- Email: [email protected]
Where is the locus in opioid withdrawal?
REVIEW 41 Bito, H., Deisseroth, K. and Tsien, R. W. (1996) Cell 87,1203-1214 42 SteIzer, A. and Shi, H. (1994) Neuroscience 62,8X-828 43 Chen, Q. X. and Wong, R. K. S. (1995) J. Physiol. 482,353-362 44 Martina, M., Mozrzymas, J. W., Boddeke, H. W. G. M. and Admowledpemeets Cherubini, E. (1996) Neurosci. L&t. 215,95-98 I would lhketo thank 45 Boddeke, H. W. G. M., Meigel, I., Boeijinda, I’., ArbuckIe, J. and SusanJones,David Docherty, R. J. (1996) Br. J Pharmacol. 118,1836-1840 Armstrong.BrianPemno 46 Docherty, R. J., Yeats, J. C., Bevan, S. and Boddeke, H. W. G. M. and PaulKellyfor helpful commentsand critical (1996) Ppiigers Arch. 431,828-837 readingof the 47 Hardwick, J. C. and Parsons, R. L. (1996) 1. Neurophysiol. 76, manuscript.and to thank 3609-3616 BrianPerrinoand Rlchatd 48 DunIap, K., Luebke, J. I. and Turner, T. J. (1995) Trends Neurosci. 18, Twnforprovidmg me 89-98 with manuswptsprior to 49 Armstrong, D. L. (1989) Trends Neurosci. 12,117-122 publication Theauthor’s researchwas supported 50 Levitan, I. B. (1994) Annu. Rev. Physiol. 56,193-212 51 Hosey, M. M., Borsotto, M. and Lazdunski, M. (1986) Pm. Nutl. bythe NIH intramural programme Acad. Sci. U. S. A. 9083,3733-3737
Whereis the locusin opioidwithdrawal? MacDonald J. Christie, John T. Williams, Peregrine B. Osborne and Clare E. Bellchambers Identification
of neuroadaptations
regions that generate withdrawal understanding
in specific brain is crucial for
and perhaps treating opioid dependence.
It has been widely proposed that the locus coeruleus (LC) is the nucleus that plays the primary causal role in the expression of the opioid withdrawal
syndrome.
MacDonald Christie, John Williams, Peregrine Osborne and Glare Bellchambers interpretation
of the literature on which it is based are at
best controversial. M. J. Christie, Associate Professor. Department of Pharmacology, The University of Sydney, NSW 2006. Australia, J. T. Williams, Senior Scientist. Vellum Institute. Oregon Health Sciences University, Portland. OR 97201, USA, P. B. Osborne, Postdoctoral Research MOW.
Department of Physiology and Pharmacology, University of Oueensland. Brisbane. OLD 4072. Australia, and C. E. Bellchambers, Research Student. Department of Pharmacology. The University of Sydney. NSW 2006. Australia.
134
believe that this view and the
Here, they suggest an alternative
view in which regions close to the LC such as the periaqueductal
grey, as well as other brain structures
which are independent
of the LC noradrenergic
system,
play a more important role in the expression of the opioid withdrawal
syndrome.
Dependence on opioid drugs is widely believed to result from neuroadaptations located in multiple neural systems. Identification of adaptive mechanisms occurring in brain regions that generate the expression of withdrawal is central to understanding opioid dependence. Some systems, such as the mesolimbic dopaminergic system, are involved in motivational aspects of dependence such as wanting or craving, while others are thought to mediate aversive components characterized by many of the signs of the opioid withdrawal syndrome’-3. The locus coeruleus (LC) has been the most thoroughly studied of these regions and it has been proposed to play the principal, causal role in the expression of many withdrawal sign+. Indeed, the view that the LC is central to the mechanisms of opioid dependence appears as an
TiPS - April 1997 (Vol. 18)
52 Lai, Y., Peterson, B. 2. and CatteralI, W. A. (1993) I. Neurochem. 61,
1333-1339 53 Imredy, J. I’. and Yue, D. T. (1994) Neuron 12,1301-1318 54 Marrion, N. V. (1996) Neuron 16,163-173 55 Chen, T-c., Law, B., Kondratyuk, T. and Rossie, S. (1995) i. Biol. Chem. 270,7750-7756 56 Momayezi, M. et al. (1987) 1. Cell Bioi. 105, 181-189 57 RenstrGm, E., Ding, W-G., Bokvist, K. and Rorsman, P. (1996) Neuron17,5X3-522 53 Antoni, F. A. et al. (1995) J Biol. Chem. 270,2805~28061 59 Liu, J-P., Sim, A. T. R. and Robinson, P. J. (1994) Science265,97Ck973 60 Robinson, I? J, Liu, J-P., Powell, K. A., Fykse, E. M. and Siidhof, T. C. (1994) Trends Neurosci. 17,348-353 61 Enan, E. and Matsumura, F. (1992) Biochetn. Pharmacol. 43, 1777-1784 62 Hughes, R. L. (1990) Neu, Engi. 1. Med. 323,420-421 63 Lyson, T. et al. (1993) Circ. Res. 73,596-602
orthodoxy in commentaries8 and pharmacological texts9J0.The validity of this hypothesis is an important issue as it underpins a cellular model of withdrawal which is largely based on observations made in the LC. It is our view that the experimental evidence relating to this hypothesis is equivocal and that the emphasis given to the LC has detracted from recognizing the potential of other brain regions and cellular mechanisms as being more likely candidates for initiating and driving the withdrawal response. This review examines the experimental evidence relating the LC to the opioid withdrawal syndrome, particularly several issues that remain controversial. One contentious issue is whether an intact, functioning LC is a requirement for withdrawal, and whether the normal behavioural functions of the LC are consistent with a role in producing signs of opioid withdrawal. Controversies also surround the extent to which withdrawal activation recorded in single LC neurones arises from adaptive changes in the cells themselves or, alternatively, in response to extrinsic afferent input ultimately driven by neurones that are withdrawing from opioids elsewhere in the brain. Conflicting experimental evidence has also raised questions concerning the validity of mechanisms that have been proposed to contribute to withdrawal activation in single LC neurones. Finally, alternative interpretations suggesting involvement of adaptations in other brain regions in the initiation and expression of withdrawal behaviour are considered. Is the LC necessary for the expression of withdrawal behaviour? Studies utilizing microinjections of opioid antagonists into discrete brain regions have implicated several regions in expression of withdrawal signs. This approach has suggested major involvement of the LC and periaquaductal grey (PAG) in expression of withdrawal behaviour, but also the ventral tegmental area, central nucleus of the amygdala, nucleus raphe magnus, hypothalamic nuclei, nucleus accumbens and spinal cord2J1J2.In the most extensive behavioural studies to date, microinjections of methylnaloxonium into the LC
0 1997, Elsevier Science Ltd
SO165-6147(97)01045-6
REVIEW
of conscious, morphine-dependent rats produced a large number of withdrawal signsii; similar results were reported for the LC using naloxonei3. However, microinjection studies are often inconclusive because actions at nearby sites cannot readily be ruled out when high concentrations of antagonists and large injection volumes are used. These limitations can be controlled for, to some extent, if microinjections to regions in the vicinity of the nucleus in question are examined systematically and found to produce negative results. This has not been resolved in the case of the LC. Indeed, Maldonado and co-workers” reported that the next most sensitive region to elicit withdrawal behaviour was the PAG, which is more or less contiguous with the LC. Bozarthn also reported that withdrawal behaviour elicited by systemic naloxone was less severe after chronic microinfusion of morphine in the PAG in close proximity to the LC than in more rostra1 areas of the PAG. It should be noted that the sensitivity of the PAG to opioid receptor antagonists could have been greatly underestimated in some studies because it is an anatomically and functionally heterogeneous region? some subdivisions of the PAG might have been very sensitive to naloxone and others completely insensitive. Therefore, these studies have not clearly demonstrated that the generation of withdrawal behaviour can be localized to the LC as opposed to, or in addition to, nearby regions. Systematic microinjections of opioid receptor antagonists specifically targeted at the LC, discrete functional columns of the nearby PAG and other regions in the vicinity of the LC could resolve this issue. Lesions should provide more direct evidence for a necessary role of a particular region in the expression of withdrawal behaviour. However, the effects of lesions are difficult to interpret if damage to the region is incomplete, or if there is substantial damage to surrounding areas and fibres of passage. These problems are pronounced with electrolytic lesions. Chemical lesions using selective neurotoxins do not suffer the shortcomings of electrolytic lesions because they are capable of destroying discrete neurochemical classes of neurones (or their terminals) with little effect on surrounding neurones or fibres of passage. Chemical lesions of the LC have suggested that there is no involvement of the LC in withdrawal behaviour. Extensive (>95% depletion of cortical noradrenaline) lesions to ascending noradrenergic fibres from the LC using 6-hydroxydopamine injections into the median forebrain bundle failed to attenuate any signs of opioid withdrawall6. This study clearly ruled out involvement of ascending noradrenergic projections from the LC, but did not exclude descending noradrenergic fibres. Another neurotoxin, DSP4 [N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine], produces extensive, selective lesions to all (ascending and descending) noradrenergic terminals which arise from the LC, while sparing fibres arising from other catecholamine-containing cell groupsi7. Treatment with DSP4 prior to induction of dependence failed to attenuate
any withdrawal signs precipitated by systemic naloxonei*. Chronic depletion of catecholamines using other pharmacological methods (6-hydroxydopamine and cl-methyl-para-tyrosine) also failed to inhibit naloxoneprecipitated withdrawali9. These studies suggest that noradrenergic projections arising from the LC are not necessary for the expression of most signs of opioid withdrawal behaviour. Electrolytic lesions of the LC were reported to inhibit many of the signs of opioid withdrawal induced by intracerebroventricular methylnaloxonium2°. The lesions produced -60% depletion of cortical and hippocampal noradrenaline (i.e. they were incomplete). An alternative explanation for inhibition of withdrawal behaviour is that electrolytic lesions destroyed fibres of passage which are important for the expression of withdrawal behaviour, for example, many of the descending fibres which arise from midbrain regions such as the PAG course close to the LC (Ref. 21). Furthermore, medullary adrenergic and noradrenergic fibres which project to the midbrain, and could be important for withdrawal, traverse the lateral boundary of the LC (Ref. 22). It is also possible that the positive effects of electrolytic lesion studies were due to destruction of nearby PAG neurones, or their afferents and efferents. Unfortunately, the effects of lesions in regions in the vicinity of the LC have not yet been systematically examined. Until these controversies can be more thoroughly resolved (e.g. by using discrete excitotoxin lesions in the LC, adjacent PAG and other areas) it would be prudent to conclude that evidence from lesion studies suggest that the LC IIS not directly involved and, therefore, not necessary for the expression of withdrawal behaviour.
