Opioid modulation of magnocellular neurosecretory cell activity

Neuroscience Research 36 (2000) 97 – 120 www.elsevier.com/locate/neures

Review article

Opioid modulation of magnocellular neurosecretory cell activity Colin H. Brown *, John A. Russell, Gareth Leng Laboratory of Neuroendocrinology, Department of Biomedical Sciences, Uni6ersity of Edinburgh, Hugh Robson Building, Edinburgh, EH8 9XD, UK Received 12 October 1999; accepted 15 November 1999

Abstract Magnocellular neurosecretory cells of the hypothalamic supraoptic and paraventricular nuclei secrete the hormones, oxytocin and vasopressin, into the systemic circulation from the posterior pituitary gland. Oxytocin is important for parturition and is essential for lactation. Vasopressin regulates body fluid homeostasis. The secretion of these hormones is altered in response to peripheral stimuli that are conveyed via projections from other parts of the brain. Endogenous opioid peptide systems interact with the magnocellular neurosecretory system at several levels to restrain the basal secretion of these hormones as well as their secretory responses to various physiological stimuli. The inhibition of basal secretion can occur at the level of the neurosecretory terminals where endogenous opioids inhibit the release of oxytocin, and at the cell bodies of magnocellular cells to modulate the activity pattern of vasopressin cells. The responses of the magnocellular neurosecretory system to physiological stimuli are also regulated by these mechanisms but in addition probably also by pre-synaptic inhibition of afferent inputs to magnocellular cells as well as direct effects on the cell bodies of afferent input cells to modulate their activity. Here, we review the mechanisms and functional consequences of opioid interactions with oxytocin and vasopressin cells. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Dependence; Dynorphins; Morphine; Opioid receptors; Oxytocin; Paraventricular nucleus; Supraoptic nucleus; Vasopressin

1. Introduction The importance of circulating oxytocin in parturition and lactation (Russell and Leng, 1998), and of vasopressin in body fluid homeostasis, are well established. In mammals, oxytocin is important for the timing of parturition (Antonijevic et al., 1995a) and is essential for lactation (Nishimori et al., 1996; Young et al., 1996). Studies on transgenic oxytocin knock-out mice demonstrate that parturition can proceed in the absence of oxytocin whereas, without oxytocin to induce mammary myoepithelium contraction, milk let-down will not occur in response to suckling (Nishimori et al., 1996; Young et al., 1996). Vasopressin mediates antidiuresis and vasoconstriction to conserve body water,

* Corresponding author. Tel.: +44-131-650-3271; fax: +44-131650-3711. E-mail address: [email protected] (C.H. Brown)

maintaining plasma osmolarity, and to sustain blood pressure against hypovolemia. In the rat at least, oxytocin acts upon the kidney in conjunction with vasopressin, to promote natriuresis (Verbalis et al., 1991), and also releases atrial natriuretic factor from the heart (Haanwinckel et al., 1995). The activity of magnocellular neurosecretory cells is sensitive to inhibition by both exogenous and endogenous opioids. The interactions between opioids and magnocellular neurosecretory cells are not the same for oxytocin and vasopressin cells and can be altered by physiological status and by the activity of the cells themselves. Oxytocin and vasopressin magnocellular neurosecretory cells are among the most extensively studied cells in the brain; their discrete anatomical organisation, largely within the hypothalamic supraoptic and paraventricular nuclei, and the ability to determine the output of the population of cells as plasma hormone concentrations make these cells open to a wide range of experimental manipulations. Endogenous opioid pep-

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tides modulate the activity of many cell types within the nervous system; among these are the systems controlling oxytocin and vasopressin secretion from the posterior pituitary gland (neurohypophysis). Activity in oxytocin and vasopressin cells is entirely dependent upon synaptic input and exogenous opioids inhibit many of the excitatory stimuli for magnocellular neurosecretory cells against which they have been tested. This inhibition can be exerted directly on the magnocellular cells, pre-synaptically upon their afferent inputs, or at the cell bodies of their afferent inputs to modulate their activity. However, some endogenous opioid influences appear to be active only under certain physiological conditions. While activity in magnocellular cells per se depends upon synaptic inputs, the patterning of activity in vasopressin cells is strongly influenced by the intrinsic membrane properties of the cells and we have recently demonstrated that these too may provide a mechanism through which opioids modulate the activity of vasopressin cells. Here, the interactions of endogenous opioid systems with oxytocin and vasopressin cells are reviewed with particular reference to the functional consequences of these interactions to their responses to physiological stimuli.

2. The magnocellular neurosecretory system Oxytocin and vasopressin are synthesised by magnocellular neurosecretory cells located mainly in the supraoptic and paraventricular nuclei in the hypothalamus and in a number of smaller accessory cell groups between these two nuclei. The supraoptic nucleus contains essentially only magnocellular cells whereas the paraventricular nucleus also contains parvocellular cells that project to the median eminence where they secrete corticotrophin-releasing hormone or vasopressin (Bloom et al., 1982; Tramu et al., 1983) that act synergistically to control adrenocorticotrophic hormone secretion from the anterior pituitary (Gillies et al., 1982). There are also centrally-projecting parvocellular oxytocin and vasopressin cells in the paraventricular nucleus that are involved in the modulation of social and reproductive behaviours (Landgraf, 1995). Each magnocellular cell sends a single axon to the neurohypophysis from where they secrete either oxytocin or vasopressin. Each axon gives rise to 2000 – 10 000 neurosecretory endings organised in an en passant arrangement within the neurohypophysis. Action potentials generated in the cell bodies open voltage-dependent Ca2 + channels when they arrive in the nerve terminals and the consequent Ca2 + entry triggers exocytosis of neurosecretory vesicles to release oxytocin or vasopressin (and co-produced peptides) into the extracellular space which then diffuse into the general circulation through fenestrated capillaries in the neurohypophysis

(Hatton, 1990; Armstrong, 1995). Activation of opioid receptors can alter Ca2 + influx into the perikarya and terminals to modulate the activity of magnocellular cells and hormone release at the neurohypophysis (see Sections 2.3 and 2.7). Magnocellular cells possess between one and three dendrites that are also anatomically organised. In the supraoptic nucleus, most of these are confined within the nucleus, projecting to the ventral surface of the nucleus where they form a layer of dendritic bundles within the ventral glial lamina that also contains glial cell bodies and processes. In the paraventricular nucleus a large proportion of dendrites projects medially towards the subependymal region of the third ventricle (Hatton, 1990). The dendrites contain a large accumulation of neurosecretory granules that release oxytocin and vasopressin into the extracellular space by exocytosis, which are important in modulating the activity of the neurones themselves (Ludwig, 1998). Magnocellular cells also synthesise peptides other than oxytocin and vasopressin and so dendritic as well as systemic oxytocin and vasopressin secretion is likely to be accompanied by co-secretion of other neuropeptides. Modulation of dendritic release is another mechanism by which endogenous opioid peptides may affect the activity of magnocellular neurosecretory cells because the opioid antagonist, naloxone, increases oxytocin release in vivo in pregnancy (Douglas et al., 1995b) as well as electrically-stimulated oxytocin release within the supraoptic nucleus in vitro (Ingram et al., 1996). Magnocellular cells receive inputs from various forebrain and brainstem regions, most notably the subfornical organ (SFO), median preoptic nucleus (MnPO) and organum vasculosum of the lamina terminalis (OVLT) [collectively referred to as the region anterior and ventral to the third ventricle (AV3V region)], olfactory bulb, amygdala, septum and hypothalamus in the forebrain and the nucleus tractus solitarii (NTS) and ventrolateral medulla (VLM) in the brainstem (Cunningham and Sawchenko, 1991). Opioid receptors have been localised to many of these areas (Mansour et al., 1988) and these provide another level at which endogenous opioid peptides may interact with the magnocellular secretory system.

2.1. Opioid receptor classification The analgesic, soporific and euphoric effects of extracts of the opium poppy, Papa6er somniferum, have been known for many hundreds of years. In 1803, Setu¨rner isolated the main active constituent alkaloid from crude opium, morphine. The stereospecific requirements for analgesic activity of opioids such as morphine led to the postulation of the existence of specific receptors for these drugs. Initially, the demonstration of differing pharmacological profiles of activity

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led to the identification of three types of opioid receptor; m, k and s, for which morphine, ketocyclazocine and N-allylnormetazocine were the prototypic agonists (Gilbert and Martin, 1976). However, the effects of N-allylnormetazocine are not antagonised by the general opioid antagonist, naloxone, and so the s-receptor is no longer considered to be an opioid receptor. Extensive pharmacological studies have since led to the definition of three ‘classical’ opioid receptor types: m, k and d (Table 1) which have all now been cloned (Evans et al., 1992; Kieffer et al., 1992; Chen et al., 1993; Meng et al., 1993; Minami et al., 1993; Thompson et al., 1993). These receptors are members of the seven transmembrane domain G-protein-coupled receptor family. While there is pharmacological evidence for the existence of several other opioid receptors (o, i, l, z) these have not been extensively characterised and the genes encoding these postulated opioid receptors have yet to be cloned. Each of the three opioid receptor types, m, k and d, contain about 400 amino acids with approximately 60% homology between the three receptor types and about 90% homology for each type between species. Each receptor is coded for by a single gene: MOR-1 (m), KOR-1 (k) and DOR-1 (d). However, pharmacological studies indicate the existence of further sub-types of each of the three opioid receptors that may arise from post-translational modification of the gene product. Two splice variants of the MOR-1 receptor have been cloned that have similar pharmacology but differ in the temporal profile of agonist-induced receptor internalisa-

