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The Role of Alpha-2 Adrenoceptors in the Regulation of Oxytocin Neurones in the Suckled Rat
Brain Research Bulletin, Vol. 44, No. 2, pp. 193–197 Copyright © 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/97 $17.00 1 .00
PII S0361-9230(97)00115-9
The Role of Alpha-2 Adrenoceptors in the Regulation of Oxytocin Neurones in the Suckled Rat ALEX BAILEY,1 GEOFF CLARKE AND JONATHAN WAKERLEY Department of Anatomy, School of Medical Sciences, University of Bristol, University Walk, Bristol, BS8 1TD, UK [Received 3 March 1997; Accepted 15 May 1997] ABSTRACT: The role of alpha-2 adrenoceptors in the milkejection reflex was investigated by making electrophysiological recordings from oxytocin neurones in the supraoptic nucleus of urethane-anaesthetised rats. Systemic administration of the alpha-2 adrenoceptor antagonist, idazoxan (0.5 mg/kg, IV), temporarily suppressed OT cell bursting activity, while having no consistent action on basal neuronal activity. Clonidine (25 mg/ kg, IV) caused an immediate increase in the frequency and amplitude of oxytocin cell bursting, coincident with a fall in basal activity. A higher dose of clonidine (50 mg/kg, IV), inhibited both bursting and basal activity. These results indicate that alpha-2 adrenoceptors are essential for the normal functioning of the milk-ejection reflex and may be involved in the facilitatory and inhibitory regulation of suckling-evoked bursting in oxytocin neurones. © 1997 Elsevier Science Inc.
project directly to the magnocellular nuclei, including the bed nuclei, septum, and the dorsal vagal complex [14,23]. This study examines the possible significance of alpha-2 adrenoceptor-mediated transmission in the milk-ejection reflex by investigating the effects of systemic administration of the alpha-2 adrenoceptor agonist, clonidine, and antagonist, idazoxan, on the electrophysiological activity of SON OT neurones in the urethane anaesthetised lactating rat. A preliminary account of some of these results has already been described [1]. MATERIALS AND METHODS Experiments were performed on lactating Wistar rats (280 –390 g) taken from the departmental colony, between days 8 –12 postpartum. Animals were housed individually with their litter and maintained under controlled environmental conditions with food and water available ad lib. Animals were separated from all but one of their litter overnight and subsequently anaesthetised with a single intraperitoneal injection of urethane (1.2 g/kg, ethyl carbamate, Sigma, UK). A jugular vein and mammary gland were cannulated for drug administration and intramammary pressure recording respectively. All animals received a single dose (10 mg/kg, IV) of the short-acting analgesic, fentanyl (Janssen Pharmaceutical, UK) before being placed in a stereotaxic frame and prepared for recording from the SON, as previously described [2]. One hour before recording all animals received an injection of propranolol (1 mg/kg IV, Inderal, Zeneca, UK) to suppress stress-related inhibition of the milk-ejection reflex [21]. Extracellular recordings were made in the SON with glass micropipettes (5–10 MV) filled with 0.5 M sodium chloride. Magnocellular cells of the SON were identified by antidromic invasion following electrical stimulation of the pituitary stalk and cancellation of the antidromic potential by collision with a spontaneous orthodromic potential. All recordings were stored on tape (Store 4, Racal, UK) and subsequently analysed by computer (Modulog, Grafitek Ltd, UK). Cells were classified as oxytocinergic if they displayed a characteristic high-frequency burst of activity immediately prior to a rise in intramammary pressure associated with a milk-ejection response [25]. Clonidine and idazoxan were only injected after recording neuronal activity for a minimum of 20 min, during which time two
KEY WORDS: Clonidine, Idazoxan, Milk-ejection reflex, Supraoptic nucleus.
INTRODUCTION The milk-ejection reflex is triggered by the suckling stimulus of the young, which evokes repetitive synchronous bursting of magnocellular oxytocin (OT) neurones within the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei [25]. These synchronous bursts result in a pulsatile pattern of OT release leading to a transient rise in intramammary pressure characteristic of a milk-ejection response. Precise details of the neural control of OT cell bursting during milk-ejection are not altogether clear, but magnocellular OT cells of the PVN and SON receive a substantial number of axosomatic and axodendritic noradrenergic contacts [8,20], and suckling increases the turnover of noradrenaline in the PVN and SON [5]. Furthermore, it has been reported that 6-hydroxydopamine lesions prevent suckling-induced OT release [5], indicating the involvement of noradrenergic inputs in the reflex excitation of OT neurones, probably originating from the A2 noradrenergic cell group of the brainstem [6]. Whereas the effects of alpha-1 and beta adrenoceptor manipulation on milk-ejection have been studied in detail [5], there is little evidence concerning the role of alpha-2 adrenoceptors. Alpha-2 adrenoceptor binding sites are found within the PVN and SON and, to a lesser extent, the posterior pituitary, and in regions that
1 Requests for reprints should be addressed to Dr. A. R. T. Bailey, at his present address: Department of Physiology, University Medical School, Teviot Place, Edinburgh. EH8 9PP, UK.
