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Peripheral withdrawal recruits distinct central nuclei in morphine-dependent rats
Neuropharmacology 41 (2001) 574–581 www.elsevier.com/locate/neuropharm
Peripheral withdrawal recruits distinct central nuclei in morphinedependent rats A. Hamlin a, K.M. Buller a, T.A. Day a, P.B. Osborne b
a, b,*
a Department of Physiology and Pharmacology, The University of Queensland, Brisbane Qld 4072, Australia Prince of Wales Medical Research Institute, University of New South Wales, Barker Street, Randwick NSW 2031, Sydney, Australia
Received 6 October 2000; received in revised form 18 May 2001; accepted 25 June 2001
Abstract This study examined if brain pathways in morphine-dependent rats are activated by opioid withdrawal precipitated outside the central nervous system. Withdrawal precipitated with a peripherally acting quaternary opioid antagonist (naloxone methiodide) increased Fos expression but caused a more restricted pattern of neuronal activation than systemic withdrawal (precipitated with naloxone which enters the brain). There was no effect on locus coeruleus and significantly smaller increases in Fos neurons were produced in most other areas. However in the ventrolateral medulla (A1/C1 catecholamine neurons), nucleus of the solitary tract (A2/C2 catecholamine neurons), lateral parabrachial nucleus, supramamillary nucleus, bed nucleus of the stria terminalis, accumbens core and medial prefrontal cortex no differences in the withdrawal treatments were detected. We have shown that peripheral opioid withdrawal can affect central nervous system pathways. Crown Copyright 2001 Published by Elsevier Science Ltd. All rights reserved. Keywords: Morphine dependence; Substance withdrawal syndrome; Naloxone methiodide; Proto-oncogene proteins c-fos
1. Introduction Opioid withdrawal can be precipitated peripherally in rodents by injecting morphine-dependent animals with hydrophilic opioid antagonists that effectively do not cross the blood-brain barrier (Brown and Goldberg, 1985). This produces gut hypermotility, secretomotor stimulation (diarrhoea, lacrimation, rhinorhoea and salivation) and other visceral responses but virtually none of the physical signs that are precipitated when opioid antagonists are injected into the brain (Schulteis and Koob, 1996). ‘Peripheral withdrawal’ also does not have the aversive behavioural effects that are elicited by systemic withdrawal (precipitated with antagonists that enter the brain) such as reductions in spontaneous locomotion, suppression of operant responding and conditioned place aversions (Hand et al., 1988; Mucha, 1989).
* Corresponding author. Tel.: +612-9382-7934; fax: +612-93822722. E-mail address: [email protected] (P.B. Osborne).
Based on these whole animal studies it could be assumed that central nervous system pathways are not activated by peripheral opioid withdrawal. However it has been reported recently that peripheral withdrawal can activate the dorsal horn of the rat sacral spinal cord, causing an increase in neurons that express the inducible transcription factor c-fos (Rohde et al., 1997a). In the brain, systemic opioid withdrawal markedly increases cfos and ‘acute’ Fos-related antigens (FRAs; e.g. Fos B, FRA-1 and FRA-2)(Nestler and Aghajanian, 1997; Harlan and Garcia, 1998) but the extent to which these inducible transcription factors are affected by peripheral withdrawal has not been determined. In this study, we have addressed this question by using the hydrophilic quaternary opioid antagonist naloxone methiodide to precipitate withdrawal in morphine-dependent rats. We demonstrate that peripheral withdrawal significantly increases Fos-immunoreactive (Fos-IR) neurons in many brain areas, and the pattern of recruitment is distinct from that seen when opioid withdrawal is precipitated systemically.