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tion (Koch et al., 1998). Splice variants have also been reported for the DOR-1 and KOR-1 gene (GaveriauxRuff et al., 1997). Recent evidence suggests that these differing pharmacological profiles might also arise as a result of receptor dimerisation to form homodimers as has been demonstrated for d-receptors (Cvejic and Devi, 1997) or heterodimers as has been demonstrated for d- and k-receptors (Jordan and Devi, 1999). The supraoptic nucleus contains m- and k- but not d-opioid receptors (Sumner et al., 1990). k-receptors have been visualised on the cell bodies of dissociated supraoptic nucleus neurones (Soldo and Moises, 1998) as well as in vasopressin-containing neurosecretory vesicles within the supraoptic nucleus and neurohypophysis (Shuster et al., 1999). While the location of m-receptors in relation to magnocellular cells has not been visualised, electrophysiological effects on isolated supraoptic nucleus cells indicate that m-receptors are present on the somata of these cells (Soldo and Moises, 1998) and in vivo actions of morphine support the existence of functional m-receptors pre-synaptically on afferent inputs to oxytocin cells (Onaka et al., 1995b).

2.2. Endogenous opioid peptides The knowledge about high affinity selective binding sites for opioid alkaloids prompted the search for endogenous ligands for these receptors (Table 1). In 1975 the pentapeptides, met5-enkephalin and leu5enkephalin, were isolated and shown to possess potent opioid agonist activity (Hughes et al., 1975). These

Table 1 Opioid and opioid-like receptor classification and pharmacologya Receptor type

Endogenous agonists

Agonist precursors

Synthetic agonists

Synthetic antagonists

m-receptor

Endomorphin-1 Endomorphin-2

Unknown

DAMGO

Naloxoneb CTOP

b-endorphinc

Pro-opiomelanocortin

d-receptor

Met -enkephalin Leu5-enkephalin

Pro-enkephalin A

DPDPE

Naltrindole ICI174864

k-receptor

Dynorphin A1–8 Dynorphin A1–17 Dynorphin B a-neoendorphin b-neoendorphin

Pro-dynorphind

U50,488H U69,593 Dynorphin A1–13

Nor-binaltorphimine

Met5-enkephalin-Arg-Phe Met5-enkephalin-Arg-Gly-Leu

Pro-enkephalin A

Nociceptin/orphanin FQ

Pro-nociceptin

ORL1 a

5

[phe1c(CH2-NH)Gly2]nociceptin(1–13)NH2e

The major opioid and opioid-like receptor types with their proposed endogenous agonists, the precursor peptides from which the endogenous agonists are derived, their selective synthetic agonists and antagonists. b Naloxone is most potent at m-receptors but is also active at d- and k-receptors; the decreasing order of potency for naloxone at m-, d- and k-receptors is approximately in the ratio 1:15:40. c Although b-endorphin is usually considered to be a m-agonist, it is equipotent at d-receptors. d All peptides derived from pro-dynorphin possess activity at m- and d- as well as k-receptors. e [phe1c(CH2-NH)Gly2]nociceptin(1–13)NH2 was originally marketed as a nociceptin antagonist but is now considered to be a partial agonist.

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peptides were later shown to be derived from the precursor peptide, pro-enkephalin A (Noda et al., 1982). Subsequently, many other endogenous opioid peptides with differing pharmacological profiles have been identified, most notably b-endorphin (Li and Chung, 1976) which is derived from pro-opiomelanocortin (POMC) (Nakanishi et al., 1979) and the dynorphins (A1 – 8,1 – 17) (Goldstein et al., 1979; Tachibana et al., 1982) which are derived from pro-dynorphin (pro-enkephalin B) (Kakidani et al., 1982). All of these classical opioid peptides contain the peptide structure of either met5- or leu5-enkephalin at their N-terminus. The classical endogenous opioid peptides display only weak selectivity between each of the opioid receptor types; b-endorphin for the m-receptor, dynorphin A1 – 17 for the k-receptor and met5- and leu5-enkephalin for the d-receptor. Recently, the tetrapeptides, endomorphin-1 and -2 have been characterised and these are highly selective for the m-receptor for which they have been proposed to be the endogenous ligands (Zadina et al., 1997). The endomorphins are structurally dissimilar to other endogenous opioid peptides in that they exhibit no homology to the pentapeptides, met5- and leu5-enkephalin. The precursor(s) from which the endomorphins are derived has yet to be identified.

2.3. Mechanisms of opioid actions In all systems studied to date, opioid receptors couple to their effector mechanisms solely through Gi/o proteins (Childers, 1991). Initially, it was thought that m- and d-receptors exerted their inhibitory actions through activation of inwardly-rectifying K+ conductances whereas k-receptors acted through inhibition of voltage-activated Ca2 + conductances. However, it has become clear that the effector mechanisms employed by each of the receptors is cell-type specific because m-receptor activation can also inhibit voltage-activated Ca2 + conductances and k-receptor activation can also activate inwardly-rectifying K+ conductances (Grudt and Williams, 1995). In addition to activation/inhibition of ion channels, opioid effector mechanisms also include actions mediated by second messenger systems such as inhibition of adenylyl cyclase (Childers, 1991) as well activation of phopholipase C (Smart et al., 1995) and mitogen-activated protein kinase (Fukuda et al., 1997). Since the receptors themselves are likely to possess the same properties between cell types, the specificity of action is probably conferred by differential expression of the G-protein subunits (a, b and g) in the particular Gi/o-protein to which the receptors couple in each cell type. Since the supraoptic nucleus contains m- and k-, but evidently not d-opioid receptors (Sumner et al., 1990) much of the work to elucidate the mechanisms of actions on supraoptic cells has focused on the effects of

m- and/or k-receptor activation, although there are some reports of d-opioid modulation of magnocellular cell activity (Tsushima et al., 1993a). Direct application of naloxone into the supraoptic nucleus reverses inhibition of oxytocin cell firing rate by systemic injection of the m-opioid agonist, morphine (Ludwig et al., 1997a), indicating that this inhibition is mediated by receptors within the supraoptic nucleus. m-agonists were initially shown to be more effective in inhibiting oxytocin than vasopressin cells in vitro (Wakerley et al., 1978) and intracellular recording showed that morphine inhibits the firing rate of magnocellular neurosecretory cells without altering membrane potential or input resistance (Ogata and Matsuo, 1984). More recently, it has been shown that m-receptor activation inhibits N- and P-type Ca2 + currents in isolated supraoptic nucleus neurones (Soldo and Moises, 1998), suggesting that the m-opioid inhibition of activity may be a direct action on the cells themselves. m-agonists also increase K+ conductances in supraoptic or paraventricular nucleus magnocellular cells (Wuarin and Dudek, 1990; Muller et al., 1999). Thus, it appears that m-receptor activation probably inhibits the activity of magnocellular cells through both activation of K+ conductances and inhibition of Ca2 + conductances (Fig. 1). Application of naloxone alone also modulates the amplitude and activation kinetics of K+ conductances indicating that there can be tonic activation of m-receptors by endogenous opioids in slice preparations (Muller et al., 1999). The reports concerning the mechanisms of action of k-opioids on the membrane properties of magnocellular cells are not entirely consistent. k-opioid inhibition of oxytocin cell firing rate is also reversed by direct application of naloxone into the supraoptic nucleus (Ludwig et al., 1997a), suggesting that k-opioid inhibition of magnocellular cell activity is primarily mediated by receptors within the nucleus; consistent with this, k-opioids also inhibit the firing rate of supraoptic nucleus cells recorded from slice preparations (Inenaga et al., 1990). k-agonists reduce post-synaptic potentials and Ca2 + components of action potentials in supraoptic nucleus cells (Inenaga et al., 1994). At least some of these effects are exerted directly on the magnocellular cells themselves because k-opioids also inhibit voltageactivated Ca2 + currents in dispersed cultured supraoptic nucleus cells (Mason et al., 1988). However, more recently it has been shown that neither k- nor d-opioid receptor activation inhibits N- and P-type Ca2 + currents in isolated supraoptic nucleus neurones (Soldo and Moises, 1998) and that U50,488H decreases K+ conductances recorded from putative oxytocin cells as well as having differential effects on K+ currents in putative vasopressin cells, increasing the transient A current but decreasing the delayed rectifier current (Muller et al., 1999). It is difficult to reconcile these

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Fig. 1. Schematic representation of membrane potentials determining activity in magnocellular neurosecretory cells. The diagram shows the potential sites where opioids and nociceptin may modulate excitability of magnocellular neurosecretory cells. m-, k- and ORL1-receptors have been visualised in the supraoptic nucleus and activation of these receptors alters K+ and Ca2 + -currents in magnocellular neurosecretory cells (see Section 2.3 for full details). These actions may modulate excitability by (1) pre-synaptic inhibition of afferent inputs; (2) hyperpolarisation of the post-synaptic membrane through activation of K+-channels; and/or (3) reduction of post-spike excitability by reducing depolarising after-potential amplitude by reducing Ca2 + entry. In addition to the effects on the membrane conductances associated with action potential activity shown in the figure, opioid receptor activation can also induce a persistent K+ conductance to cause a small, prolonged membrane hyperpolarisation that reduces the excitability of the cells.

