KAZUMASA HONDA, HIDEO NEGORO, TETSUJI FUKUOKA, TAKASHI HIGUCHI AND KIYOSHI UCHIDE

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1 Endocrinol. Japon. 1985, 32 (1), Effect of Microelectrophoretically Applied Acetylcholine, Noradrenaline, Dopamine and Serotonin on the Discharge of Paraventricular Oxytocinergic Neurones in the Rat KAZUMASA HONDA, HIDEO NEGORO, TETSUJI FUKUOKA, TAKASHI HIGUCHI AND KIYOSHI UCHIDE Department of Physiology, Fukui Medical School, Matsuoka, Fukui, Abstract The effects of microelectrophoretic applications of neurotransmitter substances and their antagonists on the activity of paraventricular oxytocinergic neurones were studied in urethane anesthetized lactating rats. Oxytocinergic neurones were identified by their antidromic response to the stimulation of the neurohypophysis and by their characteristic high frequency discharge of action potentials approximately s before reflex milk ejection. Acetylcholine (ACh) excited the majority (75%) of paraventricular oxytocinergic neurones, and none of the cells was inhibited in its activity by ACh. In about half of the oxytocinergic cells, atropine and hexamethonium reduced the number of action potentials during the burst discharge preceding reflex milk ejection. Noradrenaline (NE), dopamine (DA) and serotonin (5-HT) reduced the activity of most (75-100%) of oxytocinergic neurones, and none of the cells was excited by these catecholamines. These results suggest that paraventricular oxytocinergic neurones receive excitatory cholinergic inputs and inhibitory noradrenergic, dopaminergic and serotonergic inputs. The response of antidromically identified paraventricular and supraoptic neurosecretory cells to microelectrophoretically applied putative neurotransmitter substances has been examined in many laboratories (Arnauld et al., 1983; Barker et al., 1971; Bioulac et al., 1978; Dreifuss and Kelly, 1972; Moss et al., 1971, 1972). Both the supraoptic nucleus and the magnocellular part of the Received October 18, 1984 paraventricular nucleus contain two major cell groups; oxytocin and vasopressin-secreting neurones (Rhodes et al., 1981). Furthermore, recent studies suggested that these nuclei also contained cell bodies of neurones other than oxytocin- and vasopressin-secreting neurones (Cuello and Kanazawa, 1978; Elde and Hafelt, 1978; Finley et al., 1981; Hafelt et al., 1977; Sar et al., 1978; Swanson and Sawchenko, 1983). Thus, functionally different cell groups in these nuclei might be included in early studies. The results of early microelectrophoretic studies were rather confusing probably due to the failure to identify these cell groups. The present experiments were undertaken to examine the response of identified