Is withdrawal excitation of LC neurones in vim intrinsic, or is it secondary to afferent activity? Excitation of neurones at action potential frequencies exceeding normal basal levels has been considered to be a cellular sign of withdrawal because the acute actions of opioids are generally inhibitory in neurone+. Excessively increased action potential activity following systemic injections of naloxone into dependent animals has been observed in the cerebral cortex24,several hypothalamic nucleizs, the LC (see below), the rostra1 ventrolatera1 medulla (RVLM) including the nucleus reticularis paragigantocellularis26 (PGi) and the dorsal horn of the spinal cord 27.Again, the most thoroughly studied region is the LC where the duration of the withdrawal precipitated by systemic injections of opioid receptor antagonists is correlated with increased action potential activity of LC neurones in viva from control frequencies of <1 Hz up to -5 Hz (Refs 28-32). However, it is difficult to identify whether intrinsic neuroadaptations in the LC caused these effects because intrinsic excitation cannot be isolated from increased activity of afferents after systemic naloxone. Biochemical indices of increased neural activity, such as induction of c-Fos immunoreactivity or mRNA and glucose utilization, have also suggested excitation of the
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REVIEW LC, PAG and many other brain regions during withdrawal, including all of the aforementioned regions*as. These indices provide supportive evidence for excitation of neuronal groups during withdrawal but taken alone do not establish whether the observed activation of many of these regions is involved in either the initiation or expression of withdrawal behaviour, or is perhaps secondary to withdrawal activation of other groups of neurones. The latter complication has been controlled for to some extent in some brain regions by examining c-Fos expression after precipitating withdrawal under anaesthesias3. If withdrawal excitation is due to intrinsic neuroadaptations, that is, if biochemical adaptations within a population of neurones play a role in the initiation of withdrawal, then it should also occur when antagonists are directly applied to neurones in vim. Aghajaniar+ reported a twofold increase in action potential frequency following microiontophoresis of naloxone directly into the LC of dependent animals. In contrast, Akaoka and Aston-Jones32 reported no increase following pressure application of antagonists directly into the nucleus of dependent animals. More recently, the latter authors noted a very modest increase using lower concentrations of methylnaloxone than in previous experiments39. It should be noted that it is very difficult to resolve whether the small activation produced by local injections of opioid receptor antagonists was due to intrinsic neuroadaptations occurring in LC neurones or stimulation of transmitter release from afferents impinging on LC neurones. The discrepancies between the effects of systemic and locally applied antagonists are almost certainly explained by increased activity of afferents to the LC during systemically induced withdrawal. Lesions to the PGi, the major source of excitatory afferents to the LC, profoundly attenuated withdrawal activation of the LC (Ref. 30), as did microinjection of excitatory amino acid antagonists directly into the LC (Ref. 32). Thus, it is not yet clear how much, if any, intrinsic excitation of LC neurones occurs during withdrawal.
Is the proposed role of the LC in withdrawal behaviour consistent with the known physiological functions of the region? It is unclear whether withdrawal behaviour is consistent with established functional roles of the LC in untreated animals. Although early studies40suggested that electrical stimulation of the LC in monkeys mimicked some features of withdrawal behaviour, particularly anxiety, more recent studies have questioned this. The best established behavioural function of the LC is in arousal and vigilance 4143.Phasic activation of LC neurones, often at high frequencies (up to 10 Hz), is strongly associated with alerting and orienting responses in behaving animals 41,@.High tonic discharge frequencies have been associated with vigilance42. These studies suggest that a high discharge frequency of LC neurones need not be associated with behaviours consistent with opioid withdrawal. Indeed, electrical stimulation of the
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LC in humans was also reported to produce a general alerting effect together with a general sense of well being, without any subjective or behavioural manifestations of anxiety or discomforP. It remains possible that these behavioural functions are consistent, to a limited extent, with aspects of opioid withdrawal such as hyperarousal, sensory response deficits41 and perhaps anxiey2, but presumably not with the majority of withdrawal signs expressed by animals.
Is a role for the LC established in vitro? Although the in vivo case for a role of the LC in initiating withdrawal is weak, it might be strengthened if neuroadaptations capable of generating withdrawal (i.e. intrinsic excitation of LC neurones) could be demonstrated in vitro. Nestler and co-worker@ have proposed a model in which LC neurones in morphine-dependent rats undergo withdrawal excitation in response to compensatory changes in CAMP protein phosphorylation pathways. The evidence cited in support of this model is: (1) in brain slices taken from morphine-dependent rats, the frequency of spontaneous action potentials of LC neurones is higher than in control preparations; (2) in control LC neurones spontaneous firing depends on a persistent Na+ current that is activated by a CAMPdependent mechanism and is, therefore, inhibited by opioids; (3) chronic opiate treatment results in an upregulation of the CAMP pathway; (4) dependent slices are more sensitive to increases in the spontaneous firing rate caused by CAMP analogues; and (5) recovery from withdrawal activation parallels the time course of the recovery of the upregulated CAMPpathway when measured in vivo. As discussed below, serious controversies surround the first two of these points which are critical.
Withdrawal excitation of LC neurones in vitro? Electrophysiological studies in brain slices have generally not found any withdrawal excitation of LC neurones [point (1) above]. Neither Andrade et u1.‘r5, using extracellular electrodes, nor Christie et aL46 using intracellular electrodes found withdrawal activation in LC neurones within brain slices. Kogan et aL4’, using extracellular recording, reported an increase of -0.6 Hz in the firing of LC neurones from dependent slices, two to eight hours after slice preparation. The authors explained the discrepancy with earlier studies in terms of the large sample number in their study and the improved quality of slice preparations (but the latter cannot be quantified using extracellular electrodes). Large numbers of neurones were indeed sampled, but a very small number of morphine-treated animals (three) was used. The number of animals sampled is important because the action potential activity of neurones can vary considerably depending on the quality of tissue preparations. More importantly, experiments were not carried out to discriminate intrinsic spontaneous activity from afferent synaptic drive which is present in brain slices, and might have caused the small increases reported.