effects of k-receptor activation on K+-currents with the observed effects on firing rate. However, it is clear that regardless of the effect of k-agonists on particular channel types expressed by magnocellular cells, the consequence of magnocellular cell k-receptor activation is consistently to inhibit the activity of these cells. The synthetic k-opioid peptide, dynorphin A1 – 13, has also been shown to inhibit evoked and spontaneous EPSPs and IPSPs in magnocellular cells (Inenaga et al., 1994), raising the possibility that k-receptor activation may also reduce activity in magnocellular cells by presynaptic inhibition of afferent inputs. Another distinctive aspect of k-opioid inhibition of oxytocin cell firing rate is that it is not affected by i.c.v. pertussis toxin [which inactivates Gi/o proteins (Kaslow and Burns, 1992)] injection at doses that eliminate the inhibitory actions of morphine (Pumford et al., 1993a), suggesting that m- and k-opioid inhibition of oxytocin cell activity are mediated by different mechanisms. This k-opioid inhibition may not be mediated by pertussis toxin sensitive Gi/o-proteins but perhaps by direct actions on adenylate cylcase as has been shown for isletactivating protein (Katada and Ui, 1982). An intriguing possibility for the mechanisms of opioid actions on magnocellular cells, is that these may be mediated in part by effects on dendritic release of peptides because naloxone increases stimulated oxytocin release from isolated supraoptic and paraventricular nuclei in vitro (Ingram et al., 1996) but not in vivo

(Munro et al., 1994) except in pregnancy (Douglas et al., 1995b). It is becoming increasingly clear that dendritically-released peptides are important in the regulation of the activity of magnocellular cells (Ludwig, 1998).

2.4. Endogenous opioid peptide projections to magnocellular neurosecretory cells Apart from a few neurones in the nucleus tractus solitarii (NTS), POMC neurones in the brain are almost all localised to a single aggregation of cells within the hypothalamic arcuate nucleus. These neurones project to many brain regions where they secrete the m-opioid agonist, b-endorphin. b-endorphin fibres have been visualised in the supraoptic nucleus (Knigge and Joseph, 1982; Sawchenko et al., 1982) and there is electrophysiological evidence for an inhibitory projection from the arcuate nucleus to the supraoptic nucleus (Leng et al., 1988b) (Fig. 2). However, at least part of this inhibitory input is not mediated by opioids as it is not blocked by the opioid antagonist, naloxone. Perikarya immunoreactive for the newly-identified m-agonists, endomorphin1 and -2, have been visualised in the posterior hypothalamus (Martin-Schild et al., 1999) and both peptides are present in the supraoptic nucleus (Zadina et al., 1997) and so these may be important modulators of magnocellular cell activity.

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Some parvocellular cells of the paraventricular nucleus synthesise enkephalins (Ceccatelli et al., 1989) as do cells in the arcuate nucleus and ventromedial hypothalamus (Harlan et al., 1987) as well as NTS cells in the brainstem (Sawchenko et al., 1990) (Fig. 2). d-receptors, for which enkephalins are the endogenous ligands, have not been visualised in the supraoptic nucleus. However, the extended enkephalin products of the proenkephalin gene, met5-enkephalin-Arg-Phe and met5enkephalin-Arg-Gly-Leu are k-agonists (Boersma et al., 1994) and so pro-enkephalin cells may be able to influence magnocellular cells through this receptor type. In addition, cells containing these k-opioid receptor ligands and dynorphin B are found in the NTS and some of these cells project to the hypothalamus (Riche et al., 1990), providing another opoid projection that may influence the activity of magnocellular cells.

2.5. Co-localisation of opioids with noradrenergic projections to the magnocellular nuclei The noradrenergic cells of the A1 cell group in the VLM, the A2 cell group in the NTS and the A6 cell group in the locus coeruleus (LC) provide major afferent inputs to the supraoptic and paraventricular nuclei and are involved in mediating peripheral influences upon neurohypophysial hormone secretion. A1 cells project preferentially to vasopressin cells (Beroukas et al., 1989; Iwai et al., 1989; Shioda and Nakai, 1996) while most of the direct inputs from the NTS to the supraoptic nucleus are to oxytocin cells (Raby and Renaud, 1989). A6 cells project predominantly to the parvocellular cells of the paraventricular nucleus (Sawchenko and Swanson, 1982).

Fig. 2. Endogenous opioid peptides and opioid receptors in the magnocellular neurosecretory system. Schematic representation of supraoptic nucleus oxytocin (left) and vasopressin (right) cells showing the origin of opioid peptides and the location of opioid receptors in relation to the magnocellular neurosecretory system. (1) Enkephalins (ENK) are co-localised with noradrenaline (NA) in ascending inputs from the NTS end with oxytocin in oxytocin cells (along with dynorphin); these enkephalins (or extended enkephalin peptides) may activate post-synaptic m-receptors (or may feedback to inhibit release of excitatory NA through pre-synaptic auto-inhibition via m-receptors) to inhibit oxytocin cells. (2) b-endorphin (bEND) cells (probably in the arcuate nucleus) project to the supraoptic nucleus to directly inhibit magnocellular cells; but (3) may also activate pre-synaptic m-receptors to inhibit noradrenaline (and perhaps glutamate) release. (4) Glutamatergic and GABAergic inputs to magnocellular cells are also inhibited by activation of pre-synaptic k-receptors (for clarity, this is shown only for the vasopressin cell). (5) Dynorphin (DYN) and k-receptors are both co-localised with vasopressin in neurosecretory granules in the dendrites and axon terminals of magnocellular cells (for clarity, this is shown in the cell body); dynorphin released from dendrites may inhibit vasopressin cells through the k-receptors exposed in the dendritic membrane upon fusion of the neurosecretory granule membrane to the cell membrane. (6) Both oxytocin and vasopressin cells express k-receptors on their terminal membranes through which exogenous k-agonists can inhibit hormone release. However, only the release of oxytocin is restrained by endogenous k-agonists under basal conditions, although it appears that dynorphin released from vasopressinergic terminals may restrain oxytocin secretion under certain physiological conditions.

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The NTS appears to influence vasopressin release via projections to the parabrachial nucleus, the LC and the VLM (Day and Sibbald, 1989; Chan et al., 1994) and electrical stimulation of the A1 region excites both oxytocin and vasopressin cells in the paraventricular nucleus (Kannan et al., 1984). While activation of a1-adrenoreceptors excites vasopressin cells and stimulates vasopressin release (Moss et al., 1971; Bhargava et al., 1972; Arnauld et al., 1983; Day et al., 1985b; Brooks et al., 1986; Randle et al., 1986a,b; Yamashita et al., 1987a), excitation of magnocellular cells by A1 cell group stimulation may result from the actions of co-transmitters, possibly ATP (Day et al., 1993), because it is prevented by neurotoxic lesion of hypothalamic noradrenergic nerve terminals (Day and Renaud, 1984; Day et al., 1984) but not by noradrenergic antagonists (Day et al., 1990). Noradrenaline can also inhibit vasopressin release (Honda et al., 1985), probably by a pre-synaptic feedback mechanism mediated via a2-autoreceptors (Kimura et al., 1984; Brooks et al., 1986; Yamashita et al., 1988; Khanna et al., 1993), or by b-adrenoreceptors on the vasopressin cells themselves (Day et al., 1985b; Takano et al., 1989). In addition to enkephalins (Ceccatelli et al., 1989), other peptides such as neuropeptide Y (NPY) (Everitt et al., 1984; Sawchenko et al., 1985), galanin (Levin et al., 1987) and substance P (Bittencourt et al., 1991) are co-expressed in some noradrenergic cells of the A1 and A2 projections to the magnocellular cells; these peptides may also act as co-transmitters with noradrenaline because both neuropeptide Y (Day et al., 1985a) and galanin (Papas and Bourque, 1997) excite magnocellular cells by a direct action and substance P stimulates vasopressin release in vivo (Chowdrey et al., 1990). However, it is possible that enkephalins from noradrenergic terminals (or their extended peptides, metenkephalin-Arg-Phe and met-enkephalin-Arg-Gly-Leu) act in an auto-inhibitory feedback manner to restrain further release of noradrenaline. Although the influence of opioids on the noradrenergic inputs to oxytocin and vasopressin cells has not been studied in detail, it is clear that the A2 projection from the NTS at least is subject to presynaptic inhibition through m-receptors (Onaka et al., 1995b).

2.6. Co-localisation of opioid peptides with oxytocin and 6asopressin Oxytocin cells synthesise enkephalins (Martin and Voigt, 1981; Young and Lightman, 1992) and both vasopressin and oxytocin cells also synthesise the k-agonist, dynorphin (Watson et al., 1982; Levin and Sawchenko, 1993; Eriksson et al., 1996). Dynorphin is co-packaged with vasopressin in neurosecretory granules in the neural lobe (Whitnall et al., 1983) and the

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k-receptor at which dynorphin is active has recently been visualised in the membrane of vasopressinergic neurosecretory vesicles in the cell bodies and axon terminals (Shuster et al., 1999). The co-release of endogenous opioids from the neurohypophysis appears to restrain the secretion of oxytocin but not vasopressin. In addition, it is probable that these opioids are also released from the dendrites into the extracellular space within the magnocellular nuclei and may then modulate the activity of magnocellular cells at this locus.

