X T and that an analogue which acts as an antagonist in the uterus 414 blocked the excitatory effect of OXT. Thus the receptors for O X T which are present in the rat SON probably resemble the uterine oxytocic receptors. Similar result was obtained in rat hippocampal neurons 1~. The weak response by A V P might be mediated through such O X T receptors. However, we could not exclude the existence of VP receptors in the rat SON neurons. A previous report t claimed that the response by LVP was mediated through V2-type VP receptors in guinea pig SON neurons. These findings suggest that the population and type of receptors to neurohypophysial hormones are different in the rat and guinea pig. Moos et al. provided evidence that application of exogenous O X T released O X T from isolated magnocellular nuclei by a Ca2+-dependent mechanism in vitro ~2. It is possible that O X T released within the SON exerts a positive feedback on its own release via recurrent axon-collaterals of O X T cells ~s or via dendrites as for dopamine in the substantia nigra 5. Thus O X T may contribute to the synchronization of SON cells during, for example, milk ejection. We would like to express our appreciation to Dr. R.E.J. Dyball for help in the preparation of this manuscript and to Dr. M. Manning for supplying an oxytocin analogue. This work was supported in part by Grants-in-Aid for Scientific Research 60304042 and 60304045 from the Ministry of Education, Science and Culture of Japan.

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