REVIEW Since the withdrawal-induced excitation of LC neur- measured at -60mV under voltage ~lamps(+~*(i.e. an ones was largely blocked by lesions in the vicinity of the inhibitory action). Secondly, S-Br-S-AMP, an analogue PGi, or by glutamate receptor antagonists in vim, it seems that does not activate protein kinase A, decreased the logical to extend those experiments to the slice prepar- afterhyperpolarization following the action potential, ation. In spite of the fact that most afferents to the LC are thus increasing excitabilityss, suggesting actions of CAMP severed during the preparation of brain slices, sponta- analogues which are independent of protein kinase A. neous release of neurotransmitters continues from termi- Finally, the augmentation of spontaneous neurotransmitnals and can be modulated by opioids@. There are both ter release by agents that increase cAMPdependent pre- and postsynaptic opioid receptor-mediated mecha- mechanisms may result in synaptically driven activity51, nisms that could regulate glutamatergic, or other, neuro- which cannot be resolved in studies of action potential fretransmitter tone. For example, it is known that K opioid quencies. There is, therefore, no consensus on the presence receptors mediate presynaptic inhibition of glutamate or role of a cAMJ?-dependent cation current in the LC. The postulated opioid modulation of a CAMPrelease in the LC (Refs 49,50), but it is not known to what extent this K-receptor-mediated regulation of glutamate dependent cation current in LC neuronesbo [point (2) Opioid receptor agorelease is affected during withdrawal. Postsynaptically, above] has also been challenged61,6*. interactions between CAMP-dependent mechanisms and nists were proposed to increase a K+current and simultaboth AMPA (cY-amino-3-hydroxy-5-methyl-4- isoxazole neously decrease a &MB-dependent cation current, thus, propionic acid) and NMDA (N-methyl-o-aspartate) making the opioid reversal potential more negative than receptors have been reported in other cells51s2.Further- the K+equilibrium potential (Ex).Support for this hypothmore, desensitization of synaptically located NMDA esis came from experiments with BaCl, (to reduce the K+ receptors by a Ca2+-dependent phosphatase (calcineurin) conductance) and partial Na+ substitution (to reduce the was overcome by activation of CAMP-dependent protein cation current)m, although others disputed the interpretakinase (WA) in cultured hippocampal neuroness*. A tion of these experiment@. An alternative interpretation potential consequence of the upregulation of the CAMP is that opioids only increase a K+ conductance but a subsystem after chronic morphine treatment could, therefore, stantial proportion of the current arises from dendrites be an altered postsynaptic sensitivity to synaptically which are poorly controlled in voltage clamp experireleased glutamate. However, altered sensitivity to gluta- ment&@. The most convincing experiment supporting mate may be difficult, or impossible to reliably detect with the latter possibility used iontophoresis to apply opioids the super-fusion and/or pressure application of glutamate close to the soma of impaled neurones, or at more remote in the slice preparation53, making it difficult to resolve pre- site@; when applied at remote sites the reversal potential and postsynaptic effects. Knowledge of the effects of was more negative than E, and similar to that obtained glutamate antagonists on the spontaneous firing of LC with superfusion, but when opioids were applied near the neurones in slices withdrawn from morphine might begin soma the reversal potential was very close to the expected to resolve this issue. Blockers of synaptic transmission, value for E,. such as tetrodotoxin and cadmium, might also resolve Given the controversies surrounding a CAMPwhether naloxone causes a depolarization of LC neurones dependent cation current in the LC and the proposed during withdrawal which is distinct from the simple effect of opioids on this current, it seems premature to reversal of persistent actions of morphine in the tissue. extend the interpretation to an augmented CAMPEvidence supporting the existence of an Na+ current dependent current during morphine withdrawal. The regulated by intracellular levels of CAMPin LC is contro- only experiments cited in support of the CAMPhypothesis versial [point (2) above]. Experiments using whole cell come from an extracellular study47, which showed an patch recording have established that the firing rate of enhanced response of LC neurones to superfusion with SLC neurones can be regulated by manipulations directed Br-&Ml’ during withdrawal. Interpretation of that result, at the CAMP pathway54,s5.Intracellular application of however, did not take into consideration a number of inhibitors of CAMP-dependent processes decreased or potential pre- and postsynaptic sites and mechanisms that abolished activity, whilst CAMPanalogues and other acti- may have been responsible for such an augmented effect vators augmented activityH35.However, from such stud- (see above). Moreover, if an enhanced CAMP-dependent ies of action potential frequency it is not possible to isolate cation current were present during withdrawal, then a the current(s) responsible for the changes in firing. Direct shift in the reversal potential of opioid-induced memevidence for CAMI regulation of an Na+ current has come brane currents should be observed. Contrary to this from studies that have used membrane permeable CAMP possibility, Christie et al.&found no change in the opioidanalogues and stimulators, such as forskolin, in conjunc- induced conductance or reversal potential in LC neurones tion with extracellular and intracellular recordmg47,~,55. following chronic morphine treatment. However, interpretation of such experiments is not at all In conclusion, there are several lines of evidence inconstraightforward and requires consideration of several sistent with the proposed chain of causality linking adapother observations. Firstly, forskolin, in contrast to CAMP tations in LC neurones with the expression of withdrawal analogues, had small and variable effects on spontaneous behaviou~7 (also see Box 1): (1) the proposed mechafiring and most often caused an outward current nisms connecting hypertrophy of the CAMP signalling
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REVIEW Box 1. Are the effects of clonidine evidence for a role of the locus coeruleus in the expression of withdrawal? The ability of cl,-adrenoceptor agonists, especially clonidine, to alleviate withdrawal signs in animals and humans1 has been widely cited as implicating the locus coeruleus (LC) in expression of withdrawal for the following reasons: clonidine acts acutely on a,-adrenoceptors in the LC to produce hyperpoIarization* and inhibit neurotransmitter release3; application of clonidine directly within the LC inhibits action potential activity in animals during withdrawal in vivd. However, this evidence does not prove that a role exists for the LC in expression of withdrawal because inhibition of action potential activity in the LC during withdrawal does not necessarily cause inhibition of the expression of withdrawal behaviour. Clonidine acts on cr,-adrenoceptors in numerous regions of the central and peripheral nervous systems, many of which could be involved in the withdrawal alleviating effects of the drug. cu,-adrenoceptors are widely co-localized with p-opioid receptors6 and produce their effects by a similar mechanism, for example, via opening of the same population of K+ channels’. Moreover, cw2adrenoceptor agonists produce similar actions to p-opioid agonists in other regions implicated in the expression of withdrawal behaviour, including the periaquaductal grey (PAG) (Ref. 8) and dorsal horn of the spinal cords. Indeed, cl,-adrenoceptor agonists also overcome sympathetic nervous system mediated signs of withdrawal when injected intrathecallylo and the excitation of neurones in the PAG during withdrawal
in vitrdl.
Potentially, microinjection studies could resolve
whether or not the LC is crucial for the withdrawal
cascade with excitability of LC neurones are controversial; (2) excitation of LC neurones during withdrawal in vitro is controversial, and much of the withdrawal induced excitation of LC neurones observed in vim following systemic injection of antagonists is driven by afferents; (3) chemical lesions of the LC do not abolish withdrawal behaviour; and (4) many of the somatic signs of withdrawal are not consistent with the known behavioural functions of the LC. Where is the biochemical locus of withdrawal? Rebound of adenylate cyclase activity during withdrawal was first demonstrated by Sharma et al.63and has long been considered a candidate for a role in generating opioid withdrawal (reviewed by Collier, Ref. 64). Although adaptations in this signalling system have consistently been demonstrated in the LC and other region@, they do not appear to be important for cellular mechanisms of opioid withdrawal in the LC. In contrast to the LC, opioids have been shown to affect a variety of ionic conductances in other neurones, and this has clearly been shown to be mediated by inhibition of CAMPformation in at least one case6. Augmented production of CAMP during withdrawal could, therefore, be of importance for initiation of withdrawal behaviour in some groups of
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alleviating actions of clonidine (but see caveats discussed in text). However, direct microinjections of very high concentrations of clonidine (more than five orders of magnitude greater than that required to abolish action potential activity in LC neurones in z&d) within the LC of freely behaving animals inhibited few naloxone precipitated withdrawal signs (diarrhoea, ptosis, weight loss, and wet dog shakes only at the highest doses)‘*. It has, therefore, yet to be resolved whether the withdrawal alleviating effects of clonidine are due to its effects on the LC directly, on regions other than the LC, or on various brain regions which express both a2-adrenoceptors and CL-opioidreceptors. References 1 Redmond, D. E.,Jr andKrystal,J. H. (1984)Annu. Rev. Neurosci. 7,443478 2 Williams,J. T., Henderson,G. and North, R. A. (1985) Neurostience14,95-101 3 Starke, K. and Montel, H. (1973) Neurophu~ucology 12, 1073-1080 4 Aghajanian, G.K. (1978)Nature276,186-188 5 Nicholas,A. P., Hokfelt,T. andPie&one, V. A. (1996)Trends PharmacoZ. Sci. 17,245-2.55 6 Young,W.S. andKuhar,M.J. (1980)Proc.NON. Ad. Sci. U. S. A. 77,1696-1700 7 North,R. A. andWilliams,J. T. (1985)J. Pkysiol.364,2652&l 8 Vaughan,C. W.,Bandler,R.andChristie,M.J. (1996)J. Pkysio2. 490,373-381 9 North,R.A. andYoshimura, M.(1984)J. Physiol. 349,655 10 Buccafi.~~~o, J. J. (1990)BrainRes. 513,8-14 11 chieng,B. andChristie,M.J. (1996)J. Neurosci. 15,712&7136 12 Taylor,J. R., &worth, J. D., Garcia,E. J.* Roth, R. H. and Redmond, D. E.,Jr (1988)Psychopharmacology 96,121-134