0028-3908/01/$ - see front matter. Crown Copyright 2001 Published by Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 8 - 3 9 0 8 ( 0 1 ) 0 0 1 0 1 - 0
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2. Methods Adult male Wistar rats (350–500 g) were used in all of the experiments. Two types of pretreatment (vehicle or morphine) were combined with three types of withdrawal treatment (saline, naloxone methiodide and naloxone). This gave six experimental groups that included four control groups (vehicle/saline, n=4; vehicle/naloxone methiodide, n=4; vehicle/naloxone, n=7; and morphine/saline, n=4), a peripheral withdrawal group (morphine/naloxone methiodide, n=7) and a systemic withdrawal group (morphine/naloxone, n=8). The treatment schedules were administered to batches of four rats in no particular sequence. The pretreatment consisted of three subcutaneous injections (48 h apart) of a slow release vehicle ± morphine HCl (50 mg/kg, Sigma pharmaceuticals). The vehicle was an emulsion of 42.5% paraffin oil and 7.5% mannide manooleate (Sigma-Aldrich) in a NaCl solution (0.9%) containing 311 nM NaOH to which 25 mg/ml of morphine HCl was added. The emulsion was warmed to 35°C and 2 ml/kg was injected subcutaneously. Then 48 h after the final pretreatment injection, vehicle or morphine treated rats were administered a withdrawal injection of normal saline, naloxone methiodide (5 mg/kg s.c.; Research Biochemicals Inc.) or naloxone (5 mg/kg s.c., SigmaAldrich). Withdrawal injections were administered to each rat in a batch of four at 30 min intervals from 10 a.m. onwards. The animals were withdrawn in their individual home cage and then over a 15 min period were monitored for the following opioid withdrawal signs: diarrhoea, wet dog shakes, teeth chattering, ptosis, and rearing or digging. About 2 h after precipitating withdrawal, rats were deeply anaesthetised (sodium pentobarbitone 50 mg/kg i.p.) and transcardially perfused with 2% sodium nitrite solution followed by 4% formaldehyde in 0.1 M phosphate buffer (pH 7.4). The brains were then removed postfixed and processed for immunohistochemistry using a dual immunoperoxidase technique described previously (Smith and Day, 1993). Brains taken from concurrently treated batches of rats were processed simultaneously. A 1:4 series of forebrain sections were processed for Fos (1:100,000 for 48 h; rabbit polyclonal antibody, Santa Cruz Biotechnology) and a 1:5 series of midbrain and hindbrain sections were processed for Fos and tyrosine-hydroxylase (1:25,000; mouse polyclonal antibody, Incstar). Unilateral counts of Fos-IR neurons were made in coronal sections spaced at 160 µm intervals in cortex, forebrain and hypothalamus, and at 250 µm intervals in midbrain, pons and medulla. Equality of the six group means in each structure was compared using a one-way analysis of variance. Unplanned comparisons among pairs of group means were made using the Tukey HSD test for unequal sample sizes, or using the Games–Howell procedure where the
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raw and transformed data failed Levine’s test for homogeneity of variance.