2.7. Opioid actions at the neurohypophysis Opioid receptors in the neurohypophysis are almost exclusively of the k-receptor type (Herkenham et al., 1986), although some m-receptors may also be present (Bunn et al., 1985; Sumner et al., 1990). Stimulated release of oxytocin and vasopressin from isolated neurohypophysial nerve terminals is inhibited by k-agonists (Zhao et al., 1988a), indicating that neurohypophysial k-receptors are functionally coupled to effector mechanisms, probably through suppression of Ca2 + currents (Rusin et al., 1997). However, naloxone alone increases oxytocin release from such preparations to a much greater extent than vasopressin release (Zhao et al., 1988b), an effect that may be mediated, in part, by inhibition of the stimulatory effects of noradrenaline released within the neurohypophysis (Zhao et al., 1988c) probably by A2 cells that project to the neurohypophysis (Garten et al., 1989). Thus, it appears that, despite the presence of functional k-opioid receptors (Shuster et al., 1999) and dynorphin (Whitnall et al., 1983) within vasopressinergic neurosecretory vesicles, endogenous opioid peptide restraint of hormone release at neurohypophysial terminals is restricted to oxytocin rather than vasopressin cells (Fig. 2). While the d-agonists, the enkephalins, are co-localised with oxytocin in magnocellular cells (Martin and Voigt, 1981; Young and Lightman, 1992), antagonism of d-receptors does not alter stimulated oxytocin release from the neurohypophysis in vivo or in vitro (Bicknell et al., 1985b). However, there is some evidence that enkephalins may be involved in morphological reorganisation of axon terminals in the neurohypophysis (Blanco et al., 1994), although the mechanism by which this is achieved is unclear. Unlike their failure to alter N- and P-type Ca2 + currents at the cell bodies of magnocellular cells (Soldo and Moises, 1998), k- (but not m- or d-) agonists reduce L-, N- and P/Q-type Ca2 + currents in isolated neurohypophysial terminals (Rusin et al., 1997). The differential expression of Ca2 + channel sub-types in the cell bodies and axon terminals of magnocellular cells (Fisher and Bourque, 1996) may account for the different mechanisms of action of k-opioids in reducing the activity of the cells in the hypothalamus and in inhibiting the release of hormone from the neurohypophysis.

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2.8. Opioid-like peptides Screening of cDNA and genomic libraries led to the discovery of a new receptor that bears a high homology to the k-opioid receptor, the opioid receptor like-1 (ORL1) receptor (Mollereau et al., 1994). Since initially no endogenous ligand was known for ORL1, it also was named the ‘orphan opioid receptor’. Despite the high degree of structural homology between ORL1 and opioid receptors, opioid ligands (including naloxone) have very low affinity for this receptor. The endogenous ligand for ORL1 was then discovered to be the heptadecapeptide, nociceptin/orphanin FQ (OFQ) (Meunier et al., 1995; Reinscheid et al., 1995) which, similarly to the opioid peptides, is derived from a larger precursor peptide, pre-pro-nociceptin. The gene encoding pre-pronociceptin is structurally very similar to the opioid peptide genes (Mollereau et al., 1996b), and nociceptin has homology with dynorphin A1 – 17. This, and the ability to confer opioid affinity by mutation of the ORL1 receptor (Mollereau et al., 1996a; Meng et al., 1998), supports the hypothesis that the ORL1, MOR-1, DOR-1 and KOR-1 all belong to the same family (Table 1). The classical ‘message’ at the amino terminal of opioid peptides is the YGGF amino acid sequence; Y is substituted by F in nociceptin, explaining its lack of affinity for opioid receptors. Nociceptin, its precursor peptide and ORL1 receptors have all been localised in the hypothalamus, including the supraoptic nucleus (Wick et al., 1994; Nothacker et al., 1996; Neal et al., 1999). In addition, nociceptin and ORL1 mRNA are present in regions that project to the supraoptic nucleus such as the LC and NTS (Neal et al., 1999). However, nociceptin is not present in the pituitary gland (Neal et al., 1999). Nociceptin inhibits the activity of supraoptic nucleus vasopressin and oxytocin cells in vitro and this inhibition is not reversed by application of the opioid antagonist, naloxone. Nociceptin induces phasic firing in fast-firing supraoptic nucleus cells in vitro, and this is dependent on intracellular Ca2 + , perhaps indicating an inhibitory action on the Ca2 + -dependent depolarising after-potentials that sustain firing during bursts. However, nociceptin also inhibits the firing of magnocellular cells independently of extracellular or intracellular Ca2 + (Doi et al., 1998), consistent with the observation that in guinea-pig supraoptic nucleus cells nociceptin activates an inwardly-rectifying (outward) K+ conductance, hyperpolarising the cells (Slugg et al., 1999); it has been suggested that such activation of an inwardly-rectifying K+ conductance might induce phasic firing in fastfiring neurones by hyperpolarising the cells to a level where Ih is activated, thus activating the Ca2 + conductances underlying the depolarising after-potential (Slugg et al., 1999). Closure of gap junctions, perhaps by inhibition of cAMP generation, has been proposed

as another mechanism by which nociceptin inhibits firing, slowly increasing membrane resistance and hyperpolarising the cells (Slugg et al., 1999).

2.9. Endogenous anti-opioid peptides? Cholecystokinin-8-sulphate (CCK) has been shown to have functional anti-opioid actions in foot-shock analgesia (Faris et al., 1983) and is also co-localised with oxytocin in some magnocellular cells (Levin and Sawchenko, 1993). CCK receptors are present in the supraoptic and paraventricular nuclei (Day et al., 1989) and their density within the magnocellular nuclei is up-regulated with physiological and pharmacological stimuli such as salt loading (Legros et al., 1990; Hinks et al., 1995), water deprivation (O’Shea and Gundlach, 1995) and chronic morphine treatment (Munro et al., 1998). CCK induces oxytocin and vasopressin release from isolated neurohypophysial nerve terminals and from hypothalamo-neurohypophysial explants (Bondy et al., 1989; Jarvis et al., 1995). As well as actions on magnocellular cell terminals, CCK also has powerful actions at the level of the cell bodies; CCK depolarises supraoptic nucleus cells in vitro (Jarvis et al., 1992), induces the expression of Fos protein in magnocellular cells (Ludwig et al., 1997b) and CCK injection into the supraoptic nucleus causes local and systemic release of oxytocin (Neumann et al., 1994). While CCK clearly directly excites both magnocellular oxytocin and vasopressin cells (Fig. 3), it has not been demonstrated that CCK release from oxytocin cells (or any other source) modulates the inhibitory action of opioids on magnocellular cells. The failure of i.c.v. CCK to mimic the effects of the opiate antagonist naloxone in precipitating morphine withdrawal behaviours (Maldonado et al., 1994) suggests that CCK may act as a functional opioid antagonist only in particular systems.

2.10. Acti6ation of oxytocin cells by systemic cholecystokinin In contrast to central CCK administration, systemic injection of CCK selectively increases the firing rate of oxytocin cells and either does not affect or inhibits the firing rate of vasopressin cells (Renaud et al., 1987) to increase neurohypophysial oxytocin release (Verbalis et al., 1986). Systemic CCK acts via CCK-A receptors located on gastric vagal afferent fibres (Verbalis et al., 1986; Luckman et al., 1993b; Miller et al., 1993) and in the area postrema. This excitation is mediated by brainstem noradrenergic inputs, because CCK induces Fos expression in the NTS and VLM (Luckman, 1992) and ablation of the area postrema (which overlies the NTS), or of central noradrenergic neurones, reduces systemic CCK-induced stimulation of oxytocin secretion (Carter and Lightman, 1987; Onaka et al., 1995a), whereas

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Fig. 3. CCK does not antagonise morphine actions on magnocellular neurosecretory cells. The graphs show extracellular ratemeter recordings (averaged in 15 s bins) of the activity of supraoptic nucleus magnocellular neurosecretory cells (identified by antidromic activation from the posterior pituitary) recorded from urethane-anaesthetised (1.25 g/kg, i.p.) female Sprague-Dawley rats. (A) A vasopressin cell (identified by inhibition following 20 mg/kg CCK i.v.; not shown) that was inhibited from continuous into phasic firing by i.c.v. morphine (MOR) administration (200 ng). This cell was transiently excited by i.c.v. injection of 5 ng CCK. Nevertheless, the second injection of CCK (5 ng, i.c.v.) did not prevent morphine inhibition of the cell whereas 5 mg/kg i.v. naloxone (NLX) reversed the morphine-induced inhibition. (B) A continuously-active oxytocin cell (identified by a transient excitation following administration of 20 mg/kg CCK i.v.: (IV CCK) recorded from a morphine-dependent rat (10 – 50 mg/h i.c.v. morphine over 5 days). 50 ng i.c.v. CCK (ICV CCK) had little effect on the firing rate of the cell while naloxone caused a dose-dependent excitation (NLX1,2,3: 50, 500, 5000 mg/kg i.v., respectively), indicative of withdrawal excitation. The lack of effect of i.c.v. CCK on the firing rate of the cell either before or after naloxone shows that CCK did not antagonise the effects of the on-going i.c.v. morphine infusion.