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or more milk-ejection–related bursts occurred. Clonidine was administered at doses of either 25 mg/kg or 50 mg/kg, and idazoxan was given at a dose of 0.5 mg/kg. Mean basal firing rate (with high-frequency bursts excluded), the frequency and amplitude of milk-ejection related bursts were determined for a 20-min pre- and postinjection interval for each cell. Statistical analysis was performed using a repeated measures one-way analysis of variance followed by a Student–Newman–Keuls test with significance taken as p , 0.05. Data are expressed as mean 6 SEM. RESULTS Recordings were obtained from 28 SON OT cells that were all antidromically identified and displayed regular high frequency bursts, each followed by a milk-ejection response. At the lower dose (25 mg/kg), clonidine caused an immediate increase in burst frequency in all six cells tested (Fig. 1), although in some cells this increase was transient and was followed by an inhibition. The increase in burst frequency was also accompanied by an increase in mean burst amplitude (from 46.9 6 5.8 to 63 6 5.5 spikes/burst, p , 0.05). Most (5 of 6) cells subsequently showed one or more “double” bursts following clonidine injection, whereby the interburst interval was less than 30 s, leading to two separate milkejection responses. Following clonidine injection there was a reduction in basal firing rate (from 0.6 6 0.2 to 0.1 6 0.2 spikes/s, p , 0.05) and this effect persisted long after the facilitatory effect on bursting activity had diminished (Fig. 1). In 10 cells tested with the higher (50 mg/kg) clonidine dose there was, by contrast, an inhibition of bursting activity (Fig. 2), which persisted for 20 –30 min before bursts resumed. The interruption of bursting following clonidine was accompanied by a prolonged (over an hour) reduction in basal firing (from 0.9 6 0.2 to 0.1 6 0.1 spikes/s, p , 0.05). No cells displayed “double bursts” following administration of the higher dose of clonidine. Systemic injection of idazoxan completely prevented bursting in all six cells tested (Fig. 3), and the mean duration of the effect was 44.8 6 5.8 min. It was notable that burst amplitude was diminished when bursts were first resumed. Idazoxan had a variable effect on the basal firing of OT cells, and overall there was no immediate change in mean firing rate, although activity did gradually decline over the subsequent hour of recording. In six further antidromically activated SON OT cells clonidine failed to inhibit the background activity in the presence of idazoxan. Thus, mean firing rate before drug administration was 0.8 6 0.4 spikes/s, compared to 1.0 6 0.3 spikes/s following injection of 50 mg/kg clonidine in the presence of idazoxan (0.5 mg/kg). Because idazoxan itself blocked bursting activity, this precluded any experiments to examine interactions of this antagonist with the effect of clonidine on bursting of OT cells.
FIG. 1. The effect of clonidine (25 mg/kg IV) on the activity of SON OT neurones during suckling. (A) An example of the basal activity (continuous graph) and bursting responses (vertical bars) of an SON OT neurone for 20 min before and 60 min after clonidine administration. (B) Composite data of the mean basal firing rate of all SON cells tested with this dose of clonidine (n 5 6). (C) The mean frequency of milk-ejection related bursts of these cells (n 5 6).
DISCUSSION Systemic injection of idazoxan inhibited suckling-induced bursting of SON OT neurones indicating that alpha-2 adrenoceptor transmission is an essential step in the activation of the milkejection reflex. Consistent with this, clonidine (25 mg/kg) had a facilitatory effect on the milk-ejection reflex, with a significant increase in the mean frequency and amplitude of bursts, confirming earlier reports that urethane-anaesthetised rats treated with xylazine, an alpha-2 adrenoceptors agonist, display rapid milkejection responses [21]. The timing of milk-ejection bursts is thought to be organised upstream of OT neurones [24], and the unusual clustering of milk-ejection responses (double bursts) observed in some animals following clonidine, supports an action on
afferent pathways. Such a conclusion would be in agreement with results from in vitro recordings of SON neurones, where excitatory responses are readily evoked by alpha-1, but not alpha-2, adrenoceptor agonists [9]. Given that the predominant effect of clonidine is to suppress central noradrenaline release [4], it might be argued that the facilitatory effect of clonidine resulted from elimination of noradrenergic inhibition of bursting activity, especially because there is evidence for beta-adrenoceptor–mediated inhibition of the milk-ejection reflex [21]. However, the animals employed in this study were pretreated with the beta-adrenoceptor antagonist, propranolol, so this would seem unlikely. It should be further noted
EFFECT OF CLONIDINE ON THE MILK-EJECTION REFLEX
195 reflex. The contrasting effect of low and high doses of clonidine on the milk-ejection reflex has parallels in relation to the effect of clonidine on the anxiety state, with low doses being anxiolytic and high doses anxiogenic [19]. High doses of clonidine are known to cause transient changes in cardiovascular parameters [2], although similar cardiovascular changes are also seen with the lower dose of clonidine (Bailey, unpublished), so this is unlikely to provide an explanation for this result. One explanation for the contrasting effects of clonidine at the different doses is that the primary drug action at a given site is supplemented by secondary actions as the concentration of the drug increases. It is also possible that the phenomenon results from regional differences in the density and affinity of alpha-2 adrenoceptors, so that different doses of clonidine produce different effects according to the local concentration achieved in the context of the ongoing noradrenergic tone. The inhibitory action of clonidine on the milk-ejection reflex may operate through disruption of the noradrenergic input to OT cells, which is thought to be essential for their excitation during suckling [5]. One source of these noradrenergic afferents is the A2 cell
FIG. 2. The effects of clonidine (50 mg/kg IV) on the activity of SON OT neurones during suckling. (A) An individual record of an SON OT neurone. (B) The mean basal firing rate for all the recorded cells (n 5 10). (C) The mean frequency of bursts for 20 min before, and 60 min after, drug administration.