3. Results 3.1. Behaviour Rats were monitored over a period of 15 min for somatic and behavioural signs of opioid withdrawal. In morphine-dependent rats both naloxone and naloxone methiodide injections (5 mg/kg s.c.) caused diarrhoea but only naloxone injections caused wet dog shakes, teeth chattering, ptosis, rearing, digging, and escape behaviours. Injections of naloxone, naloxone methiodide or saline did not cause withdrawal signs in vehicletreated rats, nor were they observed in morphine-dependent rats injected with saline. 3.2. Fos expression The antibody used was specific for the Fos protein and does not cross react with FRAs. Fos-IR neurons were counted in sixteen brain structures in which the number of Fos-IR neurons was increased by systemic opioid withdrawal with naloxone (Fig. 1). The mean number of Fos-IR neurons counted in each structure for the six treatment groups are presented in Table 1. An overall difference in the means of the six treatment groups was detected in each structure (One-way ANOVA, P ⬍0.001). Relatively low numbers of Fos-IR neurons were induced by the four control treatments (i.e. vehicle/saline, vehicle/naloxone, vehicle/naloxone methiodide, and morphine/saline) and in most structures there was no significant difference in the means (Table 1). However, in the central nucleus of the amygdala, Fos-IR neurons in the vehicle/naloxone group were increased relative to all other control groups (Tukey HSD, P⬍0.05). Differences amongst some control means were also detected in the parvocellular hypothalamic paraventricular nucleus (vehicle/naloxone ⬎ vehicle/saline, morphine/saline); medial prefrontal cortex (morphine/saline ⬎ vehicle/saline) and supramamillary nucleus (vehicle/naloxone, vehicle/naloxone methiodide ⬎ vehicle/saline). However, these results were ambiguous as there were overlaps in the sets of control means that were not significantly different. Peripheral withdrawal (morphine/naloxone methiodide) increased Fos-IR neurons in comparison to each of the four control groups in all of the structures except the locus coeruleus (morphine/naloxone methiodide = vehicle/saline, vehicle/naloxone, vehicle/naloxone methiodide, morphine/saline; Games–Howell, P⬎0.05) and the ventrolateral periaqueductal gray (PAG) (morphine/naloxone methiodide = morphine/saline Tukey HSD, P⬎0.05; morphine/naloxone methiodide ⬎
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Fig. 1. Fos-immunoreactivity in the lateral parabrachial nucleus (LPB) and locus coeruleus (LC) in morphine-dependent rats injected with saline, naloxone (systemic withdrawal) or naloxone methiodide (peripheral withdrawal). Calibration bars = 400 µm.
Table 1 Mean (±SEM) number of Fos-IR neurons within different brain structures (values are total counts from evenly spaced sections through each structure identified using the Atlas of Paxinos and Watson) Vehicle treated
Morphine dependent
Structurea (no. of sections counted)
Saline (n=4)
Naloxone methiodide (n=4)
Naloxone (n=7) Saline (n=4)
MPC (3) ACB, shell (5) ACB, core (5) BST, laterodorsal (4) BST, ventromedial (4) BST, ventrolateral (2) CEA (5) PVN, magnocellular (2) PVN, parvocellular (2) SUM (3) VTA (7) PAG, ventrolateral (4) LPB (4) A6 (LC) (5) A2/C2 (NTS) (17) A1/C1 (VLM) (17)
13±5.2b 20±5 15±2.4 5±1.8 49±12.3 34±6.2 11±3d 3±0.4 3±0.9e 2±0.6*f 5±0.8 35±11.6 35±6.7 0h 7±0.6 9±0.9
11±3.9 21±6 11±3.7 27±6.1 38±9.4 29±3.4 27±10.8d 3±0.3 5±1.6 6±0.3f 9±0.6 31±5.2 33±4.3 0h 9±0.9 7±0.9
8±1.3 34±4.3 19±3 19±6 52±13.5 19±5.1 167±30.1*d 5±0.5 9±0.7*e 5±0.6f 5±0.9 38±5.4 36±4.4 1±0.5h 10±1.1 9±0.5
31±3.9*b 41±18.4 32±16.2 7.1±3.8 16±6.5 10±0.6 20±11d 4±1.4 2±0.7e 5±1.1 7.8±1 50±17g 56±7.3 0h 6±1 7±0.6
Naloxone methiodide (n=7)b
Naloxone (n=8)
131±12.2 251±19.3 149±11.9 80±8.3 202±15.8 128±10.7 487±28.2 35±4.5 200±22.3 42±5.2 24±2.8 104±15 (6)nsg 294±32.8 12±4.6 (6)nsh 52±5.4 59±7.7
156±9.3 525±19.4*c 173±18.4 115±17.6 270±21.7 192±20.3 1182±86.8*c 136±9.5*c 481±37.4*c 57±2.9 90±8.3*c 277±34 (7)*c 280±18.3 149±40.2 (7)*c 107±21 90±13.2
a MPC, medial prefrontal cortex; ACB, nucleus accumbens; BST, bed nucleus of the stria terminalis; CEA, central nucleus of the amygdala; PVN, paraventricular hypothalamic nucleus; SUM, supermammillary nucleus; VTA, ventral tegmental area; PAG, periaqueductal gray; LC, locus coeruleus; NTS, nucleus of the solitary tract; VLM, ventrolateral medulla. b P⬍0.05 vs. vehicle/saline. c P⬍0.05 vs. morphine/naloxone methiodide. d P⬍0.05 vs. vehicle/saline, vehicle/naloxone methiodide, morphine/saline. e P⬍0.05 vs. vehicle/saline, morphine/saline. f P⬍0.05 vs. vehicle/naloxone methiodide, vehicle/naloxone. g P⬎0.05 vs. morphine/naloxone (P⬍0.05 vs. vehicle/saline, vehicle/naloxone methiodide, vehicle/naloxone). h P⬎0.05 vs. vehicle/saline, vehicle/naloxone methiodide, vehicle/naloxone, morphine/saline.