lesion of the forebrain inputs to oxytocin cells does not (Blackburn and Leng, 1990). While both the A1 and A2 cell groups are activated by systemic CCK, none of the A1 cell group neurones that are activated project to the supraoptic nucleus (Onaka et al., 1995a). Finally, CCKinduced excitation of oxytocin cells is accompanied by increased noradrenaline release within the supraoptic nucleus (Kendrick et al., 1991), and in the paraventricular nucleus, CCK-induced excitation of oxytocin cells can be prevented by iontophoretic administration of the a-adrenoreceptor antagonist phentolamine (Ueta et al., 1993). The release of noradrenaline in the supraoptic nucleus induced by systemic CCK can be prevented by direct administration of morphine into the supraoptic nucleus (Onaka et al., 1995b), indicating that the nora-

drenergic terminals which mediate CCK-induced excitation of oxytocin cells are sensitive to modulation by opioids (Fig. 2). Similarly, systemic injections of morphine block CCK-induced oxytocin release and CCKinduced Fos expression in the supraoptic nucleus, but do not prevent the CCK-induced Fos expression in the brainstem (Onaka et al., 1995a). In virgin rats, the opioid antagonist naloxone enhances systemic CCK-induced oxytocin release (Flanagan et al., 1988) as does the k-antagonist, norbinaltorphimine (Leng et al., 1997). This enhancement of secretion results from blockade of endogenous opioid peptide actions on k-receptors on the oxytocin nerve terminals as it is not accompanied by an increased firing rate of oxytocin cells (Leng et al., 1992).

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2.11. Opioid interactions with the oxytocin system in pregnancy Oxytocin released from the neurohypophysis is important in the timing of parturition, although recent studies on transgenic oxytocin knock-out mice demonstrate that parturition can proceed in its absence (Nishimori et al., 1996; Young et al., 1996). During parturition, oxytocin is released in a pulsatile pattern as a result of bursts of co-ordinated activity of oxytocin cells triggered by the passage of pups through the birth canal (Summerlee, 1981). However, oxytocin infusion will not induce parturition until the last afternoon of pregnancy (Antonijevic et al., 1995b). This is partly a consequence of relative insensitivity of the uterus to oxytocin, because oxytocin receptor expression is upregulated only in the few hours before parturition, and prior to that progesterone antagonises the action of oxytocin at its receptors (Grazzini et al., 1998). Central mechanisms prevent premature activation of the oxytocin system at parturition. On the morning of the final day of pregnancy, oxytocin cells are restrained by centrally-acting opioids (Douglas et al., 1995b). Naloxone has little effect on the activity of oxytocin cells in virgin rats, but at the end of pregnancy naloxone (but not the specific k-opioid antagonist, nor-binaltorphimine) induces Fos protein expression in oxytocin cells under basal conditions and enhances their electrical activation in response to systemic CCK (Douglas et al., 1995b). As noted above, systemic CCK acts via brainstem A2 noradrenergic neurones, while morphine has an inhibitory action pre-synaptically on noradrenergic terminals in the supraoptic nucleus (Onaka et al., 1995b). Signals from the contracting uterus are carried from the brainstem in part by noradrenergic fibres from the A2 neurones in the NTS (Antonijevic et al., 1995b). Thus it is possible that the central tonic endogenous m-opioid inhibition of oxytocin cell activity emerging towards the end of pregnancy is exerted at the presynaptic opioid receptors on the noradrenergic nerve terminals in the supraoptic nucleus. b-endorphin may be responsible for this emergent opioid tone on oxytocin cells as both the peptide content and the mRNA content of its precursor, POMC, increase in the arcuate nucleus during pregnancy (Douglas et al., 1995a). Despite this opioid tone (or perhaps because this tone is relaxed), parturition begins on the afternoon of day 21. Reinforcement of the opioid tone by injection of morphine during parturition prevents delivery of further pups for up to an hour (Luckman et al., 1993a). Similarly, the stress of environmental disturbance interrupts parturition via an opioid-mediated, naloxone-reversible inhibition of oxytocin secretion (Leng et al., 1987). The onset of parturition is linked to circadian time (Russell and Leng, 1998) and this may involve the direct input from the suprachiasmatic nucleus to the

supraoptic and paraventricular nuclei (Buijs et al., 1994; Cui et al., 1997b), or a direct retinal projection to the supraoptic nucleus may also be involved (Cui et al., 1997a). Oxytocin secretion is also strongly restrained by endogenous opioids in mid-pregnancy which allows the pituitary stores of oxytocin to accumulate for parturition (Leng et al., 1997). However, at this time opioid inhibition of oxytocin secretion is mediated by k-receptors in the pituitary. After about day 16, the k-receptors in the pituitary are down-regulated and desensitised (Douglas et al., 1993) and the pituitary content of dynorphin decreases. By term, with this auto-inhibitory mechanism less effective (Douglas et al., 1995a), the stores of oxytocin are available for ready release (Fig. 4). The increasing circulating oestrogen levels and collapse in progesterone secretion towards the end of pregnancy may underlie the activation of the central m-opioid inhibition of oxytocin cell activity in pregnancy.

2.12. The milk-ejection reflex Oxytocin is essential for suckling-induced milk ejection in lactating rats (Nishimori et al., 1996; Young et al., 1996) and is released in pulses, each a few minutes apart, as a result of brief high frequency bursts of activity co-ordinated among oxytocin cells (Belin et al., 1984). A typical milk-ejection burst contains approximately 100 spikes and lasts 1–2 s (Wakerley and Lincoln, 1973; Lincoln and Wakerley, 1974; Wakerley and Ingram, 1993), releasing a bolus of about 1 mU (2 pmoles) of oxytocin into the circulation. The amount of oxytocin released by each burst is larger than would be expected by simply scaling up the amount of oxytocin released by a single spike; this ‘frequency facilitation’ of release results from a progressive increase in extracellular [K+] which depolarises the axons allowing later spikes to depolarise more of the terminal field (Dyball et al., 1988; Leng et al., 1988a) and from summation of Ca2 + entry into the terminals to augment exocytosis (Jackson et al., 1991; Jackson, 1993). As well as bursts being efficient for oxytocin release (Bicknell, 1988), the pulsatile release of oxytocin which results causes a near-maximal increase of intramammary pressure, without desensitising peripheral oxytocin receptors as does a continuous infusion (Bicknell, 1988). Opiates inhibit the milk ejection reflex. Initially it was believed that this inhibition by morphine resulted from actions at the nerve terminals in the neurohypophysis because milk-ejection bursts were found to persist in the face of morphine inhibition of milk ejection itself (Clarke et al., 1979). However, the lack of m-receptors and the insensitivity of nerve terminals in the posterior pituitary to morphine, relative to the abundance of k-receptors and potency of k-agonists (Russell et al.,

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1993), and the effectiveness of m- and k-agonists in inhibiting the firing of oxytocin cells led to further analysis. Milk-ejection bursts in oxytocin cells may be more resistant to opioid inhibition than basal activity as it has been shown that high frequency stimulation of inputs to oxytocin cells results in an increased oxytocin release that is relatively resistant to morphine inhibition (Bull et al., 1994). Nevertheless, milk ejection bursts in some oxytocin cells are reduced, or indeed eliminated, by morphine while others are unaffected (Pumford et al., 1991); this reduction in the number of bursting cells and in the amplitude of bursts in others may prevent release of sufficient oxytocin to induce milk ejection in the mammary gland. In addition, OT cells possess electrical properties favouring the expression of short spike trains and some of these properties are enhanced during lactation, including a greater degree of Ca2 + influx per action potential (Stern and Armstrong, 1996). These altered electrical properties may make the high frequency firing that they favour more resistant to opioid blockade. In intact animals, oxytocin cells fire in bursts only during suckling or parturition (Richard et al., 1991)

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and in lactating rats there is an increased noradrenergic innervation of oxytocin cells (Michaloudi et al., 1997). Nevertheless, recent evidence demonstrating behaviour similar to bursting from oxytocin cells in organotypic culture suggests that local inputs from glutamatergic cells may also contribute to burst generation (Jourdain et al., 1998). Bursts are co-ordinated by dendro-dendritic interactions between oxytocin cells, although other brain areas modulate the reflex (Ingram et al., 1995). While bursts are co-ordinated between oxytocin cells, there is no evidence to suggest the existence of specific ‘pacemaker’ cells that initiate bursts. Oxytocin is released from magnocellular cell dendrites (Pow and Morris, 1989), depolarises oxytocin cells (Yamashita et al., 1987b) and increases Ca2 + liberation from intracellular stores (Lambert et al., 1994). These effects probably reflect a receptor-mediated action because oxytocin cells express oxytocin receptor mRNA (Yoshimura et al., 1993). In lactating rats oxytocin binding sites have been localised to the dendrites (Freund-Mercier et al., 1994). During suckling oxytocin is released within the supraoptic nucleus (Moos et al., 1989; Neumann et al., 1993a,b) and i.c.v. or intra-supraoptic nucleus oxytocin

Fig. 4. Changes in endogenous opioid peptide interactions with oxytocin cells over pregnancy and parturition. In virgin animals, oxytocin (OT) cells express both m- and k-opioid receptors at the level of the cell body and m-receptors are probably also present on afferent nerve terminals on oxytocin cells. However, there is little, if any, activity of endogenous opioids at this level. k-receptors are also expressed on oxytocin nerve terminals in the neurohypophysis where feedback from opioid peptides co-secreted with oxytocin inhibits further oxytocin release. In mid-pregnancy, the auto-inhibitory mechanisms are activated in the neurohypophysis, restraining the release of oxytocin and hence increasing the accumulation of pituitary oxytocin stores. By the end of pregnancy, the neurohypophysial auto-inhibitory mechanisms are down-regulated, allowing the release of oxytocin. However, at this time, the activity of oxytocin cells is restrained by endogenous opioid actions mediated by m-receptors on the cell body (or axon terminals of afferent inputs). A reduction in this central opioid tone may contribute to the enhanced secretion of oxytocin evident during parturition. Dendrites omitted for clarity.