neurones, but these have not yet been established. Enhanced cAMI? formation could also mediate indirect actions that result from the metabolism of cAMP to adenosine, followed by adenosine transport into the extracellular space where it acts on adenosine receptors&. Such a mechanism could contribute to withdrawal, in an as yet uncharacterized manner, in some neural circuits. Hypertrophy of the CAMPcascade is probably not the only important adaptation to occur after chronic treatment with opioids. Opioids have been shown to produce excitatory effects via several biochemical cascades such as mobilization of intracellular Ca2+and protein kinase C, as well as several ionic channels including voltage-gated K+ channels and L-type Ca2+ channels67. Adaptations to these mechanisms could be important for the initiation of withdrawal in some groups of neurones, but in most cases they have not been adequately explored in those brain regions thought to be important for the expression of withdrawal behaviour. Where is the anatomical locus of withdrawal? Jf the LC is not involved in the initiation or expression of withdrawal then alternative hypotheses need to be considered. Alternatives should incorporate the unequivocal observation that LC neurones are strongly
REVIEW
Table 1. Evidence for a role of different brain regions in opioid withdrawal Region
Evidence
LC
PAG
PGi
Dorsal horn
PVN
t t _ t
t t ne ne
ne ne ne ne
ne ne ne ne
ne ne ne ne
t f -
ne ne ne
t ne ne
t t ne
t ne +
+ ne -
t t t
ne ne ne
ne ne ne
t ne ne
t t
t
ne
ne
Behavioural Microinjection of antagonists in dependent animals Chronic microinfusion of morphine plus systemic antagonists Chemical lesion of region Electrolytic lesion of region
Electrophysiological Withdrawal Withdrawal Withdrawal
excitation excitation excitation
Electrophysiological
(in vi14 (above control) after systemic antagonist after locally applied antagonists after lesioning afferents
(in vitro)
after antagonists in brain slices Withdrawal excitation in presence of neurotransmission Adaptation of membrane properties identified Withdrawal
excitation
blockers
Biochemical Markers of increased activity (c-fos, glucose Hypertrophy of CAMP cascade Translocation
of protein
utilization)
kinase C
a
t
t
t
t
t
ne
ne
t
ne
Different Indices have implrcated a large number of brain regions in the initiation and expression of opioid withdrawal (see text) but information on the role of many of these regions is limited to one or two measures. The table summarizes whether critical evidence has been obtained and supports (for), negates (against), is equivocal (equiv.) or has not examined (ne) the role of regions that have been most thoroughly studied in opioid withdrawal. t. for; -, against, f, equivocal; LC. locus coeruleus; PAG, periaqueductal grey; PGi. nucleus paragigantocellularis; PVN, hypothalamic paraventricular nucleus. aThis negative result is dubious because, contrary to all other studies (see text), no increase in c-Fos expression was found in this region by the same authors.
activated during withdrawal in uivo, as well as the positive results of some microinjection and lesion studies in the region. It seems reasonable to hypothesise that neuroadaptations develop in afferent systems acting through the PGi to produce withdrawal excitation of LC neurones. Biochemical measures (c-Fos expression) of neuronal activation35 and hypertrophy of the CAMPsignalling cascade4 have been reported to occur in the PGi following chronic morphine treatment. Increased action potential activity in viva of presumed adrenergic (but not other) RVLM neurones has also been reported during withdrawal precipitated by systemic naloxone injection@. However, it is not yet clear whether withdrawal activation in this region is due to neuroadaptations within PGi neurones, or is driven by adaptations in sensory, or possibly other, afferents; one potential source of afferent stimulation is the dorsal horn of the spinal cord. Microinjection of opioid antagonists into the PGi of dependent rats could perhaps resolve this issue. Increased action potential activity in vivo27, c-Fos immunoreactivity38, and adenylate cyclase activity@ in the dorsal horn have been associated with withdrawal. Translocation of protein kinase C has also been suggested as a potential mechanism to account for withdrawal in dorsal horn neurones69. As with the PGi, it is not yet clear whether intrinsic properties of dorsal horn neurones are responsible for initiation of withdrawal, although studies of the effects of intrathecal injections of a,-adrenoceptor agonists suggest that this might be the case. The PAG, which has been implicated in expression of withdrawal (see below) is another source of afferents
to the PGi (Refs 70,71) and could play an indirect role in withdrawal excitation of the LC. Withdrawal-induced excitation of PAG neurones could be responsible for some of the positive effects of microinjection and lesion studies cited above because of its close proximity to the LP. There is considerable evidence that neurones in the caudal part of the ventrolatera1 PAG, which are located within -1mm of the LC, undergo withdrawal excitation both in vivo and in vitro. Microinjection studies suggest involvement of the PAG in expression of withdrawal behaviour (see above). Moreover, the known roles of the PAG in regulation of autonomic and somatic components of defensive behaviour and nociceptive responses’s are generally consistent with a number of signs of withdrawal. Expression of c-Fos is also elevated in the ventrolateral PAG of freely behaving35-37and anaesthetizedsb rats following withdrawal. Withdrawal excitation of PAG neurones occurs in vitro, differs both quantitatively and qualitatively from simple reversal of the acute effects of morphine, is attenuated by cr,-adrenoceptor agonists, and is not affected by blockers of synaptic neurotransmission7*. These results suggest that adaptations within PAG neurones could be responsible for initiation and expression of some withdrawal signs. The precise ionic and biochemical mechanisms underlying these adaptations are not yet clear. Microinjections of the serine/threonine kinase inhibitor, H7, into the PAG attenuated many signs of withdrawal72 which might implicate protein kinases A, C or others in the adaptive process. However, adenylate cyclase activity was reported
not to be elevated in the
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REVIEW PAG during withdrawal@ (but it is not clear which subdivisions were sampled because the same workers, in contrast to all other studies, also reported no activation of c-Fos in the PAG). Although some microinjection studies have suggested that the PAG plays a lesser role than the LC in the expression of withdrawal”, such experiments have ignored the organization of functional columns along the rostrocaudal axis of the PAG. Neurones within the caudal ventrolateral PAG are interconnected with a number of brain regions15 implicated in the expression of withdrawal including amygdaloid nucleiu, the nucleus accumbenW3, several hypothalamic areas25 as well as the PGi (see above). Neuroadaptations in these regions have not yet been intensively studied in cellular physiological and biochemical studies but they are reasonable candidates for involvement in the initiation and expression of withdrawal behaviour. It is possible that much of the expression of withdrawal behaviour arises from neuroadaptations distributed among several elements of neural networks which include and impinge on the PAG, PGi and dorsal horn of the spinal cord. However, specific adaptations that contribute to the expression of withdrawal behaviour in these neurones have not yet been well characterized (see Table 1). Selected references 1 Bozarth, M. A. and Wise, R. A. (1984)Science224,516-517 2 Koob, G. F. and Bloom, F. E. (1988)Science242,715-723 3 Di Chiara, G. and North, R. A. (1992) Trends Pharmcol. Sci. 13, 185-193 4 Nestler, E. J. (1992)I. Neurosci.12,2439 5 Nestler, E. j. (1996)Neuron 16,897-900 6 Nestler, E. I. Hooe. B. T. and Widnell. K. L. (1993) Neuron 11. 995-1006 I’ ’ 7 Self, D. W. and Nestler, E. J. (1995) Annu. Rev. Neurosci. 18, 463-495 8 Hyman, S. E. (1996)Science 273,611-612 9 Rang, H. I’., Dale, M. M. and Ritter, J. M. (1995)Pharmacology (3rd edn), pp. 625-626,Churchill Livingstone 10 Neal, M. J. (1992) Medical Pharmacologyaf a Glance (2nd edn), pp. 64-65, Blackwell Scientific 11 Maldonado, R., Stinus, L., Gold, L. H. and Koob, G. F. (1992) J. Pharmacol. Exp. Ther.261,669-677 12 Calvino, B., Lagowska, J. and Ben-Ari, Y. (1979) Brain Res. 177, 19-34 13 Esposito, E., Kruszewska, A., Ossowaka, G. and Samanin, R. (1987) Psychopharmacology 93,393-396 14 Bozarth, M. A. (1994)Newrosci. Biobehav.Rev. 18,373-383 15 Bandler, R. and Shipley, M. T. (1994)TrendsNeurosci.17,379-389 16 Britton, K. T., Svensson, T., Schwartz, J., Bloom, F. E. and Koob, G. F. (1984)LifeSci. 34,133-139 17 Fritschy, J-M. and Grzanna, R. (1989)Neuroscience30,181-197 18 Chieng, B. and Christie, M. J. (1995)Neurosci.Lett. 186,37-40 19 Blasig, J., Herz, A. and Grams& C. (1975)Naunyn-Schmiedeberg’s Arch. Pharmacol. 286,325-336 20 Maldonado, R. and Koob, G. F. (1993)Brain Res. 605.128-138 21 HolsteEe, G; (1991)Prof. Brain ies. S?, 307-421 22 Herbe;, H. Ad Saper,?. B. (1992)J. Camp.Neural. 315,34-52 23 North, R. A., Williams, 1.T., Surorenant, A. and Christie, M. J. (1987) Proc. fiat2. Acad. Sci. U.-S. A. 8415487-5491 24 Fry, J. P. and Zieglgansberger, W. (1980) Br. J. Pharrnacol. 68, 585-592 25 Russell, J. A., Leng, G. and Bicknell, R. J. (1995)Exptl. Physiol. 80, 307-340 26 Baraban, S. C., Stometta, R. L. and Guyenet, P. G. (1995)Brain Res. 676‘245-257 27 Johnson, S. M. and Duggan, A. W. (1981) Neurophannacology20, 1033-1038 I
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Whereis the locusin opioidwithdrawal? MacDonald J. Christie, John T. Williams, Peregrine B. Osborne and Clare E. Bellchambers Identification
of neuroadaptations
regions that generate withdrawal understanding
in specific brain is crucial for
and perhaps treating opioid dependence.