A. Hamlin et al. / Neuropharmacology 41 (2001) 574–581
vehicle/saline, vehicle/naloxone, vehicle/naloxone methiodide; Tukey HSD, P⬍0.05). Systemic withdrawal (morphine/naloxone) increased Fos-IR neurons in comparison to the four control groups in all of the structures. Fig. 2 illustrates the differences between the control treatments and the effects of systemic and peripheral withdrawal by plotting the means of the pooled control groups together with the means of the morphine/naloxone methiodide and morphine/naloxone groups. The increase in Fos-IR neurons caused by peripheral withdrawal relative to systemic withdrawal is illustrated in Fig. 3 by plotting the normalised difference between the morphine/naloxone and vehicle/naloxone means, and the morphine/naloxone methiodide and vehicle/naloxone methiodide means. The data are ranked in order of the structure least affected by peripheral withdrawal, the locus coeruleus, to the structure most affected, the lateral parabrachial nucleus. No difference was detected between the means of the systemically and peripherally
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Fig. 3. Systemic withdrawal (open circles) and peripheral withdrawal (closed circles) represented as the differences between the antagonist/morphine and antagonist/vehicle treatments. The data has been normalised using the mean of the morphine/naloxone group in each structure and ranked according to the magnitude of the peripheral withdrawal effect. Bars are the SE of the difference between the means.
Fig. 2. Mean number of Fos-IR neurons counted in the pooled control groups and in the peripheral withdrawal and systemic withdrawal groups. Asterisks indicate a significant difference between peripheral withdrawal and systemic withdrawal groups (P⬍0.05). Error bars are the SE of the means.
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withdrawn groups in several structures (Table 1; Fig. 2). These included medullary regions that relay and process peripheral sensory information such as the lateral parabrachial nucleus, A1/C1 catecholamine neurons in the ventrolateral medulla (VLM) and A2/C2 catecholamine neurons in the nucleus of the solitary tract (NTS) (Fig. 4). However there was also no difference in midbrain and forebrain structures that included the core of the accumbens, medial prefrontal cortex, supramammillary nucleus and the bed nucleus of the stria terminalis (BST).
Fig. 4. Fos-IR neurons in morphine-dependent rats injected with naloxone (left panels) or naloxone methiodide (right panels). Shown in the micrographs are transverse sections containing the nucleus accumbens (ACB), central nucleus of the amygdala (CEA), PVN, NTS and VLM. The lighter cytoplasmic staining in the NTS and VLM sections is tyrosine hydroxylase immunoreactivity, and in the CEA and PVN sections corresponds to immunoreactivity for corticotrophin releasing factor (calibration bars = 500 µm).