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antagonist administration blocks the milk-ejection reflex (Richard et al., 1991; Lambert et al., 1993). Thus, dendritic oxytocin release is likely to be necessary for the milk-ejection reflex to occur, and the major target of this oxytocin may also be the dendrites of oxytocin cells, perhaps to bring the background firing rate of non-bursting cells into the range that favours the occurrence of bursts (Brown and Moos, 1997) as well as pre-synaptic modulation of inputs to the oxytocin cells (Brussaard et al., 1996; Kombian et al., 1997). It is expected that endogenous opioids co-produced in oxytocin cells would be co-released from their dendrites. However, while naloxone increases electrically-stimulated dendritic oxytocin release in vitro (Ingram et al., 1996), it does not augment suckling-induced peripheral oxytocin release in conscious rats (Hartman et al., 1987). This indicates that endogenous opioid peptides are not tonically active on oxytocin cells during lactation, or are not able to effectively inhibit milk-ejection bursts.

2.13. Morphine dependence in oxytocin neurones

Fig. 5. Morphine withdrawal excitation of oxytocin cells. (A) The firing rate (averaged in 10 s bins) of a supraoptic nucleus oxytocin cell (excited after 20 mg/kg CCK i.v.) recorded from a morphine-dependent rat, showing a profound and sustained morphine-withdrawal excitation after injection of 5 mg/kg naloxone (NLX). (B) The left hand panel shows a plot of the mean inter-spike intervals (9 S.E.M., in 10 ms bins) of nine oxytocin cells recorded from morphine-dependent rats before (closed circles) and after (open circles) administration of naloxone as in A. Note the shift to the left of the modal inter-spike interval. The right-hand panel shows a plot of the hazard function of the mean inter-spike intervals of the nine cells. This gives the rate of cell firing per 10 ms after a spike (at time 0), given that another spike has not occurred earlier and is a measure of excitability. Immediately after an action potential (both before and after withdrawal) firing within 20 ms of a spike is minimal; this is a result of the hyperpolarising after-potential that follows each spike. After morphine withdrawal (open circles) the cells not only display a higher probability of firing a second action potential at any time after an action potential (reflecting the increased activity after withdrawal) but there is also a greater probability of action potentials firing in the period 20– 250 ms after each action potential relative to longer intervals; this increased excitability is deduced to result from the influence of depolarising after potentials following withdrawal. (C) The panels show the firing rate of oxytocin cells recorded from morphine-dependent rats after administration of naloxone (5 mg/kg, i.v.): antagonism at post-synaptic a1-adrenoreceptors by i.c.v. injection of the a1-adrenoreceptor antagonist, WB4101 (WB: left-hand panel), causes a dose-dependent, reversible inhibition of oxytocin cell firing rate in morphine-withdrawn rats (closed circles: 25 ng; open circles: 50 ng). Similarly, pre-synaptic inhibition of noradrenaline release by i.c.v. administration of the a2-adrenoreceptor agonist, clonidine (CLON), also causes

After chronic i.c.v. administration of morphine or U50,488H, oxytocin cells develop tolerance to these opioids (Russell et al., 1995; Brown et al., 1998a), reflecting a reduced sensitivity to inhibition by the m- or k-agonists; in the case of morphine, this may result from m-receptor down-regulation (Sumner et al., 1990). While tolerance develops to both of these opioids, there is no cross-tolerance between their effects, at least from m- to k-receptors (Pumford et al., 1993b), suggesting that the mechanisms of tolerance to each of the drugs are specific to the respective receptors at which each drug is active. While oxytocin cells develop tolerance to both morphine and U50,488H, oxytocin cells develop dependence upon morphine (Bicknell et al., 1988) but not U50,488H (Brown et al., 1998a). Morphine dependence involves changes in the cells such that they require the continued presence of morphine to function apparently normally, and is revealed by a rebound hyper-excitation after withdrawal of morphine acutely precipitated by naloxone administration (Fig. 5A). This causes an approximate 100-fold increase in oxytocin release (Bicknell et al., 1988), primarily as a result of increased oxytocin cell firing rate, potentiated by frequency-facili-

a dose-dependent inhibition of oxytocin cell firing rate in morphinewithdrawn rats (closed circles: 40 ng; open circles: 80 ng). Thus, morphine-withdrawal excitation of oxytocin cells involves changes in the intrinsic properties of these cells but for these changes to be expressed functionally as an increase in activity requires on-going activity in excitatory synaptic inputs, including noradrenergic synapses.

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tation of release and naloxone antagonism at opioid receptors in the neurohypophysis (Leng et al., 1989b). k-opioid mechanisms on oxytocin cells remain intact during morphine withdrawal because the k-agonist U50,488H can fully suppress activity after morphine withdrawal. Oxytocin heteronuclear RNA and immediate early gene expression in the supraoptic and paraventricular nuclei are also increased during withdrawal excitation (Russell et al., 1995; Jhamandas et al., 1996) as is dendritic oxytocin release (Russell et al., 1992a) which contributes to the withdrawal excitation of oxytocin cells (Brown et al., 1997). Administration of naloxone directly into the supraoptic nucleus of morphine-dependent rats also evokes withdrawal excitation of oxytocin cells (Ludwig et al., 1997a), indicating that the mechanisms underlying morphine withdrawal reside within the nucleus. Withdrawal-induced increases in Fos protein expression by oxytocin cells can also be precipitated by systemic naloxone after chronic infusion of morphine into the supraoptic nucleus (Johnstone et al., 2000) indicating that not only are the mechanisms that underlie withdrawal localised to the supraoptic nucleus but also the mechanisms underlying dependence itself. Many central neurones express m-receptors (Mansour et al., 1995) but few have been shown to develop morphine dependence (Nye and Nestler, 1996) and oxytocin cells are the only identified peptidergic neurones which have been shown to do so (Russell et al., 1995). Neurones of the A2 cell group in the NTS express Fos protein after naloxone-precipitated morphine withdrawal (Stornetta et al., 1993) some of which project to the supraoptic nucleus (Murphy et al., 1997). Acute morphine administration acts on the terminals of A2 afferent inputs to reduce noradrenaline release into the supraoptic nucleus (Onaka et al., 1995b). Thus, withdrawal excitation of oxytocin cells may result in part from increased activity in afferent noradrenergic inputs, augmented by escape from pre-synaptic inhibition of noradrenaline release with the supraoptic nucleus. However, the proportion of A2 cells that project to the supraoptic nucleus and that are activated by withdrawal is small in comparison to those that express Fos protein after systemic CCK (Onaka et al., 1995a) and the increase in noradrenaline release within the supraoptic nucleus during morphine withdrawal is also modest (Murphy et al., 1997), being less than that after systemic CCK administration (Onaka et al., 1995b) which is a much weaker stimulus for oxytocin release than morphine withdrawal and does not induce morphine withdrawal excitation in oxytocin cells (Brown et al., 1996). There is no change in the response of oxytocin cells to systemic CCK before and after morphine withdrawal excitation, suggesting that the noradrenergic input mediating the CCK response is not fully activated during morphine withdrawal and that mor-