2 128 HONDA et al. Endocrinol. Japon. February 1985 oxytocinergic neurones to microelectrophoretic application of ACh, NE, DA or 5-HT. Materials and Methods Adult Wistar rats weighing g at days 9-13 of lactation were used. After h separation from their young, the mother rats were anesthetized with urethane at 1.1g/kg b. w., injected ip. The teat duct of an inguinal mammary gland was cannulated with a stainless steel tube (0.7mm outer diameter) and connected to a pressure transducer (Nihonkoden, LPU-0.1 A) to record intramammary pressure. A flexible cannula (Silastic, Dow Corning) was inserted into the right atrium through the right jugular vein to inject synthetic oxytocin (Atonin-O, Teikoku Hormone Mfg. Co.). A side-by-side stimulating electrode of stainless steel wire was inserted into the neurohypophysis in order to identify the paraventricular neurosecretory cells by antidromic stimulation. The stimulating electrode was considered to be in the neurohypophysis when an abrupt following stimulation (a 4s pulse train at 50 Hz, duration of pulse; 0.5ms, amplitude of current; 1mA). Eight to 11 pups that had been separated from their mother for 16-18h were applied to the nipples. Extracellular recordings were then started. Extracellular recordings were obtained through a micropipette filled with 0.5M sodium acetate (impedance 5-20 megohm, tip diameter 1-2ƒÊm) fixed to a 7-barreled micropipette (Akaishi et al., 1981) containing solutions of the following substances for microelectrophoresis: L-glutamate (Nakarai, 0.5 M, ph 6.4), acetylcholine chloride (Nakarai, 1.0 M, ph 5.0), atropine sulfate (Nakarai, 1.0 M, ph 5.4), hexamethonium chloride (Nakarai, 1.0 M, ph 6.9), noradrenaline hydrochloride (Nakarai, 1.5 M, ph 4.5), dopamine hydrochloride (Nakarai, 1.5 M, ph 4.5), 5-hydroxytryptamine hydrochloride (Sigma, 1.0 M, ph 5.4) and sodium chloride (Nakarai, 0.15 M, ph 7.0) for current control and current balancing. Since glutamate is known to have a nonspecific exciting action on the nerve cells (Curtis, 1965, Curtis and Crawford, 1969), it was used to confirm the effectiveness of the multibarreled micropipette. The tip diameter and the resistance in the barrels of the multibarreled micropipette were 3-6ƒÊm and megohm, respectively. Microelectrophoretic application of these substances was achieved with a micro-iontophoresis unit (Dia Medical System Co., DPI- 30 FA). The applied current intensities ranged from 1 ~10-9 to 200 ~10-9 A. When the drug application caused a significant change in the firing activity, a current of equal magnitude was passed through the 0.15 M NaCl-filled barrel as a control. Any cells that were affected by the current control were discarded from the data. Action potentials were amplified with a microelectrode amplifier (Nihon Kohden, MEZ-8201), displayed on an oscilloscope (Nihon Kohden, VC-10), and then counted with a pulse counter (Nihon Kohden, ET 612 J) whose output was plotted on an ink-writing recorder (Nihon Kohden, WI-641 G). The change in the mean firing rate of each neurone during drug application was analyzed by Student's t-test. When the p value was smaller than 0.05, the response of the neurone to the drug was considered to be positive. The effect of drug application on the neurosecretory burst preceding reflex milk ejection was considered to be positive if the drug application caused more than a 20% change in the number of action potentials per burst compared to that before the application. increase in the intramammary pressure occured Results Forty-two neurones in the paraventricular nucleus were antidromically identified as neurosecretory cells. Of these, 20 cells displayed a characteristic burst of accelerated activity approximately 15-20s before reflex milk ejection induced by suckling stimuli and were identified as oxytocinergic cells (Lincoln and Wakerley, 1975; Poulain et al., 1977). The response of these oxytocinergic cells to microelectrophoretically applied glutamate, ACh, NE, DA or 5-HT was tested. Glutamate facilitated the activity in all of the 13 cells tested (1-100nA). In nine out of 12 cells activity was facilitated by the application of ACh (5-100 na, one case; 200 na), and the remaining 3 cells were unresponsive (2-200 na), Activity of none of the cells was inhibited by ACh. On the other hand, NE, 5-HT and DA decreased the activity. NE decreased the activity in 5 out of 6 cells (2-30 na), and the activity of the remaining

3 Vol.32, No.1 OXYTOCIN CELL AND NEUROTRANSMITTERS 129 one was not changed by NE (15 na). Theactivity of three out of 4 cells decreased during the application of 5-HT (5-100 na) and the remaining cell was unresponsive to 5-HT (200 na). The activity of all of the 5 cells tested decreased when DA was applied (2-30 na). The activity of none of the cells was facilitated when NE, DA or 5-HT was applied (Table 1, Fig. 1). A B C D Fig. 1. Polygraph records showing the responses of paraventricular oxytocinergic neurones to microelectrophoretic applications of ACh, NE, DA and 5-HT. A: Excitation by application of ACh. B, C and D: Inhibitions by applications of NE, DA and 5-HT, respectively. The numbers refer to application current in nanoamperes. Of the putative transmitters tested in this experiment, ACh alone exerted an exciting effect on the activity of oxytocinergic cells. We therefore examined the effects of microelectrophoretic application of atropine and hexamethonium on burst discharges preceding reflex milk ejection induced by suckling stimuli (Table 2, Fig. 2). The drugs were applied from a few minutes after a milk ejection till the following milk ejection. Atropine reduced the burst discharge in 5 out of 9 oxytocinergic cells (10-110nA). In the remaining 4 cells, the burst discharge was unaffected by atropine. The basal firing rate was reduced by atropine in 6 out of 10 cells (2-30nA). The activity of one cell was increased (5nA) and that of 3 cells was not changed by atropine (10-110nA). In 4 out of 8 cells, the burst discharges were reduced by hexamethonium (2-20nA), and the activity of the remaining 4 cells did not change during the burst discharge (5-110nA). When hexamethonium was applied, basal activities were reduced in 2 cells (10 and 20nA), increased in 1 cell (5nA), and unchanged in the remaining 5 cells (2-110nA). Discussion The results of the present series of experiments clearly indicated that paraventricular oxytocinergic neurones were excited by Table 1. The responses of paraventricular oxytocinergic cells to several microelectrophoretically applied putative neurotransmitters