that a similar increase in milk-ejection responses following low doses of clonidine is observed in animals that have not been propranol pretreated (Clarke, unpublished), so it is improbable that this facilitatory effect was in any way related to propranalol itself, and a more plausible explanation might be that clonidine acts by potentiating pathways that are facilitatory to the milk-ejection reflex. For example, there are pathways between the magnocellular nuclei and the bed nucleus of the stria terminalis [22], a structure rich in alpha-2 adrenoceptors [23], and thought to exert important modulatory influences on the milk-ejection reflex [12]. Therefore, by binding within this region and altering local noradrenaline levels, clonidine may facilitate the bursting activity of magnocellular OT neurones. Whereas a facilitatory effect was seen with low doses of clonidine, at a higher dose clonidine inhibited the milk-ejection
FIG. 3. The effect of idazoxan (0.5 mg/kg) on the activity of SON OT neurones during suckling. (A) An individual record of an SON OT neurone. (B) The mean basal firing rate for all cells tested (n 5 6). (C) The mean frequency of bursts for 20 min before and 60 min after drug administration.
196 group of the dorsal vagal complex [6], and systemically administered clonidine is known to strongly inhibit the activity of these neurones [16]. Clonidine also causes presynaptic suppression of noradrenaline release in the magnocellular nuclei [15], and this would further serve to prevent any noradrenaline-dependent excitation. Indeed, it has been demonstrated that clonidine can prevent excitation of vasopressinergic magnocellular neurones in response to stimulation of their noradrenergic afferents [11], and a similar effect could occur in the noradrenergic input to OT neurones, thereby accounting for the inhibition of bursting with the higher dose of clonidine. Given the present evidence for the importance of alpha-2 adrenoceptors in the milk-ejection reflex, as well as previous evidence concerning beta-adrenoceptors [21], the question arises as to what is the precise role of the natural adrenoceptor ligand, noradrenaline, given that this will activate both receptor subtypes. At the level of the magnocellular nuclei, there is evidence that noradrenaline itself has a predominantly excitatory effect when directly applied to oxytocin neurones [26]. However, determining the complete role of the natural ligand in the milk-ejection reflex will require detailed information about the predominant adrenoceptor subtypes at all the sites at which noradrenergic transmission operates during suckling-induced activation of oxytocin neurones, and such detailed information is currently unavailable. At both doses clonidine had an inhibitory action on the basal firing of OT neurones, and this effect could be blocked by idazoxan. It has been reported previously that clonidine similarly inhibits SON vasopressin neurones [2], although the duration of the effect was shorter than with OT neurones. It is interesting that the suppression of basal firing observed with the low dose of clonidine occurred in the face of a facilitatory effect on bursting activity. This is in line with previous observations that basal firing can be eliminated in OT neurones without preventing their bursting activity in response to suckling [3], and supports the view that the two types of activity are controlled by entirely separate mechanisms. Because in vitro electrophysiological studies [9] suggest that clonidine has no effect at the level of the magnocellular nuclei, it would seem that the inhibition of basal firing was brought about through modulation of afferent pathways. Lesion studies indicate that the excitatory drive underlying spontaneous firing in SON neurones depends mainly on afferents arising from rostral rather than caudal structures [13], and consistent with this, the spontaneous firing of SON neurones does not seem to be generated by noradrenergic excitation from the brainstem [7]. Instead, basal firing probably depends upon glutaminergic excitation [18] and inhibitory GABA inputs may also be important [17]. The fact that clonidine can prevent glutamate release from synaptic terminals, whereas GABAergic terminals are unaffected [10], provides an attractive explanation for the suppression of background firing observed in this study. In summary, suckling-induced OT cell bursting activity was inhibited following blockade of alpha-2 adrenoceptors and, similarly, bursting activity was facilitated following stimulation of alpha-2 adrenoceptors by systemic clonidine administration. Interestingly, the facilitatory effect of clonidine occurred simultaneously with a suppression of OT cell basal firing rate, underlying the complex regulation of OT cell activity. At a higher dose clonidine inhibited both the basal and bursting activity of OT cells. These results suggest that alpha-2 adrenoceptors play an essential role in regulating the milk-ejection reflex and may be involved in the facilitatory and inhibitory regulation of suckling-evoked bursting in OT neurones.