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phine-dependent and -withdrawn oxytocin cells are not hypersensitive to noradrenergic activation. Finally, neurotoxic lesion of central catecholaminergic cells at the start of morphine treatment does not affect withdrawal excitation of oxytocin cells (Brown et al., 1998b) and so the noradrenergic input to oxytocin cells is unlikely to be primarily responsible for the increased activity evident in oxytocin cells during morphine withdrawal. Nevertheless, acute interruption of the noradrenergic input to oxytocin cells with an a1-adrenoreceptor antagonist (Brown et al., 1998b) or an a2-adrenoreceptor agonist (Fig. 5C) attenuates withdrawal excitation. However, basal activity is also inhibited by such treatments in morphine-naı¨ve rats and blockade of a1adrenoreceptors does not prevent Fos expression in the supraoptic nucleus during morphine withdrawal (Johnstone et al., 2000), despite the inhibition of the excitation of firing rate. These effects in dependent rats reveal a requirement for active excitatory synaptic inputs in dependent cells for the expression of withdrawal excitation as an increase in electrical activity. Similarly, with regard to other inputs, acute lesion of the AV3V region silences oxytocin cells in morphinenaı¨ve rats (Leng et al., 1989a) and reduces, but does not prevent, withdrawal excitation of oxytocin cells (Russell et al., 1992b). Glutamate is an important transmitter in the AV3V input to oxytocin cells and acute central administration of the AMPA/kainate antagonist, DNQX also decreases withdrawal excitation. Thus, it would appear that full withdrawal excitation in oxytocin cells requires their afferent inputs but the activity in these inputs is not necessarily increased during withdrawal as there is little increase in Fos protein expression following withdrawal in AV3V cells (or brainstem noradrenergic cells) projecting to the supraoptic nucleus (Murphy et al., 1997). The mechanisms involved in morphine dependence and withdrawal excitation involve mediation by Gi/o proteins because withdrawal excitation is much reduced by pertussis toxin injection into the supraoptic nucleus at the start of morphine treatment (unpublished observations). Thus, withdrawal excitation of supraoptic oxytocin cells probably results primarily from intrinsic mechanisms when the chronic occupancy of m-receptors on the oxytocin cells is interrupted by naloxone. The decrease in the modal inter-spike interval evident after withdrawal (Fig. 5B) indicates an increased probability of a further spike firing 30–150 ms after the previous spike. The mechanisms underlying this increased postspike excitability are as yet unknown but, given its similarity to the post-spike excitability of vasopressin cells that can be attributed to the presence of a Ca2 + dependent non-synaptic post-spike depolarising afterpotential (DAP) (Bourque et al., 1986), it is possible that an increase in DAP amplitude in oxytocin cells

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may contribute to withdrawal excitation. This is supported by the observation that the L-type Ca2 + -channel blocker, verapamil, given centrally or into the supraoptic nucleus, reduces morphine withdrawal excitation of oxytocin cells (Blackburn-Munro et al., 2000). However, the apparent increase in DAP amplitude after withdrawal excitation may not simply result from a direct action on the conductances underlying the DAP itself but rather from a reduction of the slow after-hyperpolarisation (AHP) that normally masks the DAP in oxytocin cells (Greffrath et al., 1998). While these intrinsic mechanisms may underlie withdrawal excitation of oxytocin cells, they nevertheless require synaptic input to be expressed as increased activity; removal of any one excitatory input may reduce the overall excitability of the cells in a non-specific manner to limit the ability of the cell to respond to the withdrawal stimulus with an increase in firing rate (Leng et al., 1999). In contrast to oxytocin cells, vasopressin cells do not develop morphine dependence; during withdrawal there is no net change in the firing rate of vasopressin cells, although the firing pattern within bursts indicates increased excitability (Bicknell et al., 1988). This may reflect a change in DAPs similar to that postulated for oxytocin cells but because DAPs are normally of predominant importance in regulating phasic firing, the change in activity is minimal in comparison to oxytocin cells.

2.14. Modulation of phasic acti6ity in 6asopressin cells by k-opioid receptor acti6ation The sensitivity of vasopressin secretion to inhibition by k-opioids, and the consequent diuretic effects, are well-established. The diuretic effects of k-agonists were attributed initially to effects in the kidney to attenuate the anti-diuretic response to vasopressin, and subsequently also to actions at the neurohypophysis to reduce secretion of vasopressin (Slizgi and Ludens, 1982; Leander, 1983). However, vasopressin release from isolated neurohypophysial preparations is not much altered by k-opioid agonists or antagonists (Bicknell et al., 1985a; Bondy et al., 1988; Falke, 1988) and k-agonists that cross the blood-brain-barrier are more potent at promoting diuresis than those that do not (Brooks et al., 1993). This suggests that k-agonist inhibition of basal vasopressin secretion in the intact animal results from a reduction in the activity of vasopressin cells. This was confirmed by recording inhibition of vasopressin (and oxytocin) neurone firing rate after systemic k-agonist injection (Pumford et al., 1993b); this is reversed by systemic nor-binaltorphimine or by local naloxone administration into the supraoptic nucleus (Ludwig et al., 1997a). k-agonist inhibition of vasopressin cell activity is not simply a pharmacological

phenomenon because application of a k-antagonist alone into the supraoptic nucleus increases the activity of vasopressin cells (Brown et al., 1998a), indicating that an endogenous k-opioid restrains the activity of vasopressin cells under basal conditions, as has previously been suggested from experiments on vasopressin secretion (Wells and Forsling, 1991). Magnocellular neurosecretory cell axon terminals cannot sustain intrinsic repetitive firing (Bourque, 1990) and so vasopressin secretion is primarily determined by action potentials initiated at the cell bodies. Unlike oxytocin cells, vasopressin cells each fire in robust ‘phasic’ bursts under basal conditions (Wakerley et al., 1978). Again, unlike the bursting behaviour of oxytocin cells in suckling and parturient rats, vasopressin cell bursts typically last much longer (20–40 s) at a slower firing rate (5–10 spikes/s) and are separated by shorter intervals (of about 10–20 s) during which the cells are normally silent (Fig. 6A). As there is no synchronisation of bursts between vasopressin cells (Leng and Dyball, 1983), systemic vasopressin release is continuous rather than pulsatile. Nevertheless, phasic activity is of physiological importance as it increases the efficiency of hormone release by making optimal use of the terminal membrane properties. Intra-supraoptic nucleus administration of a k-receptor antagonist increases the duration of phasic bursts (Brown et al., 1998a), indicating that termination of phasic bursts in vasopressin cells involves endogenous activation of k-receptors within the supraoptic nucleus. Furthermore, desensitisation of supraoptic nucleus k-receptors by chronic k-agonist administration into the nucleus induces tolerance to acute inhibition of firing rate by U50,488H but nevertheless eliminates phasic activity in vasopressin cells (Brown et al., 1998a), indicating that normallyfunctioning k-receptors are not only involved in, but are essential for, the expression of phasic activity by vasopressin cells. Systemic administration of a k-antagonist after chronic k-agonist treatment induces a consistent increase in the activity of vasopressin cells (Brown et al., 1998a), but only to firing rates normally seen in vasopressin cells recorded from untreated rats. Thus, this excitation is unlikely to represent a withdrawal hyperexcitation analogous to that seen for oxytocin cells challenged with chronic morphine infusion and so it appears that vasopressin cells develop tolerance to, but not dependence upon, U50,488H. The source of the endogenous k-agonist that restrains the activity of vasopressin cells is currently uncertain. However, dynorphin is also present in vasopressin cells at the highest levels seen in the brain (Molineaux et al., 1982; Watson et al., 1982). The supraoptic nucleus contains a high density of k-receptors (Sumner et al., 1990) and these have recently been localised to the same neurosecretory vesicles that contain vasopressin (Shus-

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Fig. 6. Modulation of phasic activity by endogenous k-opioids. (A) The firing rate (averaged in 10 s bins) of a supraoptic nucleus vasopressin cell recorded in vivo. This cell was initially inhibited by i.c.v. infusion of the k-agonist, U50,488H (50 mg/h). Similarly to all other vasopressin cells tested, this cell recovered some activity despite sustained U50,488H infusion, indicating the development of acute tolerance to the inhibitory actions of U50,488H. The inset panels show the instantaneous frequency of firing of the cell in the first 5 min of recording (left) and in the 5 min during U50,488H infusion when the cell was most active (right). The spontaneous activity before the i.c.v. U50,488H infusion is clearly organised into phasic busts, but in the face of U50,488H the activity displays an irregular pattern despite the continued presence of some high-frequency events. (B) Plot of the hazard function of the cell shown in A prior to (filled circles) and during (open circles) i.c.v. U50,488H infusion. The lower firing rate per 10 ms in the face of the U50,488H infusion reflects the reduced activity of the cell at this time. The influence of depolarising after-potentials during phasic activity in the control period is demonstrated by the greater probability of a spike firing in the period 20– 250 ms after the preceding spike relative to longer intervals; this post-spike excitability is eliminated upon recovery of activity during the ongoing U50,488H infusion, indicating that depolarising after-potentials are still inhibited despite the recovery of activity.

ter et al., 1999) (and presumably dynorphin). Thus, dendritic release of vasopressin will be accompanied by a high local concentration of dynorphin over the cell membrane containing newly-exposed k-opioid receptors and these may be sufficient to mediate feedback inhibition of vasopressin cell activity over the time-course of bursts.