4 130 HONDA et al. Endocrinol. Japon. February 1985 Fig. 2. Polygraph records showing the responses of three paraventricular oxytocinergic neurones to microelectrophretic applications of atropine and hexamethonium. The total number of spikes per neurosecretory burst is shown beside each one. The numbers beside the names of drug refer to application current in nanoamperes. In neurone A, in- A tramammary pressure (lower trace) was indicated simultaneously with the electrical activity (upper trace). Note the complete blockade of the burst discharges by the applications of both atropine B and hexamethonium, whereas increases in intramammary C pressure occurred. Burst discharge was reduced by atropine but not by hexamethonium in neurone B, while hexamethonium effectively reduced the burst discharge but atropine did not in neurone C. Table 2. The effects of microelectrophoretically applied atropine and hexamethonium on the burst discharges preceding reflex milk e jection and the basal activity in paraventricular oxytocinergic cells ACh and inhibited by NE, DA and 5-HT, and that none of the oxytocinergic neurones was inhibited by ACh nor excited by these catecholamines. In contrast with our results, both excitatory and inhibitory effects were observed following the application of ACh or these catecholamines in early microelectrophoretic studies (Barker et al., 1971; Moss et al., 1971, 1972), in which neurosecretory neurones were identified only by the antidromic stimulation of the neurohypophysis. The results of these early microelectrophoretic studies may include the responses of the neurones other than oxytocinergic cells. Arnauld et al. (1983) have shown that supraoptic neurosecretory neurones which displayed a phasic firing pattern (putative vasopressinergic neurones) were excited by microelectrophoretic application of ACh and were inhibited by that of NE. However, they observed an inhibitory action of ACh on some of the cells which fired continuously. Neu-

5 Vol.32, No.2 OXYTOCIN CELL AND NEUROTRANSMITTERS 131 rones containing substances other than immunoreactive oxytocin and vasopressin have been found in the paraventricular and supraoptic nuclei (Cuello and Kanazawa, 1978; Elde and Hokfdelt, 1978; Finley et al., 1981; Hokfelt et al., 1977; Sar et al., 1978; Swanson and Sawchenko, 1983). Therefore, the inhibitory action of ACh and the excitatory action of NE on some of the antidromically identified neurosecretory cells in the early studies and in the results on continuous firing neurone of Arnauld et al. (1983) may reflect the responses to ACh and NE of cells other than oxytocin- or vasopressin-secreting neurones in these nuclei, which innervate the neurohypophysis. The results of the present series of experiments seem to indicate that a cholinergic mechanism is involved in the milk-ejection reflex at the level of oxytocinergic cell. However, we could not elucidate which type of receptor was involved in the occurrence of the milk ejection reflex, because the depressing effects of atropine and hexamethonium on the burst discharge of oxytocinergic cells preceding reflex milk ejection were of the same degree. Clarke et al. (1978) indicated that the milk-ejection reflex was blocked by the intravenous injection of nicotinic antagonist but not by the injection of muscarinic antagonist. The difference between our results and those of Clarke et al. (1978) may be due to the difference in the route of drug application. The results of the present experiment seem to indicate that both the muscarinic and nicotinic synapses were involved in the occurrence of the milk-ejection reflex at the level of oxytocinergic cells. However, it is known that atropine has a depressing action on the other central nervous systems (Clarke and Davies, 1973; Curtis and Phillis, 1960). Furthermore, Dreifuss et al. (1972) indicated that a much larger dose of atropine injected into the carotid artery was required to offset the effect of microelectrophoretically applied ACh than that of dihydro-Ĉ-erythroidine in the supraoptic neurosecretory cell. Thus, we cannot exclude the possibility that the depressing effect of atropine is due to its nonspecific action. It is likely that not only cholinergic synaptic transmission but also some other mechanisms are involved in the final transmission of the afferent input for milk-ejection reflex, since cholinergic blocker inhibited the burst discharge preceding reflex milk ejection in only about half of the oxytocinergic neurones. Recently, Freund-Mercier and Richard (1984) showed that oxytocin injected into the 3rd ventricle facilitated the milk-ejection reflex and the neurosecretory burst of paraventricular oxytocinergic neurones. Furthermore, Theodosis et al. (1981) and Hatton and Tweedle (1982) have demonstrated that extensive neurohal surface membrane appositions and multiple synaptic contacts are increased in the supraoptic nucleus during lactation. Therefore the final neuronal transmission of milk-ejection reflex seems to be a complicated phenomenon. In in vitro experiments, it was indicated. that NE facilitated the activity of supraoptic neurosecretory cells probably through the activation of a-adrenergic receptors (Wakerley et al., 1983; Randle et al., 1984). It was also indicated in the studies using slice preparation that DA increased the firing rate of the neurones which fired in a continuous manner in the supraoptic nucleus (Mason, 1983 a, b). Although there is no report of such experiments on paraventricular neurones, these results seem to be in contrast with ours. Diffusion of a microelectrophoretically applied drug may be mainly limited to the cell body, while in in vitro preparation the drug applied to the medium may reach not only the cell body but also its dendrites and interneurones. Therefore, a possible explanation of the difference between our results and those of in vitro studies is that the properties of the receptors for NE and DA which are localized to cell bodies may be different from those of recep-