4. Discussion In this paper, we report that withdrawal precipitated outside the central nervous system in morphine-dependent rats increases Fos-IR neurons in many regions of the brain. We compared the effect of peripheral opioid withdrawal to systemic withdrawal precipitated with naloxone, which is an opioid antagonist that readily crosses the blood-brain barrier. No significant difference in the number of Fos-IR neurons induced by the two treatments was detected in: A1/C1 catecholamine neurons in VLM, A2/C2 catecholamine neurons in NTS, lateral parabrachial nucleus, supramamillary nucleus, BST, nucleus accumbens core, and medial prefrontal cortex. This suggests that most, if not all, of the induction of Fos was triggered by withdrawal activation of opioiddependent neurons or tissues located outside the central nervous system. If this is the case then induction of cfos mRNA and Fos protein reported in these regions by previous studies (Hayward et al., 1990; Stornetta et al., 1993; Baraban et al., 1995; Couceyro and Douglass, 1995; Chahl et al., 1996; Nye and Nestler, 1996; Georges et al., 2000) is unlikely to be involved in the centrally mediated aversive behavioral effects or somatic signs of withdrawal (reviewed by Schulteis and Koob, 1996). In contrast, no significant difference between the controls and peripheral withdrawal was detected in A6 catecholamine neurons in the locus coeruleus. Therefore in this structure induction of Fos could entirely be due to the withdrawal activation of opioid-dependent neurons located in the central nervous system. Both peripheral and central withdrawal mechanisms appeared to contribute to the induction of Fos in the remaining brain regions examined-PAG, ventral tegmental area, magnocellular and parvocellular divisions of the paraventricular nucleus of the hypothalamus (PVN), central nucleus of the amygdala, and nucleus accumbens shell. We used the hydrophilic opioid antagonist naloxone methiodide (also known as methylnaloxonium iodide) to precipitate peripheral withdrawal in morphine-dependent rats. As reported by previous studies, this caused diarrhoea but did not induce somatic withdrawal signs that are precipitated in dependent animals by intracerebral injections of opioid antagonists (Bozarth, 1994; Rohde et al., 1997a,b). Naloxone methiodide and certain other quaternary salts of opioid antagonists are hydrophilic and have an extremely restricted ability to cross the blood-brain barrier in rat (Brown and Goldberg, 1985). This has been effectively demonstrated by the failure of quaternary naloxone to reduce supraspinal and spinal antinociception induced by morphine, even when doses as high as 300 mg/kg i.p. are used (Russell et al., 1982; Milne et al., 1990). Conditioned place-preferences induced centrally by morphine are also not affected ˚ gmo et al., 1992), and place aversions induced by (A methyl naloxone administered into the cerebral ven-
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tricles are not induced by subcutaneous injections (Hand et al., 1988). In morphine-dependent rats, peripheral injections of quaternary opioid antagonists cause virtually none of the physical signs that are precipitated by intracerebral antagonist injections (Maldonado et al., 1992) and do not induce aversive behavioral effects that are elicited by systemic withdrawal (Hand et al., 1988; Mucha, 1989; Koob et al., 1989). Intra-cerebral injection of opioid antagonists in morphine-dependent rats induce physical and behavioral signs which must be due to withdrawal activation of neural circuits within the brain. Structures that could be part of these circuits have been identified by precipitating withdrawal signs with localised antagonist microinjections. For example, somatic withdrawal signs can be elicited in morphine-dependent rats by microinjections of methylnaloxonium into the locus coeruleus, ventrolateral PAG and central nucleus of the amygdala (Maldonado et al., 1992). Similarly, in behavioural studies, i.c.v. injections of antagonists precipitate conditioned aversions (Hand et al., 1988; Mucha, 1989) and disruption of operant responding (Koob et al., 1989), whereas place aversions are formed when opioid antagonists are microinjected into the PAG, ventral tegmental area, amygdala, nucleus accumbens as well as the medial dorsal thalamus (Stinus et al., 1990). Our study has confirmed previous reports that systemic withdrawal increases Fos-IR neurons in most of these regions (reviewed by Harlan and