BAILEY, CLARKE AND WAKERLEY ACKNOWLEDGEMENT
A. R. T. Bailey was supported by the Palmer-Challons Trust.
REFERENCES 1. Bailey, A. R. T.; Clarke, G.; Wakerley, J. B. Effect of clonidine on the activity of oxytocin neurones in the suckled lactating rat. J. Physiol. (Lond). 459:485P; 1993. 2. Bailey, A. R. T.; Clarke, G.; Wakerley, J. B. Inhibition of supraoptic vasopressin neurones following systemic clonidine. Neuropharmacology 33:211–214; 1994. 3. Blackburn, R. E.; Leng, G.; Russell, J. A. Control of magnocellular oxytocin neurones by the region anterior and ventral to the third ventricle (AV3V region) in rats. J. Endocrinol. 114:253–261; 1987. 4. Buccafusco, J. J. Neuropharmacological and behavioural actions of clonidine: Interactions with central neurotransmitters. Int. Rev. Neurobiol. 33:55–107; 1992. 5. Crowley, W. R.; Armstrong, W. E. Neurochemical regulation of oxytocin secretion in lactation. Endocrinol. Rev. 13:33– 65; 1992. 6. Cunningham, E. T.; Sawchenko, P. E. Anatomical specificity of noradrenergic inputs to the paraventricular and supraoptic nuclei in the hypothalamus of the rat. J. Comp. Neurol. 274:60 –76; 1988. 7. Day, T. A.; Renaud, L. P.; Sibbald, J. R. Excitation of supraoptic vasopressin cells by stimulation of the A1 noradrenaline cell group: failure to demonstrate role for established adrenergic or amino acid receptors. Brain Res. 516:91–98; 1990. 8. Hornby, P. J.; Piekut, D. T. Catecholamine distribution and relationship to magnocellular neurones in the paraventricular nucleus of the rat. Cell Tissue Res. 238:239 –236; 1987. 9. Inenaga, K.; Dyball, R. E. J.; Okuya, S.; Yamashita, H. Characterization of hypothalamic noradrenaline receptors in the supraoptic nucleus and periventricular region of the paraventricular nucleus of the mouse in vitro. Brain Res. 369:37– 47; 1986. 10. Kamisaki, Y.; Hamahashi, T.; Okada, C. M.; Itoh, T. Clonidine inhibition of potassium-evoked release of glutamate and aspartate from rat cortical synaptosomes. Brain Res. 568:193–198; 1991. 11. Khanna, S.; Sibbald, J. R.; Day, T. A. Alpha-2 adrenoceptor modulation of A1 noradrenergic neuron input to supraoptic vasopressin cells. Brain Res. 613:164 –167; 1993. 12. Lambert, R. C.; Moos, F. C.; Ingram, C. D.; Wakerley, J. B.; Kremarik, P.; Guerne, Y.; Richard, P. Electrical activity of neurons in the ventrolateral septum and bed nuclei of the stria terminalis in suckled rats: Statistical analysis gives evidence for sensitivity to oxytocin and for relation to the milk-ejection reflex. Neuroscience 54:361–376; 1993. 13. Leng, G.; Blackburn, R. E.; Dyball, R. E. J.; Russell, J. A. Role of anterior peri-third ventricular structures in the regulation of supraoptic neuronal activity and neurohypophysial hormone secretion in the rat. J. Neuroendocrinol. 1:35– 46; 1989. 14. Livingstone, A.; Morris, B. Localisation of [3H] clonidine binding in rat neurohypophysis by means of electron-microscopic autoradiography. Cell. Tissue Res. 234:467– 469; 1986. 15. Mermet, C.; Suaud–Chagny, M. F.; Ganon, F. Electrically-evoked noradrenalin release in the rat hypothalamic paraventricular nucleus studied by in vivo electrochemistry: Autoregulation by alpha-2 receptors. Neuroscience 34:423– 432; 1990. 16. Moore, S. D.; Guyenet, P. G. Alpha-receptor mediated inhibition of A2 noradrenergic neurons. Brain Res. 276:188 –191; 1983. 17. Moos, F. GABA-induced facilitation of the periodic bursting activity of oxytocin neurones in suckled rats. J. Physiol. (Lond.). 488:103– 114; 1995. 18. Nissen, R.; Hu, B.; Renaud, L. P. Regulation of spontaneous phasic firing of rat supraoptic vasopressin neurones in vivo by glutamate receptors. J. Physiol. (Lond.) 484:415– 423; 1995. 19. Soderpalm, B.; Engel, J. A. Biphasic effects of clonidine on conflict behaviour: Involvement of different alpha-adrenoceptors. Pharmacol. Biochem. Behav. 30:471– 477; 1988. 20. Swanson, L. W.; Connelly, M. A.; Hartman, B. K. Further studies of
EFFECT OF CLONIDINE ON THE MILK-EJECTION REFLEX the fine structure of the adrenergic innervation of the hypothalamus. Brain Res. 151:165–174; 1978. 21. Tribollet, E.; Clarke, G.; Drieffuss, J. J.; Lincoln, D. W. The role of central adrenergic receptors in the reflex release of oxytocin. Brain Res. 142:69 – 84; 1978. 22. Tribollet, E.; Armstrong, W. E.; Dubois–Dauphin, M.; Dreifuss, J. J. Extra-hypothalamic afferent inputs to the supraoptic nucleus area of the rat as determined by retrograde and anterograde tracing techniques. Neuroscience 15:135–148; 1985. 23. Unnerstall, J. R.; Kopatjic, T. A.; Kuhar, M. J. Distribution of alpha-2 binding sites in the rat and human central nervous system: Analysis of some functional, anatomic correlates of the pharmacological ef-
197 fects of clonidine and related adrenergic agents. Brain Res. Rev. 7:69 –101; 1984. 24. Wakerley, J. B.; Ingram, C. D. Synchronisation of bursting in hypothalamic oxytocin neurones: Possible coordinating mechanisms. N.I.P.S. 8:129 –133; 1993. 25. Wakerley, J. B.; Clarke, G.; Summerlee, A. J. S. Milk-ejection and its control. In: Knobil, E.; Neill, J. D., ed. The physiology of reproduction, vol. 2, 2nd ed. New York: Raven Press Ltd. 1994: 1131–1177. 26. Yamashita, H.; Inenaga, K.; Kannan, H. Depolarising effect of noradrenaline on neurons of the rat supraoptic nucleus in vitro. Brain Res. 405:348 –352; 1987.