It has been widely proposed that the locus coeruleus (LC) is the nucleus that plays the primary causal role in the expression of the opioid withdrawal
syndrome.
MacDonald Christie, John Williams, Peregrine Osborne and Glare Bellchambers interpretation
of the literature on which it is based are at
best controversial. M. J. Christie, Associate Professor. Department of Pharmacology, The University of Sydney, NSW 2006. Australia, J. T. Williams, Senior Scientist. Vellum Institute. Oregon Health Sciences University, Portland. OR 97201, USA, P. B. Osborne, Postdoctoral Research MOW.
Department of Physiology and Pharmacology, University of Oueensland. Brisbane. OLD 4072. Australia, and C. E. Bellchambers, Research Student. Department of Pharmacology. The University of Sydney. NSW 2006. Australia.
134
believe that this view and the
Here, they suggest an alternative
view in which regions close to the LC such as the periaqueductal
grey, as well as other brain structures
which are independent
of the LC noradrenergic
system,
play a more important role in the expression of the opioid withdrawal
syndrome.
Dependence on opioid drugs is widely believed to result from neuroadaptations located in multiple neural systems. Identification of adaptive mechanisms occurring in brain regions that generate the expression of withdrawal is central to understanding opioid dependence. Some systems, such as the mesolimbic dopaminergic system, are involved in motivational aspects of dependence such as wanting or craving, while others are thought to mediate aversive components characterized by many of the signs of the opioid withdrawal syndrome’-3. The locus coeruleus (LC) has been the most thoroughly studied of these regions and it has been proposed to play the principal, causal role in the expression of many withdrawal sign+. Indeed, the view that the LC is central to the mechanisms of opioid dependence appears as an
TiPS - April 1997 (Vol. 18)
52 Lai, Y., Peterson, B. 2. and CatteralI, W. A. (1993) I. Neurochem. 61,
1333-1339 53 Imredy, J. I’. and Yue, D. T. (1994) Neuron 12,1301-1318 54 Marrion, N. V. (1996) Neuron 16,163-173 55 Chen, T-c., Law, B., Kondratyuk, T. and Rossie, S. (1995) i. Biol. Chem. 270,7750-7756 56 Momayezi, M. et al. (1987) 1. Cell Bioi. 105, 181-189 57 RenstrGm, E., Ding, W-G., Bokvist, K. and Rorsman, P. (1996) Neuron17,5X3-522 53 Antoni, F. A. et al. (1995) J Biol. Chem. 270,2805~28061 59 Liu, J-P., Sim, A. T. R. and Robinson, P. J. (1994) Science265,97Ck973 60 Robinson, I? J, Liu, J-P., Powell, K. A., Fykse, E. M. and Siidhof, T. C. (1994) Trends Neurosci. 17,348-353 61 Enan, E. and Matsumura, F. (1992) Biochetn. Pharmacol. 43, 1777-1784 62 Hughes, R. L. (1990) Neu, Engi. 1. Med. 323,420-421 63 Lyson, T. et al. (1993) Circ. Res. 73,596-602
orthodoxy in commentaries8 and pharmacological texts9J0.The validity of this hypothesis is an important issue as it underpins a cellular model of withdrawal which is largely based on observations made in the LC. It is our view that the experimental evidence relating to this hypothesis is equivocal and that the emphasis given to the LC has detracted from recognizing the potential of other brain regions and cellular mechanisms as being more likely candidates for initiating and driving the withdrawal response. This review examines the experimental evidence relating the LC to the opioid withdrawal syndrome, particularly several issues that remain controversial. One contentious issue is whether an intact, functioning LC is a requirement for withdrawal, and whether the normal behavioural functions of the LC are consistent with a role in producing signs of opioid withdrawal. Controversies also surround the extent to which withdrawal activation recorded in single LC neurones arises from adaptive changes in the cells themselves or, alternatively, in response to extrinsic afferent input ultimately driven by neurones that are withdrawing from opioids elsewhere in the brain. Conflicting experimental evidence has also raised questions concerning the validity of mechanisms that have been proposed to contribute to withdrawal activation in single LC neurones. Finally, alternative interpretations suggesting involvement of adaptations in other brain regions in the initiation and expression of withdrawal behaviour are considered. Is the LC necessary for the expression of withdrawal behaviour? Studies utilizing microinjections of opioid antagonists into discrete brain regions have implicated several regions in expression of withdrawal signs. This approach has suggested major involvement of the LC and periaquaductal grey (PAG) in expression of withdrawal behaviour, but also the ventral tegmental area, central nucleus of the amygdala, nucleus raphe magnus, hypothalamic nuclei, nucleus accumbens and spinal cord2J1J2.In the most extensive behavioural studies to date, microinjections of methylnaloxonium into the LC
0 1997, Elsevier Science Ltd
SO165-6147(97)01045-6
REVIEW
of conscious, morphine-dependent rats produced a large number of withdrawal signsii; similar results were reported for the LC using naloxonei3. However, microinjection studies are often inconclusive because actions at nearby sites cannot readily be ruled out when high concentrations of antagonists and large injection volumes are used. These limitations can be controlled for, to some extent, if microinjections to regions in the vicinity of the nucleus in question are examined systematically and found to produce negative results. This has not been resolved in the case of the LC. Indeed, Maldonado and co-workers” reported that the next most sensitive region to elicit withdrawal behaviour was the PAG, which is more or less contiguous with the LC. Bozarthn also reported that withdrawal behaviour elicited by systemic naloxone was less severe after chronic microinfusion of morphine in the PAG in close proximity to the LC than in more rostra1 areas of the PAG. It should be noted that the sensitivity of the PAG to opioid receptor antagonists could have been greatly underestimated in some studies because it is an anatomically and functionally heterogeneous region? some subdivisions of the PAG might have been very sensitive to naloxone and others completely insensitive. Therefore, these studies have not clearly demonstrated that the generation of withdrawal behaviour can be localized to the LC as opposed to, or in addition to, nearby regions. Systematic microinjections of opioid receptor antagonists specifically targeted at the LC, discrete functional columns of the nearby PAG and other regions in the vicinity of the LC could resolve this issue. Lesions should provide more direct evidence for a necessary role of a particular region in the expression of withdrawal behaviour. However, the effects of lesions are difficult to interpret if damage to the region is incomplete, or if there is substantial damage to surrounding areas and fibres of passage. These problems are pronounced with electrolytic lesions. Chemical lesions using selective neurotoxins do not suffer the shortcomings of electrolytic lesions because they are capable of destroying discrete neurochemical classes of neurones (or their terminals) with little effect on surrounding neurones or fibres of passage. Chemical lesions of the LC have suggested that there is no involvement of the LC in withdrawal behaviour. Extensive (>95% depletion of cortical noradrenaline) lesions to ascending noradrenergic fibres from the LC using 6-hydroxydopamine injections into the median forebrain bundle failed to attenuate any signs of opioid withdrawall6. This study clearly ruled out involvement of ascending noradrenergic projections from the LC, but did not exclude descending noradrenergic fibres. Another neurotoxin, DSP4 [N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine], produces extensive, selective lesions to all (ascending and descending) noradrenergic terminals which arise from the LC, while sparing fibres arising from other catecholamine-containing cell groupsi7. Treatment with DSP4 prior to induction of dependence failed to attenuate
any withdrawal signs precipitated by systemic naloxonei*. Chronic depletion of catecholamines using other pharmacological methods (6-hydroxydopamine and cl-methyl-para-tyrosine) also failed to inhibit naloxoneprecipitated withdrawali9. These studies suggest that noradrenergic projections arising from the LC are not necessary for the expression of most signs of opioid withdrawal behaviour. Electrolytic lesions of the LC were reported to inhibit many of the signs of opioid withdrawal induced by intracerebroventricular methylnaloxonium2°. The lesions produced -60% depletion of cortical and hippocampal noradrenaline (i.e. they were incomplete). An alternative explanation for inhibition of withdrawal behaviour is that electrolytic lesions destroyed fibres of passage which are important for the expression of withdrawal behaviour, for example, many of the descending fibres which arise from midbrain regions such as the PAG course close to the LC (Ref. 21). Furthermore, medullary adrenergic and noradrenergic fibres which project to the midbrain, and could be important for withdrawal, traverse the lateral boundary of the LC (Ref. 22). It is also possible that the positive effects of electrolytic lesion studies were due to destruction of nearby PAG neurones, or their afferents and efferents. Unfortunately, the effects of lesions in regions in the vicinity of the LC have not yet been systematically examined. Until these controversies can be more thoroughly resolved (e.g. by using discrete excitotoxin lesions in the LC, adjacent PAG and other areas) it would be prudent to conclude that evidence from lesion studies suggest that the LC IIS not directly involved and, therefore, not necessary for the expression of withdrawal behaviour.