6 132 HONDA et al. Endocrinol. Japon. February 1985 tors which are localized to dendrites. An alternative explanation is that the excitatory effects of NE and DA in in vitro studies may be mediated by the interneurones which exist inside the nucleus, since it was indicated that the relatively high proportion of presynaptic boutons on the magnocellular neurones in the paraventricular nucleus originated inside the nucleus (Kiss et al., 1983). Acknowledgement We thank Dr. R. E. J. Dyball for his helpful discussion during preparation of this manuscript. References Akaishi, T., H. Negoro and S. Kobayashi (1981). Electrophysiological evidence for multiple sites of action of angiotensin II in stimulating paraventricular neurosecretory cells in the rat. Brain Research. 220, Arnauld, E., M. Cirino, B. S. Layton and L. P. Renaud (1983). Contrasting actions of amino acids, acetylcholine, noradrenaline and leucine enkephalin on the excitability of supraoptic vasopressin-secreting neurons. Neuroendocrinol. 36, Barker, J. L., J. W. Crayton and R. A. Nicoll (1971). Noradrenaline and acetylcholine responses of supraoptic neurosecretory cells. J. Physiol. 218, Bioulac, B., O. Gaffori, M. Harris and J. D. Vincent (1978). Effects of acetylcholine, sodium glutamate and GABA on the discharge of supraoptic neurons in the rat. Brain Research. 154, Clarke, G. and J. Davis (1973). The effects of anti-parkinson drugs on cortical neurones. Br. J. Pharmac Clarke, G., C. H. D. Fall, D. W. Lincoln and L. P. Merrik (1978). Effects of cholinoceptor antagonists on the suckling-induced and experimentally evoked release of oxytocin. Br. J. Pharmac. 63, Cuello, A. C. and I. Kanazawa (1978). The distribution of substance P immunoreactive fibers in the rat central nervous system. J. Comp. Neurol. 178, Curtis, D. R. (1965). The actions of amino acids upon mammalian neurones. In: Studies in Physiology (D. R. Curtis and A. K. Mcintyre ed.), Springer, Berlin pp Curtis, D. R. and J. M. Crawford (1969). Central synaptic transmission microelectrophoretic studies. Ann. Rev. Pharmacol. 9, Curtis, D. R. and J. W. Phillis (1960). The action of procaine and atropine on spinal neurones. J. Physiol. 153, Dreifuss, J. J. and J. S. Kelly (1972). The activity of identified supraoptic neurones and their response to acetycholine applied by iontophoresis. J. Physiol. 220, Elde, R. and T. Hokfelt (1978). Distribution of hypothalamic hormones and other peptides in the brain. In: Frontiers in Neuroendocrinology vol. 5 (W. F. Ganong and L. Martini ed.), Raven Press, New York, pp Finley, J. C. W., J. L. Maderdrut and P. Petrusz (1981). The immunocytochemical localization of enkephalin in the central nervous system of the rat. J. Comp. Neurol. 198, Freund-Mercier, M. J. and P. Richard (1984). Electrophysiological evidence for facilitatory control of oxytocin neurones by oxytocin during suckling in the rat. J. Physiol. 352, Hatton, G. I. and C. D. Tweedle (1982). Magnocellular neuropeptidergic neurons in hypothalamus; Increase in membrane apposition and number of specialized synapses from pregnancy to lactation. Brain Res. Bulletin 8, Hokfelt, T., R. Elde, O. Johansson, L. Terenius and L. Stein (1977). The distribution of enkephalin-immunoreative cell bodies in the rat central nervous system. Neuroscience letters. 5, Kiss, J. Z., M. Palkovits, L. Zaborszky, E. Tribollet, D. Szabo and G. B. Makara (1983). Quantitative histological studies on the hypothalamic paraventricular nucleus in rats. II. Number of local and certain afferent nerve terminals. Brain Res. 265, Lincoln, D. W. and J. B. Wakerley (1975). Factors governing the periodic activation of supraoptic and paraventricular neurosecretory cells during suckling in the rat. J. Physiol. 250, Mason, W. T. (1983 a). Excitation by dopamine of putative oxytocinergic neurones in the rat supraoptic nucleus in vitro.: Evidence for two classes of continuously- firing neurones. Brain Res. 267,

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