PII S0361-9230(97)00115-9
The Role of Alpha-2 Adrenoceptors in the Regulation of Oxytocin Neurones in the Suckled Rat ALEX BAILEY,1 GEOFF CLARKE AND JONATHAN WAKERLEY Department of Anatomy, School of Medical Sciences, University of Bristol, University Walk, Bristol, BS8 1TD, UK [Received 3 March 1997; Accepted 15 May 1997] ABSTRACT: The role of alpha-2 adrenoceptors in the milkejection reflex was investigated by making electrophysiological recordings from oxytocin neurones in the supraoptic nucleus of urethane-anaesthetised rats. Systemic administration of the alpha-2 adrenoceptor antagonist, idazoxan (0.5 mg/kg, IV), temporarily suppressed OT cell bursting activity, while having no consistent action on basal neuronal activity. Clonidine (25 mg/ kg, IV) caused an immediate increase in the frequency and amplitude of oxytocin cell bursting, coincident with a fall in basal activity. A higher dose of clonidine (50 mg/kg, IV), inhibited both bursting and basal activity. These results indicate that alpha-2 adrenoceptors are essential for the normal functioning of the milk-ejection reflex and may be involved in the facilitatory and inhibitory regulation of suckling-evoked bursting in oxytocin neurones. © 1997 Elsevier Science Inc.
project directly to the magnocellular nuclei, including the bed nuclei, septum, and the dorsal vagal complex [14,23]. This study examines the possible significance of alpha-2 adrenoceptor-mediated transmission in the milk-ejection reflex by investigating the effects of systemic administration of the alpha-2 adrenoceptor agonist, clonidine, and antagonist, idazoxan, on the electrophysiological activity of SON OT neurones in the urethane anaesthetised lactating rat. A preliminary account of some of these results has already been described [1]. MATERIALS AND METHODS Experiments were performed on lactating Wistar rats (280 –390 g) taken from the departmental colony, between days 8 –12 postpartum. Animals were housed individually with their litter and maintained under controlled environmental conditions with food and water available ad lib. Animals were separated from all but one of their litter overnight and subsequently anaesthetised with a single intraperitoneal injection of urethane (1.2 g/kg, ethyl carbamate, Sigma, UK). A jugular vein and mammary gland were cannulated for drug administration and intramammary pressure recording respectively. All animals received a single dose (10 mg/kg, IV) of the short-acting analgesic, fentanyl (Janssen Pharmaceutical, UK) before being placed in a stereotaxic frame and prepared for recording from the SON, as previously described [2]. One hour before recording all animals received an injection of propranolol (1 mg/kg IV, Inderal, Zeneca, UK) to suppress stress-related inhibition of the milk-ejection reflex [21]. Extracellular recordings were made in the SON with glass micropipettes (5–10 MV) filled with 0.5 M sodium chloride. Magnocellular cells of the SON were identified by antidromic invasion following electrical stimulation of the pituitary stalk and cancellation of the antidromic potential by collision with a spontaneous orthodromic potential. All recordings were stored on tape (Store 4, Racal, UK) and subsequently analysed by computer (Modulog, Grafitek Ltd, UK). Cells were classified as oxytocinergic if they displayed a characteristic high-frequency burst of activity immediately prior to a rise in intramammary pressure associated with a milk-ejection response [25]. Clonidine and idazoxan were only injected after recording neuronal activity for a minimum of 20 min, during which time two
KEY WORDS: Clonidine, Idazoxan, Milk-ejection reflex, Supraoptic nucleus.
INTRODUCTION The milk-ejection reflex is triggered by the suckling stimulus of the young, which evokes repetitive synchronous bursting of magnocellular oxytocin (OT) neurones within the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei [25]. These synchronous bursts result in a pulsatile pattern of OT release leading to a transient rise in intramammary pressure characteristic of a milk-ejection response. Precise details of the neural control of OT cell bursting during milk-ejection are not altogether clear, but magnocellular OT cells of the PVN and SON receive a substantial number of axosomatic and axodendritic noradrenergic contacts [8,20], and suckling increases the turnover of noradrenaline in the PVN and SON [5]. Furthermore, it has been reported that 6-hydroxydopamine lesions prevent suckling-induced OT release [5], indicating the involvement of noradrenergic inputs in the reflex excitation of OT neurones, probably originating from the A2 noradrenergic cell group of the brainstem [6]. Whereas the effects of alpha-1 and beta adrenoceptor manipulation on milk-ejection have been studied in detail [5], there is little evidence concerning the role of alpha-2 adrenoceptors. Alpha-2 adrenoceptor binding sites are found within the PVN and SON and, to a lesser extent, the posterior pituitary, and in regions that
1 Requests for reprints should be addressed to Dr. A. R. T. Bailey, at his present address: Department of Physiology, University Medical School, Teviot Place, Edinburgh. EH8 9PP, UK.