Is withdrawal excitation of LC neurones in vim intrinsic, or is it secondary to afferent activity? Excitation of neurones at action potential frequencies exceeding normal basal levels has been considered to be a cellular sign of withdrawal because the acute actions of opioids are generally inhibitory in neurone+. Excessively increased action potential activity following systemic injections of naloxone into dependent animals has been observed in the cerebral cortex24,several hypothalamic nucleizs, the LC (see below), the rostra1 ventrolatera1 medulla (RVLM) including the nucleus reticularis paragigantocellularis26 (PGi) and the dorsal horn of the spinal cord 27.Again, the most thoroughly studied region is the LC where the duration of the withdrawal precipitated by systemic injections of opioid receptor antagonists is correlated with increased action potential activity of LC neurones in viva from control frequencies of <1 Hz up to -5 Hz (Refs 28-32). However, it is difficult to identify whether intrinsic neuroadaptations in the LC caused these effects because intrinsic excitation cannot be isolated from increased activity of afferents after systemic naloxone. Biochemical indices of increased neural activity, such as induction of c-Fos immunoreactivity or mRNA and glucose utilization, have also suggested excitation of the
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1997 (Vol. 18)
1 3 5
REVIEW LC, PAG and many other brain regions during withdrawal, including all of the aforementioned regions*as. These indices provide supportive evidence for excitation of neuronal groups during withdrawal but taken alone do not establish whether the observed activation of many of these regions is involved in either the initiation or expression of withdrawal behaviour, or is perhaps secondary to withdrawal activation of other groups of neurones. The latter complication has been controlled for to some extent in some brain regions by examining c-Fos expression after precipitating withdrawal under anaesthesias3. If withdrawal excitation is due to intrinsic neuroadaptations, that is, if biochemical adaptations within a population of neurones play a role in the initiation of withdrawal, then it should also occur when antagonists are directly applied to neurones in vim. Aghajaniar+ reported a twofold increase in action potential frequency following microiontophoresis of naloxone directly into the LC of dependent animals. In contrast, Akaoka and Aston-Jones32 reported no increase following pressure application of antagonists directly into the nucleus of dependent animals. More recently, the latter authors noted a very modest increase using lower concentrations of methylnaloxone than in previous experiments39. It should be noted that it is very difficult to resolve whether the small activation produced by local injections of opioid receptor antagonists was due to intrinsic neuroadaptations occurring in LC neurones or stimulation of transmitter release from afferents impinging on LC neurones. The discrepancies between the effects of systemic and locally applied antagonists are almost certainly explained by increased activity of afferents to the LC during systemically induced withdrawal. Lesions to the PGi, the major source of excitatory afferents to the LC, profoundly attenuated withdrawal activation of the LC (Ref. 30), as did microinjection of excitatory amino acid antagonists directly into the LC (Ref. 32). Thus, it is not yet clear how much, if any, intrinsic excitation of LC neurones occurs during withdrawal.
Is the proposed role of the LC in withdrawal behaviour consistent with the known physiological functions of the region? It is unclear whether withdrawal behaviour is consistent with established functional roles of the LC in untreated animals. Although early studies40suggested that electrical stimulation of the LC in monkeys mimicked some features of withdrawal behaviour, particularly anxiety, more recent studies have questioned this. The best established behavioural function of the LC is in arousal and vigilance 4143.Phasic activation of LC neurones, often at high frequencies (up to 10 Hz), is strongly associated with alerting and orienting responses in behaving animals 41,@.High tonic discharge frequencies have been associated with vigilance42. These studies suggest that a high discharge frequency of LC neurones need not be associated with behaviours consistent with opioid withdrawal. Indeed, electrical stimulation of the
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Tip.5 -April
1997 (Vol. 18)
LC in humans was also reported to produce a general alerting effect together with a general sense of well being, without any subjective or behavioural manifestations of anxiety or discomforP. It remains possible that these behavioural functions are consistent, to a limited extent, with aspects of opioid withdrawal such as hyperarousal, sensory response deficits41 and perhaps anxiey2, but presumably not with the majority of withdrawal signs expressed by animals.
Is a role for the LC established in vitro? Although the in vivo case for a role of the LC in initiating withdrawal is weak, it might be strengthened if neuroadaptations capable of generating withdrawal (i.e. intrinsic excitation of LC neurones) could be demonstrated in vitro. Nestler and co-worker@ have proposed a model in which LC neurones in morphine-dependent rats undergo withdrawal excitation in response to compensatory changes in CAMP protein phosphorylation pathways. The evidence cited in support of this model is: (1) in brain slices taken from morphine-dependent rats, the frequency of spontaneous action potentials of LC neurones is higher than in control preparations; (2) in control LC neurones spontaneous firing depends on a persistent Na+ current that is activated by a CAMPdependent mechanism and is, therefore, inhibited by opioids; (3) chronic opiate treatment results in an upregulation of the CAMP pathway; (4) dependent slices are more sensitive to increases in the spontaneous firing rate caused by CAMP analogues; and (5) recovery from withdrawal activation parallels the time course of the recovery of the upregulated CAMPpathway when measured in vivo. As discussed below, serious controversies surround the first two of these points which are critical.
Withdrawal excitation of LC neurones in vitro? Electrophysiological studies in brain slices have generally not found any withdrawal excitation of LC neurones [point (1) above]. Neither Andrade et u1.‘r5, using extracellular electrodes, nor Christie et aL46 using intracellular electrodes found withdrawal activation in LC neurones within brain slices. Kogan et aL4’, using extracellular recording, reported an increase of -0.6 Hz in the firing of LC neurones from dependent slices, two to eight hours after slice preparation. The authors explained the discrepancy with earlier studies in terms of the large sample number in their study and the improved quality of slice preparations (but the latter cannot be quantified using extracellular electrodes). Large numbers of neurones were indeed sampled, but a very small number of morphine-treated animals (three) was used. The number of animals sampled is important because the action potential activity of neurones can vary considerably depending on the quality of tissue preparations. More importantly, experiments were not carried out to discriminate intrinsic spontaneous activity from afferent synaptic drive which is present in brain slices, and might have caused the small increases reported.