Garcia, 1998). In contrast, peripheral withdrawal did not significantly increase Fos-IR neurons in the locus coeruleus, and caused a significantly smaller increase than systemic withdrawal in the ventrolateral PAG, central nucleus of the amygdala, ventral tegmental area, and the shell of the nucleus accumbens. Fos-IR neurons that are induced in these regions by systemic withdrawal and not peripheral withdrawal could therefore be part of the opioid-dependent central pathways that mediate physical or behavioural withdrawal signs. We propose that the pattern of Fos expression induced by peripheral withdrawal could occur as a consequence of visceral stimulation of supraspinal sensory pathways and subsequent activation of forebrain areas involved in emotional motor control. Although opioid withdrawal in the periphery has not been completely characterised, it is well established that the syndrome includes hypermotility of visceral organs such as the gut and bladder, and secretomotor effects such as diarrhoea, lacrimation and rhinorhoea (e.g. review by Schulteis and Koob, 1996). There are two main systems by which related visceral sensory information could reach the brain (reviewed by Craig, 1996; Saper, 1996). Sympathetic afferent information is carried by the visceral sensory fibres in the spinal nerves that terminate in the superficial layers of the spinal dorsal horn. From here secondary afferent neurons send projections to the intralaminar and ventro-
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posterior lateral thalamic nuclei, that also branch and supply visceral regulatory cell groups in the VLM, nucleus of the solitary tract and parabrachial nucleus. Parasympathetic afferent information is carried by visceral sensory fibres that enter the brain via the vagus, glossopharyngeal, facial and trigeminal cranial nerves and terminate in the nucleus of the solitary tract. Studies by Rohde et al. (1997a,b) report that peripheral opioid withdrawal increases Fos-IR neurons in the dorsal horn in sacral but not lumbar segments of the spinal cord, which was attributed to withdrawal activation of organs such as the colon and bladder. In the present study peripheral withdrawal elicited a corresponding increase of supraspinal Fos-IR neurons in the VLM and parabrachial nucleus, two of the sites most densely supplied with lamina I spinal cord projections (Craig, 1996), and in A2/C2 catecholamine neurons in the NTS. However peripheral withdrawal did not induce Fos in A6 catecholamine neurons in the locus coeruleus and a limited increase in the ventrolateral PAG, which also receive sympathetic and parasympathetic sensory information. We suggest this difference could be due to the nature of the sensory stimulus elicited by peripheral withdrawal. Although noxious visceral stimuli induce Fos in the ventrolateral PAG, locus coeruleus and parabrachial nucleus, Fos is also induced in the latter by non-noxious stimuli such as duodenal loading with glucose (Lanteri-Minet et al., 1993; Rodella et al., 1998; Wang et al., 1999). This is consistent with the lateral parabrachial nucleus functioning as a general visceral integration site that responds to non-noxious stimuli such as changes in thermoregulation, salivation and gastrointestinal motility (Reilly, 1999). We found that peripheral withdrawal could account for most of the induction of Fos-IR neurons caused by systemic withdrawal in the medial prefrontal cortex, accumbens core, BST, and at least part of the increase produced in the central nucleus of the amygdala. Within the context of addiction research, Fos induced by systemic opioid withdrawal in these structures is typically attributed to the expression of emotional and motivational aspects of opioid addiction (for example see reviews by Schulteis and Koob, 1996; Koob and Le Moal, 1997). However these regions also form functional networks that have a significant role in coordinating motor function in a behavioral context such as robust visceral activity that is caused by emotion and stress (McDonald et al., 1999). Within the complex formed by the orbital and medial prefrontal cortex (OMPFC), visceral/gustatory sensory information relayed by thalamus is directed to the gustatory and agranular insular cortex where it is processed and passed on to the medial prefrontal network, which in the rat encompasses the infralimbic and prelimbic cortices and acts as the primary source of visceromotor output from the OMPFC (Price, 1999). This anatomical arrangement is consistent