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or more milk-ejection–related bursts occurred. Clonidine was administered at doses of either 25 mg/kg or 50 mg/kg, and idazoxan was given at a dose of 0.5 mg/kg. Mean basal firing rate (with high-frequency bursts excluded), the frequency and amplitude of milk-ejection related bursts were determined for a 20-min pre- and postinjection interval for each cell. Statistical analysis was performed using a repeated measures one-way analysis of variance followed by a Student–Newman–Keuls test with significance taken as p , 0.05. Data are expressed as mean 6 SEM. RESULTS Recordings were obtained from 28 SON OT cells that were all antidromically identified and displayed regular high frequency bursts, each followed by a milk-ejection response. At the lower dose (25 mg/kg), clonidine caused an immediate increase in burst frequency in all six cells tested (Fig. 1), although in some cells this increase was transient and was followed by an inhibition. The increase in burst frequency was also accompanied by an increase in mean burst amplitude (from 46.9 6 5.8 to 63 6 5.5 spikes/burst, p , 0.05). Most (5 of 6) cells subsequently showed one or more “double” bursts following clonidine injection, whereby the interburst interval was less than 30 s, leading to two separate milkejection responses. Following clonidine injection there was a reduction in basal firing rate (from 0.6 6 0.2 to 0.1 6 0.2 spikes/s, p , 0.05) and this effect persisted long after the facilitatory effect on bursting activity had diminished (Fig. 1). In 10 cells tested with the higher (50 mg/kg) clonidine dose there was, by contrast, an inhibition of bursting activity (Fig. 2), which persisted for 20 –30 min before bursts resumed. The interruption of bursting following clonidine was accompanied by a prolonged (over an hour) reduction in basal firing (from 0.9 6 0.2 to 0.1 6 0.1 spikes/s, p , 0.05). No cells displayed “double bursts” following administration of the higher dose of clonidine. Systemic injection of idazoxan completely prevented bursting in all six cells tested (Fig. 3), and the mean duration of the effect was 44.8 6 5.8 min. It was notable that burst amplitude was diminished when bursts were first resumed. Idazoxan had a variable effect on the basal firing of OT cells, and overall there was no immediate change in mean firing rate, although activity did gradually decline over the subsequent hour of recording. In six further antidromically activated SON OT cells clonidine failed to inhibit the background activity in the presence of idazoxan. Thus, mean firing rate before drug administration was 0.8 6 0.4 spikes/s, compared to 1.0 6 0.3 spikes/s following injection of 50 mg/kg clonidine in the presence of idazoxan (0.5 mg/kg). Because idazoxan itself blocked bursting activity, this precluded any experiments to examine interactions of this antagonist with the effect of clonidine on bursting of OT cells.
FIG. 1. The effect of clonidine (25 mg/kg IV) on the activity of SON OT neurones during suckling. (A) An example of the basal activity (continuous graph) and bursting responses (vertical bars) of an SON OT neurone for 20 min before and 60 min after clonidine administration. (B) Composite data of the mean basal firing rate of all SON cells tested with this dose of clonidine (n 5 6). (C) The mean frequency of milk-ejection related bursts of these cells (n 5 6).
DISCUSSION Systemic injection of idazoxan inhibited suckling-induced bursting of SON OT neurones indicating that alpha-2 adrenoceptor transmission is an essential step in the activation of the milkejection reflex. Consistent with this, clonidine (25 mg/kg) had a facilitatory effect on the milk-ejection reflex, with a significant increase in the mean frequency and amplitude of bursts, confirming earlier reports that urethane-anaesthetised rats treated with xylazine, an alpha-2 adrenoceptors agonist, display rapid milkejection responses [21]. The timing of milk-ejection bursts is thought to be organised upstream of OT neurones [24], and the unusual clustering of milk-ejection responses (double bursts) observed in some animals following clonidine, supports an action on
afferent pathways. Such a conclusion would be in agreement with results from in vitro recordings of SON neurones, where excitatory responses are readily evoked by alpha-1, but not alpha-2, adrenoceptor agonists [9]. Given that the predominant effect of clonidine is to suppress central noradrenaline release [4], it might be argued that the facilitatory effect of clonidine resulted from elimination of noradrenergic inhibition of bursting activity, especially because there is evidence for beta-adrenoceptor–mediated inhibition of the milk-ejection reflex [21]. However, the animals employed in this study were pretreated with the beta-adrenoceptor antagonist, propranolol, so this would seem unlikely. It should be further noted
EFFECT OF CLONIDINE ON THE MILK-EJECTION REFLEX
195 reflex. The contrasting effect of low and high doses of clonidine on the milk-ejection reflex has parallels in relation to the effect of clonidine on the anxiety state, with low doses being anxiolytic and high doses anxiogenic [19]. High doses of clonidine are known to cause transient changes in cardiovascular parameters [2], although similar cardiovascular changes are also seen with the lower dose of clonidine (Bailey, unpublished), so this is unlikely to provide an explanation for this result. One explanation for the contrasting effects of clonidine at the different doses is that the primary drug action at a given site is supplemented by secondary actions as the concentration of the drug increases. It is also possible that the phenomenon results from regional differences in the density and affinity of alpha-2 adrenoceptors, so that different doses of clonidine produce different effects according to the local concentration achieved in the context of the ongoing noradrenergic tone. The inhibitory action of clonidine on the milk-ejection reflex may operate through disruption of the noradrenergic input to OT cells, which is thought to be essential for their excitation during suckling [5]. One source of these noradrenergic afferents is the A2 cell
FIG. 2. The effects of clonidine (50 mg/kg IV) on the activity of SON OT neurones during suckling. (A) An individual record of an SON OT neurone. (B) The mean basal firing rate for all the recorded cells (n 5 10). (C) The mean frequency of bursts for 20 min before, and 60 min after, drug administration.