REVIEW Since the withdrawal-induced excitation of LC neur- measured at -60mV under voltage ~lamps(+~*(i.e. an ones was largely blocked by lesions in the vicinity of the inhibitory action). Secondly, S-Br-S-AMP, an analogue PGi, or by glutamate receptor antagonists in vim, it seems that does not activate protein kinase A, decreased the logical to extend those experiments to the slice prepar- afterhyperpolarization following the action potential, ation. In spite of the fact that most afferents to the LC are thus increasing excitabilityss, suggesting actions of CAMP severed during the preparation of brain slices, sponta- analogues which are independent of protein kinase A. neous release of neurotransmitters continues from termi- Finally, the augmentation of spontaneous neurotransmitnals and can be modulated by opioids@. There are both ter release by agents that increase cAMPdependent pre- and postsynaptic opioid receptor-mediated mecha- mechanisms may result in synaptically driven activity51, nisms that could regulate glutamatergic, or other, neuro- which cannot be resolved in studies of action potential fretransmitter tone. For example, it is known that K opioid quencies. There is, therefore, no consensus on the presence receptors mediate presynaptic inhibition of glutamate or role of a cAMJ?-dependent cation current in the LC. The postulated opioid modulation of a CAMPrelease in the LC (Refs 49,50), but it is not known to what extent this K-receptor-mediated regulation of glutamate dependent cation current in LC neuronesbo [point (2) Opioid receptor agorelease is affected during withdrawal. Postsynaptically, above] has also been challenged61,6*. interactions between CAMP-dependent mechanisms and nists were proposed to increase a K+current and simultaboth AMPA (cY-amino-3-hydroxy-5-methyl-4- isoxazole neously decrease a &MB-dependent cation current, thus, propionic acid) and NMDA (N-methyl-o-aspartate) making the opioid reversal potential more negative than receptors have been reported in other cells51s2.Further- the K+equilibrium potential (Ex).Support for this hypothmore, desensitization of synaptically located NMDA esis came from experiments with BaCl, (to reduce the K+ receptors by a Ca2+-dependent phosphatase (calcineurin) conductance) and partial Na+ substitution (to reduce the was overcome by activation of CAMP-dependent protein cation current)m, although others disputed the interpretakinase (WA) in cultured hippocampal neuroness*. A tion of these experiment@. An alternative interpretation potential consequence of the upregulation of the CAMP is that opioids only increase a K+ conductance but a subsystem after chronic morphine treatment could, therefore, stantial proportion of the current arises from dendrites be an altered postsynaptic sensitivity to synaptically which are poorly controlled in voltage clamp experireleased glutamate. However, altered sensitivity to gluta- ment&@. The most convincing experiment supporting mate may be difficult, or impossible to reliably detect with the latter possibility used iontophoresis to apply opioids the super-fusion and/or pressure application of glutamate close to the soma of impaled neurones, or at more remote in the slice preparation53, making it difficult to resolve pre- site@; when applied at remote sites the reversal potential and postsynaptic effects. Knowledge of the effects of was more negative than E, and similar to that obtained glutamate antagonists on the spontaneous firing of LC with superfusion, but when opioids were applied near the neurones in slices withdrawn from morphine might begin soma the reversal potential was very close to the expected to resolve this issue. Blockers of synaptic transmission, value for E,. such as tetrodotoxin and cadmium, might also resolve Given the controversies surrounding a CAMPwhether naloxone causes a depolarization of LC neurones dependent cation current in the LC and the proposed during withdrawal which is distinct from the simple effect of opioids on this current, it seems premature to reversal of persistent actions of morphine in the tissue. extend the interpretation to an augmented CAMPEvidence supporting the existence of an Na+ current dependent current during morphine withdrawal. The regulated by intracellular levels of CAMPin LC is contro- only experiments cited in support of the CAMPhypothesis versial [point (2) above]. Experiments using whole cell come from an extracellular study47, which showed an patch recording have established that the firing rate of enhanced response of LC neurones to superfusion with SLC neurones can be regulated by manipulations directed Br-&Ml’ during withdrawal. Interpretation of that result, at the CAMP pathway54,s5.Intracellular application of however, did not take into consideration a number of inhibitors of CAMP-dependent processes decreased or potential pre- and postsynaptic sites and mechanisms that abolished activity, whilst CAMPanalogues and other acti- may have been responsible for such an augmented effect vators augmented activityH35.However, from such stud- (see above). Moreover, if an enhanced CAMP-dependent ies of action potential frequency it is not possible to isolate cation current were present during withdrawal, then a the current(s) responsible for the changes in firing. Direct shift in the reversal potential of opioid-induced memevidence for CAMI regulation of an Na+ current has come brane currents should be observed. Contrary to this from studies that have used membrane permeable CAMP possibility, Christie et al.&found no change in the opioidanalogues and stimulators, such as forskolin, in conjunc- induced conductance or reversal potential in LC neurones tion with extracellular and intracellular recordmg47,~,55. following chronic morphine treatment. However, interpretation of such experiments is not at all In conclusion, there are several lines of evidence inconstraightforward and requires consideration of several sistent with the proposed chain of causality linking adapother observations. Firstly, forskolin, in contrast to CAMP tations in LC neurones with the expression of withdrawal analogues, had small and variable effects on spontaneous behaviou~7 (also see Box 1): (1) the proposed mechafiring and most often caused an outward current nisms connecting hypertrophy of the CAMP signalling
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REVIEW Box 1. Are the effects of clonidine evidence for a role of the locus coeruleus in the expression of withdrawal? The ability of cl,-adrenoceptor agonists, especially clonidine, to alleviate withdrawal signs in animals and humans1 has been widely cited as implicating the locus coeruleus (LC) in expression of withdrawal for the following reasons: clonidine acts acutely on a,-adrenoceptors in the LC to produce hyperpoIarization* and inhibit neurotransmitter release3; application of clonidine directly within the LC inhibits action potential activity in animals during withdrawal in vivd. However, this evidence does not prove that a role exists for the LC in expression of withdrawal because inhibition of action potential activity in the LC during withdrawal does not necessarily cause inhibition of the expression of withdrawal behaviour. Clonidine acts on cr,-adrenoceptors in numerous regions of the central and peripheral nervous systems, many of which could be involved in the withdrawal alleviating effects of the drug. cu,-adrenoceptors are widely co-localized with p-opioid receptors6 and produce their effects by a similar mechanism, for example, via opening of the same population of K+ channels’. Moreover, cw2adrenoceptor agonists produce similar actions to p-opioid agonists in other regions implicated in the expression of withdrawal behaviour, including the periaquaductal grey (PAG) (Ref. 8) and dorsal horn of the spinal cords. Indeed, cl,-adrenoceptor agonists also overcome sympathetic nervous system mediated signs of withdrawal when injected intrathecallylo and the excitation of neurones in the PAG during withdrawal
in vitrdl.
Potentially, microinjection studies could resolve
whether or not the LC is crucial for the withdrawal
cascade with excitability of LC neurones are controversial; (2) excitation of LC neurones during withdrawal in vitro is controversial, and much of the withdrawal induced excitation of LC neurones observed in vim following systemic injection of antagonists is driven by afferents; (3) chemical lesions of the LC do not abolish withdrawal behaviour; and (4) many of the somatic signs of withdrawal are not consistent with the known behavioural functions of the LC. Where is the biochemical locus of withdrawal? Rebound of adenylate cyclase activity during withdrawal was first demonstrated by Sharma et al.63and has long been considered a candidate for a role in generating opioid withdrawal (reviewed by Collier, Ref. 64). Although adaptations in this signalling system have consistently been demonstrated in the LC and other region@, they do not appear to be important for cellular mechanisms of opioid withdrawal in the LC. In contrast to the LC, opioids have been shown to affect a variety of ionic conductances in other neurones, and this has clearly been shown to be mediated by inhibition of CAMPformation in at least one case6. Augmented production of CAMP during withdrawal could, therefore, be of importance for initiation of withdrawal behaviour in some groups of
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alleviating actions of clonidine (but see caveats discussed in text). However, direct microinjections of very high concentrations of clonidine (more than five orders of magnitude greater than that required to abolish action potential activity in LC neurones in z&d) within the LC of freely behaving animals inhibited few naloxone precipitated withdrawal signs (diarrhoea, ptosis, weight loss, and wet dog shakes only at the highest doses)‘*. It has, therefore, yet to be resolved whether the withdrawal alleviating effects of clonidine are due to its effects on the LC directly, on regions other than the LC, or on various brain regions which express both a2-adrenoceptors and CL-opioidreceptors. References 1 Redmond, D. E.,Jr andKrystal,J. H. (1984)Annu. Rev. Neurosci. 7,443478 2 Williams,J. T., Henderson,G. and North, R. A. (1985) Neurostience14,95-101 3 Starke, K. and Montel, H. (1973) Neurophu~ucology 12, 1073-1080 4 Aghajanian, G.K. (1978)Nature276,186-188 5 Nicholas,A. P., Hokfelt,T. andPie&one, V. A. (1996)Trends PharmacoZ. Sci. 17,245-2.55 6 Young,W.S. andKuhar,M.J. (1980)Proc.NON. Ad. Sci. U. S. A. 77,1696-1700 7 North,R. A. andWilliams,J. T. (1985)J. Pkysiol.364,2652&l 8 Vaughan,C. W.,Bandler,R.andChristie,M.J. (1996)J. Pkysio2. 490,373-381 9 North,R.A. andYoshimura, M.(1984)J. Physiol. 349,655 10 Buccafi.~~~o, J. J. (1990)BrainRes. 513,8-14 11 chieng,B. andChristie,M.J. (1996)J. Neurosci. 15,712&7136 12 Taylor,J. R., &worth, J. D., Garcia,E. J.* Roth, R. H. and Redmond, D. E.,Jr (1988)Psychopharmacology 96,121-134
neurones, but these have not yet been established. Enhanced cAMI? formation could also mediate indirect actions that result from the metabolism of cAMP to adenosine, followed by adenosine transport into the extracellular space where it acts on adenosine receptors&. Such a mechanism could contribute to withdrawal, in an as yet uncharacterized manner, in some neural circuits. Hypertrophy of the CAMPcascade is probably not the only important adaptation to occur after chronic treatment with opioids. Opioids have been shown to produce excitatory effects via several biochemical cascades such as mobilization of intracellular Ca2+and protein kinase C, as well as several ionic channels including voltage-gated K+ channels and L-type Ca2+ channels67. Adaptations to these mechanisms could be important for the initiation of withdrawal in some groups of neurones, but in most cases they have not been adequately explored in those brain regions thought to be important for the expression of withdrawal behaviour. Where is the anatomical locus of withdrawal? Jf the LC is not involved in the initiation or expression of withdrawal then alternative hypotheses need to be considered. Alternatives should incorporate the unequivocal observation that LC neurones are strongly
REVIEW
Table 1. Evidence for a role of different brain regions in opioid withdrawal Region
Evidence
LC
PAG
PGi
Dorsal horn
PVN
t t _ t
t t ne ne
ne ne ne ne
ne ne ne ne
ne ne ne ne
t f -
ne ne ne
t ne ne
t t ne
t ne +
+ ne -
t t t
ne ne ne
ne ne ne
t ne ne
t t
t
ne
ne
Behavioural Microinjection of antagonists in dependent animals Chronic microinfusion of morphine plus systemic antagonists Chemical lesion of region Electrolytic lesion of region
Electrophysiological Withdrawal Withdrawal Withdrawal
excitation excitation excitation
Electrophysiological
(in vi14 (above control) after systemic antagonist after locally applied antagonists after lesioning afferents
(in vitro)
after antagonists in brain slices Withdrawal excitation in presence of neurotransmission Adaptation of membrane properties identified Withdrawal
excitation
blockers
Biochemical Markers of increased activity (c-fos, glucose Hypertrophy of CAMP cascade Translocation
of protein
utilization)
kinase C
a
t
t
t
t
t
ne
ne
t
ne
Different Indices have implrcated a large number of brain regions in the initiation and expression of opioid withdrawal (see text) but information on the role of many of these regions is limited to one or two measures. The table summarizes whether critical evidence has been obtained and supports (for), negates (against), is equivocal (equiv.) or has not examined (ne) the role of regions that have been most thoroughly studied in opioid withdrawal. t. for; -, against, f, equivocal; LC. locus coeruleus; PAG, periaqueductal grey; PGi. nucleus paragigantocellularis; PVN, hypothalamic paraventricular nucleus. aThis negative result is dubious because, contrary to all other studies (see text), no increase in c-Fos expression was found in this region by the same authors.