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with a report that Fos can be induced in the prefrontal cortex by noxious visceral stimulation (Traub et al., 1996). In the rat, infralimbic cortex functions as true visceral motor cortex — in addition to providing a major descending projection through the lateral hypothalamus it also innervates the VLM, NTS, parabrachial nucleus and the PAG (Saper, 1996). Direct contacts are made onto sympathetic preganglionic neurons in these regions and in the intermediolateral column of the thoracic spinal cord. The extended amygdala, which encompasses the BST and central nucleus of the amygdala, also receives descending projections from the medial prefrontal cortex as well as ascending projections from the parabrachial nucleus, VLM and NTS (McDonald et al., 1999). Systemic opioid withdrawal can induce Fos in A1 and A2 catecholamine neurons that project to the BST (Delfs et al., 2000). These catecholamine projections have been linked to the formation of conditioned place aversions to opioid withdrawal, as these are strongly attenuated when beta-adrenoceptor antagonists is microinjected into the BST. Our data suggests that Fos induction is not a reliable marker for identifying A1 and A2 neurons that are critical for opioid withdrawal-induced aversions. This is because the Fos induction in these neurons could mostly be attributed to peripheral withdrawal, which does not cause place aversions (Hand et al., 1988). Induction of Fos in VLM catecholamine neurons could indicate the activation of autonomic motor pathways. The adrenergic C1 neurons in the rostral VLM include pre-sympathetic bulbospinal neurons that project to the intermediolateral spinal cord and are activated during reflex stimulation of sympathetic outflow (Janig, 1988). The noradrenergic A1 neurons project to both caudal VLM and hypothalamus, and participate in autonomic control as well as activation of the hypothalamicpituitary axis. This interpretation is consistent with the limited ability of peripheral withdrawal to increase FosIR neurons in the ventrolateral PAG, stimulation of which causes a well-defined pattern of responses that include behavioral hyporeactive immobility, sympathoinhibition and anti-nociception (e.g. Keay et al., 1997). We found that injections of naloxone in morphine naı¨ve rats caused induction of Fos in the central nucleus of the amygdala. This has been reported previously and attributed to block of the effects of tonically released endogenous opioids (Carr et al., 1999; Gestreau et al., 2000). Although we did detect some other antagonist effects in morphine naı¨ve rats, these results were ambiguous due to overlaps in the sets of control means that were not significantly different. In the report by Gestreau et al. (2000) naloxone injections in morphine naı¨ve rats also induce Fos-IR neurons in the caudal, intermediate and rostral divisions of the NTS; area postrema and rostral VLM; Ko¨ lliker-Fuse nucleus and supramamillary nucleus. We were unable to detect these effects although a similar trend was apparent in the supramamillary
nucleus. However in our study only catecholamine neurons in the NTS and RV were counted, and the Ko¨ llikerFuse and area postrema were not analysed. Furthermore, to increase the sensitivity of their assay, Gestreau et al. (2000) habituated their animals to handling and saline injections over a 4 day period prior to administering the experimental injections used to induce Fos. In conclusion, our study has shown that peripheral withdrawal precipitated by antagonism of opioid receptors located outside the central nervous system can increase Fos-IR neurons in the brain. The induction of Fos was widespread and was observed in a variety of brain regions that have been implicated in motivational aspects of opioid addiction. Peripheral withdrawal mechanisms are thought not to contribute to the development of addiction, which is consistent with the absence of abuse liability reported for peripherally selective opioid agonists such as the antidiarrheal agent loperamide (Jaffe et al., 1980; Korey et al., 1980). If peripheral withdrawal can alter immediate-early gene expression in the brain then it could also contribute significantly to other molecular or cellular changes that are induced in the brain when opioid withdrawal is precipitated systemically. If so, then the motivational significance of these changes in the context of addiction warrants further investigation.