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The mechanisms by which endogenous k-agonists restrain the duration of phasic bursts is also currently unknown. Phasic firing is a consequence of intrinsic membrane properties (Renaud, 1987); bursting depends on intracellular Ca2 + , and vasopressin cells contain much lower quantities of the Ca2 + binding protein, calbindin, than oxytocin cells, while immunoneutralisation of calbindin induces phasic activity in oxytocin cells (Li et al., 1995). Phasic activity is sustained by a Ca2 + -dependent non-synaptic post-spike DAP (Bourque, 1986; Bourque et al., 1998); direct blockade of the conductance underlying the DAP eliminates phasic firing (Ghamari-Langroudi and Bourque, 1998). DAPs summate to produce a plateau potential, increasing the probability of post-synaptic potentials triggering further action potentials during bursts (Bourque et al., 1998). Inactivation of DAPs plays a key roˆle in burst termination. k-receptor activation by an endogenous agonist shortens bursts (Brown et al., 1998a) and the k-agonist U50,488H reduces the amplitude of DAPs in vitro (Brown et al., 1999) and, presumptively, in vivo (Fig. 6B). Thus, it is possible that k-receptor activation inactivates DAPs, and thus the plateau potential, terminating bursts. It is attractive to speculate that dendritically-released dynorphin might modulate phasic activity, perhaps by a pre-synaptic action, as observed for the actions of dendritically-released oxytocin upon the activity of oxytocin cells (Kombian et al., 1997). Dynorphin might also act post-synaptically because U50,488H reduces voltage-activated Ca2 + -currents in isolated supraoptic nucleus cells (Mason et al., 1988). Dendritically-released vasopressin is also auto-inhibitory (Ludwig and Leng, 1997), although the effect of vasopressin on vasopressin cell activity appears to depend on the initial activity of the cell (Gouzenes et al., 1998). Thus, vasopressin and dynorphin may act together to generate phasic activity necessary for efficient release of vasopressin into the systemic circulation. Whether these effects of dynorphin and vasopressin result from pre- and/or post-synaptic actions remains to be established, although vasopressin has been shown to depress EPSC amplitude in magnocellular cells (Kombian et al., 1997).

2.15. Opioid modulation of 6asopressin cell responses to osmotic stimuli Osmoreceptors regulate sodium and water balance to maintain extracellular fluid osmotic pressure within strict physiological limits. In rats, the secretion of vasopressin (and oxytocin), plays a key role in osmoregulation through effects on diuresis and natriuresis. When plasma Na+ concentration rises, vasopressin cells respond with an increase in the proportion of time in which they are active, and when strongly activated they fire continuously (Dyball and Pountney, 1973). However, during chronic stimulation, vasopressin cells fire

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in shorter, more intense bursts (Wakerley et al., 1978). While endogenous opioids clearly modulate phasic patterning of vasopressin cells under basal conditions, it is currently unknown whether these opioids modulate phasic activity under such stimulated conditions. Salt loading reduces dynorphin immunoreactivity in the supraoptic nucleus (Meister et al., 1990; Yagita et al., 1994) and reduces the dendritic span of magnocellular cells (Dyball and Garten, 1988), suggesting that dendritic release of dynorphin may potentially be less influential during chronic stimulation of vasopressin cells, releasing the system from tonic restraint. Salt-loading by dehydration or hypertonic saline administration reduces k-receptor density in the neurohypophysis (Brady and Herkenham, 1987; Brady et al., 1988). Thus, increased release of dynorphin during chronic stimulation may down-regulate neurohypophysial k-receptors, also freeing the system from tonic restraint under stimulated conditions. However, the release of dynorphin from vasopressin cells may be more important in the regulation of oxytocin than vasopressin secretion during dehydration, preventing inappropriate oxytocin release under these conditions (Summy-Long et al., 1990). Vasopressin (and oxytocin) cells are intrinsically osmosensitive (Mason, 1980). These cells express stretchinactivated cation channels that respond to osmotically-evoked changes in cell volume to alter membrane potential (Oliet and Bourque, 1993). However, osmotically-induced depolarisations are not of sufficient magnitude to elicit action potentials and without appropriate synaptic input, vasopressin (and oxytocin) cells do not respond to systemic osmotic stimuli with an increase in firing rate (Russell et al., 1988). The major afferent inputs essential for osmoregulation of vasopressin and oxytocin release lie in the AV3V region, in the OVLT, SFO and MnPO (McKinley et al., 1996), although there is some evidence that brainstem pathways may play a roˆle in osmoregulation (Hochstenbach and Ciriello, 1995). However, neurotoxic lesions of noradrenergic cells attenuate rather than abolish the release of vasopressin in response to systemic osmotic stimuli (Lightman et al., 1984), and so noradrenergic pathways do not appear to be essential for the response to osmotic stimuli. Consistent with its inhibitory effects on basal secretion, U50,488H suppresses the plasma vasopressin response to osmotic stimulation in a dose-dependent manner (Oiso et al., 1988). Similarly, m- and k-opioid receptors mediate an inhibitory influence of endogenous opioids on osmoreceptor-mediated vasopressin secretion in response to central infusion of hypertonic saline (Wells and Forsling, 1991). This probably reflects an action at the cell body rather than the terminals as has been demonstrated in cultured hypothalamo-neurohypophysial explants (Rossi and Brooks, 1996). However,

m-, k- and d-agonists increase antidiuresis when injected into the supraoptic or paraventricular nucleus of waterloaded rats (Tsushima et al., 1987, 1993a,b) but in chronically hyponatraemic rats, the blunted responses to osmotic stimulation are corrected by naloxone (Dohanics and Verbalis, 1995), suggesting that during chronic hyponatraemia, the activity of magnocellular cells are actively inhibited by endogenous opioids. While functional k-opioid receptors are present on the terminals of both vasopressin and oxytocin cells, endogenous opioid peptide inhibition of neurohypophysial hormone release is restricted to oxytocin rather than vasopressin cells. This may serve a physiological function, allowing preferential release of vasopressin, because blocking endogenous opioid actions with naloxone during osmotic stimulation augments the rise in plasma oxytocin but not vasopressin (Hartman et al., 1986).

2.16. Opioid modulation of 6asopressin cell responses to alterations in blood pressure Neurohypophysial vasopressin release in response to changes in blood pressure is mediated by peripheral baroreceptors, cardiopulmonary volume receptors and circulating angiotensin II. In hypotension reduced input from baroreceptors and volume receptors increases vasopressin cell activity; this excitation is mediated by a direct projection from the A1 cell group in the VLM. Hypotensive and hypovolaemic stimuli activate c-fos expression in A1 cells (Day et al., 1995) and haemorrhage increases extracellular concentrations of both noradrenaline and the purine metabolite uric acid in the supraoptic nucleus (Kendrick and Leng, 1988). This excitation may, at least partly, be mediated by ATP co-localised with noradrenaline (Buller et al., 1996). Similarly to osmotic stimuli, hypovolaemic stimuli also activate cells of the SFO. However, the SFO cells activated by hypovolaemia are distinct from those activated by osmotic stimuli (Smith and Day, 1995). Hypotension activates the renin-angiotensin system and angiotensin II acts on AT1 receptors in the SFO (Bunnemann et al., 1992) to increase the activity of the angiotensinergic input to vasopressin cells and promote vasopressin release (Jhamandas et al., 1989). m- and k-antagonists injected into the supraoptic nucleus augment the hypovolaemia-induced stimulation of vasopressin secretion (Iwasaki et al., 1994), indicating that the vasopressin response to acute hypovolemia is normally restrained by activation of m- and k-receptors located within the supraoptic nucleus. There is a high density of opioid binding sites in the SFO (Atweh and Kuhar, 1977). However, morphine does not alter the activity of SFO cells in vitro (Buranarugsa and Hubbard, 1979) and while injection of a synthetic enkephalin analogue into the SFO reduces diuresis and

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natriuresis, it does not alter blood pressure (Fregoneze and Antunes-Rodrigues, 1992). Neurotoxic lesion of brainstem catecholaminergic projections to the supraoptic nucleus reduces vasopressin secretion in response to haemorrhage (Lightman et al., 1984) and this is another site where opioids might modulate the response of vasopressin cells to alterations in blood pressure. Nevertheless, it appears that the major influence of endogenous opioids on the response of vasopressin cells to hypovolaemic stimuli is mediated by an action within the supraoptic nucleus, whether this is a direct action on the magnocellular cells themselves, or on their afferent inputs or a combination of the two has yet to be determined.

3. Conclusions Endogenous opioid systems interact with the magnocellular neurosecretory system at various levels to restrain the secretion of oxytocin and vasopressin. However, these interactions are not the same for oxytocin and vasopressin cells and are not static, but display plasticity in response to changes in physiological status. Oxytocin secretion is restrained by endogenous activation of k-receptors on the neurosecretory terminals in the neurohypophysis. However, towards the end of pregnancy, this restraint is relaxed to prepare the neurosecretory system for the massive outflow of oxytocin required for parturition to progress normally. Over this time, a m-opioid inhibition emerges at the level of the cell bodies (or perhaps pre-synaptically on their ascending noradrenergic inputs), keeping the oxytocin cells quiescent, until the day of parturition when a partial ‘withdrawal’ of endogenous m-opioid tone may render the system hyper-excitable and contribute to the increased activity apparent at term. Unlike oxytocin, the secretion of vasopressin does not appear to be modulated by opioids acting at the neurohypophysis. Rather it appears that k-opioids released from the dendrites, and not the terminals, of these cells are important in modulating the phasic activity patterning of vasopressin cells, perhaps by altering the intrinsic membrane properties of the vasopressin cells or by actions on their afferent inputs. These mechanisms might serve to restrain inappropriately-excessive secretion of vasopressin in response to cardiovascular or osmotic stimuli that release vasopressin. Again, the k-opioid modulation of vasopressin cells activity is not static but is expected to develop during a phasic burst, thus acting on a much shorter time-scale than the central m-opioid modulation of oxytocin cells.

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