activated during withdrawal in uivo, as well as the positive results of some microinjection and lesion studies in the region. It seems reasonable to hypothesise that neuroadaptations develop in afferent systems acting through the PGi to produce withdrawal excitation of LC neurones. Biochemical measures (c-Fos expression) of neuronal activation35 and hypertrophy of the CAMPsignalling cascade4 have been reported to occur in the PGi following chronic morphine treatment. Increased action potential activity in viva of presumed adrenergic (but not other) RVLM neurones has also been reported during withdrawal precipitated by systemic naloxone injection@. However, it is not yet clear whether withdrawal activation in this region is due to neuroadaptations within PGi neurones, or is driven by adaptations in sensory, or possibly other, afferents; one potential source of afferent stimulation is the dorsal horn of the spinal cord. Microinjection of opioid antagonists into the PGi of dependent rats could perhaps resolve this issue. Increased action potential activity in vivo27, c-Fos immunoreactivity38, and adenylate cyclase activity@ in the dorsal horn have been associated with withdrawal. Translocation of protein kinase C has also been suggested as a potential mechanism to account for withdrawal in dorsal horn neurones69. As with the PGi, it is not yet clear whether intrinsic properties of dorsal horn neurones are responsible for initiation of withdrawal, although studies of the effects of intrathecal injections of a,-adrenoceptor agonists suggest that this might be the case. The PAG, which has been implicated in expression of withdrawal (see below) is another source of afferents
to the PGi (Refs 70,71) and could play an indirect role in withdrawal excitation of the LC. Withdrawal-induced excitation of PAG neurones could be responsible for some of the positive effects of microinjection and lesion studies cited above because of its close proximity to the LP. There is considerable evidence that neurones in the caudal part of the ventrolatera1 PAG, which are located within -1mm of the LC, undergo withdrawal excitation both in vivo and in vitro. Microinjection studies suggest involvement of the PAG in expression of withdrawal behaviour (see above). Moreover, the known roles of the PAG in regulation of autonomic and somatic components of defensive behaviour and nociceptive responses’s are generally consistent with a number of signs of withdrawal. Expression of c-Fos is also elevated in the ventrolateral PAG of freely behaving35-37and anaesthetizedsb rats following withdrawal. Withdrawal excitation of PAG neurones occurs in vitro, differs both quantitatively and qualitatively from simple reversal of the acute effects of morphine, is attenuated by cr,-adrenoceptor agonists, and is not affected by blockers of synaptic neurotransmission7*. These results suggest that adaptations within PAG neurones could be responsible for initiation and expression of some withdrawal signs. The precise ionic and biochemical mechanisms underlying these adaptations are not yet clear. Microinjections of the serine/threonine kinase inhibitor, H7, into the PAG attenuated many signs of withdrawal72 which might implicate protein kinases A, C or others in the adaptive process. However, adenylate cyclase activity was reported
not to be elevated in the
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REVIEW PAG during withdrawal@ (but it is not clear which subdivisions were sampled because the same workers, in contrast to all other studies, also reported no activation of c-Fos in the PAG). Although some microinjection studies have suggested that the PAG plays a lesser role than the LC in the expression of withdrawal”, such experiments have ignored the organization of functional columns along the rostrocaudal axis of the PAG. Neurones within the caudal ventrolateral PAG are interconnected with a number of brain regions15 implicated in the expression of withdrawal including amygdaloid nucleiu, the nucleus accumbenW3, several hypothalamic areas25 as well as the PGi (see above). Neuroadaptations in these regions have not yet been intensively studied in cellular physiological and biochemical studies but they are reasonable candidates for involvement in the initiation and expression of withdrawal behaviour. It is possible that much of the expression of withdrawal behaviour arises from neuroadaptations distributed among several elements of neural networks which include and impinge on the PAG, PGi and dorsal horn of the spinal cord. However, specific adaptations that contribute to the expression of withdrawal behaviour in these neurones have not yet been well characterized (see Table 1). Selected references 1 Bozarth, M. A. and Wise, R. A. (1984)Science224,516-517 2 Koob, G. F. and Bloom, F. E. (1988)Science242,715-723 3 Di Chiara, G. and North, R. A. (1992) Trends Pharmcol. Sci. 13, 185-193 4 Nestler, E. J. (1992)I. Neurosci.12,2439 5 Nestler, E. j. (1996)Neuron 16,897-900 6 Nestler, E. I. Hooe. B. T. and Widnell. K. L. (1993) Neuron 11. 995-1006 I’ ’ 7 Self, D. W. and Nestler, E. J. (1995) Annu. Rev. Neurosci. 18, 463-495 8 Hyman, S. E. (1996)Science 273,611-612 9 Rang, H. I’., Dale, M. M. and Ritter, J. M. (1995)Pharmacology (3rd edn), pp. 625-626,Churchill Livingstone 10 Neal, M. J. (1992) Medical Pharmacologyaf a Glance (2nd edn), pp. 64-65, Blackwell Scientific 11 Maldonado, R., Stinus, L., Gold, L. H. and Koob, G. F. (1992) J. Pharmacol. Exp. Ther.261,669-677 12 Calvino, B., Lagowska, J. and Ben-Ari, Y. (1979) Brain Res. 177, 19-34 13 Esposito, E., Kruszewska, A., Ossowaka, G. and Samanin, R. (1987) Psychopharmacology 93,393-396 14 Bozarth, M. A. (1994)Newrosci. Biobehav.Rev. 18,373-383 15 Bandler, R. and Shipley, M. T. (1994)TrendsNeurosci.17,379-389 16 Britton, K. T., Svensson, T., Schwartz, J., Bloom, F. E. and Koob, G. F. (1984)LifeSci. 34,133-139 17 Fritschy, J-M. and Grzanna, R. (1989)Neuroscience30,181-197 18 Chieng, B. and Christie, M. J. (1995)Neurosci.Lett. 186,37-40 19 Blasig, J., Herz, A. and Grams& C. (1975)Naunyn-Schmiedeberg’s Arch. Pharmacol. 286,325-336 20 Maldonado, R. and Koob, G. F. (1993)Brain Res. 605.128-138 21 HolsteEe, G; (1991)Prof. Brain ies. S?, 307-421 22 Herbe;, H. Ad Saper,?. B. (1992)J. Camp.Neural. 315,34-52 23 North, R. A., Williams, 1.T., Surorenant, A. and Christie, M. J. (1987) Proc. fiat2. Acad. Sci. U.-S. A. 8415487-5491 24 Fry, J. P. and Zieglgansberger, W. (1980) Br. J. Pharrnacol. 68, 585-592 25 Russell, J. A., Leng, G. and Bicknell, R. J. (1995)Exptl. Physiol. 80, 307-340 26 Baraban, S. C., Stometta, R. L. and Guyenet, P. G. (1995)Brain Res. 676‘245-257 27 Johnson, S. M. and Duggan, A. W. (1981) Neurophannacology20, 1033-1038 I
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