that a similar increase in milk-ejection responses following low doses of clonidine is observed in animals that have not been propranol pretreated (Clarke, unpublished), so it is improbable that this facilitatory effect was in any way related to propranalol itself, and a more plausible explanation might be that clonidine acts by potentiating pathways that are facilitatory to the milk-ejection reflex. For example, there are pathways between the magnocellular nuclei and the bed nucleus of the stria terminalis [22], a structure rich in alpha-2 adrenoceptors [23], and thought to exert important modulatory influences on the milk-ejection reflex [12]. Therefore, by binding within this region and altering local noradrenaline levels, clonidine may facilitate the bursting activity of magnocellular OT neurones. Whereas a facilitatory effect was seen with low doses of clonidine, at a higher dose clonidine inhibited the milk-ejection
FIG. 3. The effect of idazoxan (0.5 mg/kg) on the activity of SON OT neurones during suckling. (A) An individual record of an SON OT neurone. (B) The mean basal firing rate for all cells tested (n 5 6). (C) The mean frequency of bursts for 20 min before and 60 min after drug administration.
196 group of the dorsal vagal complex [6], and systemically administered clonidine is known to strongly inhibit the activity of these neurones [16]. Clonidine also causes presynaptic suppression of noradrenaline release in the magnocellular nuclei [15], and this would further serve to prevent any noradrenaline-dependent excitation. Indeed, it has been demonstrated that clonidine can prevent excitation of vasopressinergic magnocellular neurones in response to stimulation of their noradrenergic afferents [11], and a similar effect could occur in the noradrenergic input to OT neurones, thereby accounting for the inhibition of bursting with the higher dose of clonidine. Given the present evidence for the importance of alpha-2 adrenoceptors in the milk-ejection reflex, as well as previous evidence concerning beta-adrenoceptors [21], the question arises as to what is the precise role of the natural adrenoceptor ligand, noradrenaline, given that this will activate both receptor subtypes. At the level of the magnocellular nuclei, there is evidence that noradrenaline itself has a predominantly excitatory effect when directly applied to oxytocin neurones [26]. However, determining the complete role of the natural ligand in the milk-ejection reflex will require detailed information about the predominant adrenoceptor subtypes at all the sites at which noradrenergic transmission operates during suckling-induced activation of oxytocin neurones, and such detailed information is currently unavailable. At both doses clonidine had an inhibitory action on the basal firing of OT neurones, and this effect could be blocked by idazoxan. It has been reported previously that clonidine similarly inhibits SON vasopressin neurones [2], although the duration of the effect was shorter than with OT neurones. It is interesting that the suppression of basal firing observed with the low dose of clonidine occurred in the face of a facilitatory effect on bursting activity. This is in line with previous observations that basal firing can be eliminated in OT neurones without preventing their bursting activity in response to suckling [3], and supports the view that the two types of activity are controlled by entirely separate mechanisms. Because in vitro electrophysiological studies [9] suggest that clonidine has no effect at the level of the magnocellular nuclei, it would seem that the inhibition of basal firing was brought about through modulation of afferent pathways. Lesion studies indicate that the excitatory drive underlying spontaneous firing in SON neurones depends mainly on afferents arising from rostral rather than caudal structures [13], and consistent with this, the spontaneous firing of SON neurones does not seem to be generated by noradrenergic excitation from the brainstem [7]. Instead, basal firing probably depends upon glutaminergic excitation [18] and inhibitory GABA inputs may also be important [17]. The fact that clonidine can prevent glutamate release from synaptic terminals, whereas GABAergic terminals are unaffected [10], provides an attractive explanation for the suppression of background firing observed in this study. In summary, suckling-induced OT cell bursting activity was inhibited following blockade of alpha-2 adrenoceptors and, similarly, bursting activity was facilitated following stimulation of alpha-2 adrenoceptors by systemic clonidine administration. Interestingly, the facilitatory effect of clonidine occurred simultaneously with a suppression of OT cell basal firing rate, underlying the complex regulation of OT cell activity. At a higher dose clonidine inhibited both the basal and bursting activity of OT cells. These results suggest that alpha-2 adrenoceptors play an essential role in regulating the milk-ejection reflex and may be involved in the facilitatory and inhibitory regulation of suckling-evoked bursting in OT neurones.
BAILEY, CLARKE AND WAKERLEY ACKNOWLEDGEMENT
A. R. T. Bailey was supported by the Palmer-Challons Trust.
REFERENCES 1. Bailey, A. R. T.; Clarke, G.; Wakerley, J. B. Effect of clonidine on the activity of oxytocin neurones in the suckled lactating rat. J. Physiol. (Lond). 459:485P; 1993. 2. Bailey, A. R. T.; Clarke, G.; Wakerley, J. B. Inhibition of supraoptic vasopressin neurones following systemic clonidine. Neuropharmacology 33:211–214; 1994. 3. Blackburn, R. E.; Leng, G.; Russell, J. A. Control of magnocellular oxytocin neurones by the region anterior and ventral to the third ventricle (AV3V region) in rats. J. Endocrinol. 114:253–261; 1987. 4. Buccafusco, J. J. Neuropharmacological and behavioural actions of clonidine: Interactions with central neurotransmitters. Int. Rev. Neurobiol. 33:55–107; 1992. 5. Crowley, W. R.; Armstrong, W. E. Neurochemical regulation of oxytocin secretion in lactation. Endocrinol. Rev. 13:33– 65; 1992. 6. Cunningham, E. T.; Sawchenko, P. E. Anatomical specificity of noradrenergic inputs to the paraventricular and supraoptic nuclei in the hypothalamus of the rat. J. Comp. Neurol. 274:60 –76; 1988. 7. Day, T. A.; Renaud, L. P.; Sibbald, J. R. Excitation of supraoptic vasopressin cells by stimulation of the A1 noradrenaline cell group: failure to demonstrate role for established adrenergic or amino acid receptors. Brain Res. 516:91–98; 1990. 8. Hornby, P. J.; Piekut, D. T. Catecholamine distribution and relationship to magnocellular neurones in the paraventricular nucleus of the rat. Cell Tissue Res. 238:239 –236; 1987. 9. Inenaga, K.; Dyball, R. E. J.; Okuya, S.; Yamashita, H. Characterization of hypothalamic noradrenaline receptors in the supraoptic nucleus and periventricular region of the paraventricular nucleus of the mouse in vitro. Brain Res. 369:37– 47; 1986. 10. Kamisaki, Y.; Hamahashi, T.; Okada, C. M.; Itoh, T. Clonidine inhibition of potassium-evoked release of glutamate and aspartate from rat cortical synaptosomes. Brain Res. 568:193–198; 1991. 11. Khanna, S.; Sibbald, J. R.; Day, T. A. Alpha-2 adrenoceptor modulation of A1 noradrenergic neuron input to supraoptic vasopressin cells. Brain Res. 613:164 –167; 1993. 12. Lambert, R. C.; Moos, F. C.; Ingram, C. D.; Wakerley, J. B.; Kremarik, P.; Guerne, Y.; Richard, P. Electrical activity of neurons in the ventrolateral septum and bed nuclei of the stria terminalis in suckled rats: Statistical analysis gives evidence for sensitivity to oxytocin and for relation to the milk-ejection reflex. Neuroscience 54:361–376; 1993. 13. Leng, G.; Blackburn, R. E.; Dyball, R. E. J.; Russell, J. A. Role of anterior peri-third ventricular structures in the regulation of supraoptic neuronal activity and neurohypophysial hormone secretion in the rat. J. Neuroendocrinol. 1:35– 46; 1989. 14. Livingstone, A.; Morris, B. Localisation of [3H] clonidine binding in rat neurohypophysis by means of electron-microscopic autoradiography. Cell. Tissue Res. 234:467– 469; 1986. 15. Mermet, C.; Suaud–Chagny, M. F.; Ganon, F. Electrically-evoked noradrenalin release in the rat hypothalamic paraventricular nucleus studied by in vivo electrochemistry: Autoregulation by alpha-2 receptors. Neuroscience 34:423– 432; 1990. 16. Moore, S. D.; Guyenet, P. G. Alpha-receptor mediated inhibition of A2 noradrenergic neurons. Brain Res. 276:188 –191; 1983. 17. Moos, F. GABA-induced facilitation of the periodic bursting activity of oxytocin neurones in suckled rats. J. Physiol. (Lond.). 488:103– 114; 1995. 18. Nissen, R.; Hu, B.; Renaud, L. P. Regulation of spontaneous phasic firing of rat supraoptic vasopressin neurones in vivo by glutamate receptors. J. Physiol. (Lond.) 484:415– 423; 1995. 19. Soderpalm, B.; Engel, J. A. Biphasic effects of clonidine on conflict behaviour: Involvement of different alpha-adrenoceptors. Pharmacol. Biochem. Behav. 30:471– 477; 1988. 20. Swanson, L. W.; Connelly, M. A.; Hartman, B. K. Further studies of
EFFECT OF CLONIDINE ON THE MILK-EJECTION REFLEX the fine structure of the adrenergic innervation of the hypothalamus. Brain Res. 151:165–174; 1978. 21. Tribollet, E.; Clarke, G.; Drieffuss, J. J.; Lincoln, D. W. The role of central adrenergic receptors in the reflex release of oxytocin. Brain Res. 142:69 – 84; 1978. 22. Tribollet, E.; Armstrong, W. E.; Dubois–Dauphin, M.; Dreifuss, J. J. Extra-hypothalamic afferent inputs to the supraoptic nucleus area of the rat as determined by retrograde and anterograde tracing techniques. Neuroscience 15:135–148; 1985. 23. Unnerstall, J. R.; Kopatjic, T. A.; Kuhar, M. J. Distribution of alpha-2 binding sites in the rat and human central nervous system: Analysis of some functional, anatomic correlates of the pharmacological ef-
197 fects of clonidine and related adrenergic agents. Brain Res. Rev. 7:69 –101; 1984. 24. Wakerley, J. B.; Ingram, C. D. Synchronisation of bursting in hypothalamic oxytocin neurones: Possible coordinating mechanisms. N.I.P.S. 8:129 –133; 1993. 25. Wakerley, J. B.; Clarke, G.; Summerlee, A. J. S. Milk-ejection and its control. In: Knobil, E.; Neill, J. D., ed. The physiology of reproduction, vol. 2, 2nd ed. New York: Raven Press Ltd. 1994: 1131–1177. 26. Yamashita, H.; Inenaga, K.; Kannan, H. Depolarising effect of noradrenaline on neurons of the rat supraoptic nucleus in vitro. Brain Res. 405:348 –352; 1987.