Acknowledgement Supported by NHMRC project grant 971126 (P.B.O.)
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Georges, F., Stinus, L., Le Moine, C., 2000. Mapping of c-Fos gene expression in the brain during morphine dependence and precipitated withdrawal, and phenotypic identification of the striatal neurons involved. European Journal of Neuroscience 12, 4475–4486. Gestreau, C., Le Guen, S., Besson, J.M., 2000. Is there tonic activity in the endogenous opioid systems? A c-Fos study in the rat central nervous system after intravenous injection of naloxone or naloxone-methiodide. Journal of Comparative Neurology 427, 285–301. Hand, T.H., Koob, G.F., Stinus, L., Le, M.M., 1988. Aversive properties of opiate receptor blockade: evidence for exclusively central mediation in naive and morphine-dependent rats. Brain Research 474, 364–368. Harlan, R.E., Garcia, M.M., 1998. Drugs of abuse and immediate-early genes in the forebrain. Molecular Neurobiology 16, 221–267. Hayward, M.D., Duman, R.S., Nestler, E.J., 1990. Induction of the cfos proto-oncogene during opiate withdrawal in the locus coeruleus and other regions of rat brain. Brain Research 525, 256–266. Janig, W., 1988. Pre- and postganglionic vasoconstrictor neurons: differentiation, types, and discharge properties. Annual Review of Physiology 50, 525–539. Jaffe, J.H., Kanzler, M., Green, J., 1980. Abuse potential of loperamide. Clinical Pharmacology & Therapeutics 28, 812–819. Keay, K.A., Crowfoot, L.J., Floyd, N.S., Henderson, L.A., Christie, M.J., Bandler, R., 1997. Cardiovascular effects of microinjections of opioid agonists into the “Depressor Region” of the ventrolateral periaqueductal gray region. Brain Research 762, 61–71. Koob, G.F., Le Moal, M., 1997. Drug abuse: hedonic homeostatic dysregulation. Science 278, 52–58. Koob, G.F., Wall, T.L., Bloom, F.E., 1989. Nucleus accumbens as a substrate for the aversive stimulus effects of opiate withdrawal. Psychopharmacology 98, 530–534. Korey, A., Zilm, D.H., Sellers, E.M., 1980. Dependence liability of two antidiarrheals, nufenoxole and loperamide. Clinical Pharmacology & Therapeutics 27, 659–664. Lanteri-Minet, M., Isnardon, P., de, P.J., Menetrey, D., 1993. Spinal and hindbrain structures involved in visceroception and visceronociception as revealed by the expression of Fos, Jun and Krox-24 proteins. Neuroscience 55, 737–753. Milne, R.J., Coddington, J.M., Gamble, G.D., 1990. Quaternary naloxone blocks morphine analgesia in spinal but not intact rats. Neuroscience Letters 114, 259–264. Maldonado, R., Stinus, L., Gold, L.H., Koob, G.F., 1992. Role of different brain structures in the expression of the physical morphine withdrawal syndrome. Journal of Pharmacology and Experimental Therapeutics 261, 669–677. McDonald, A.J., Shammah-Lagnado, S.J., Shi, C., Davis, M., 1999. Cortical afferents to the extended amygdala. Annals of the New York Academy of Sciences 877, 309–338. Mucha, R.F., 1989. Taste aversion involving central opioid antagonism
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Peripheral withdrawal recruits distinct central nuclei in morphinedependent rats A. Hamlin a, K.M. Buller a, T.A. Day a, P.B. Osborne b
a, b,*
a Department of Physiology and Pharmacology, The University of Queensland, Brisbane Qld 4072, Australia Prince of Wales Medical Research Institute, University of New South Wales, Barker Street, Randwick NSW 2031, Sydney, Australia
Received 6 October 2000; received in revised form 18 May 2001; accepted 25 June 2001
Abstract This study examined if brain pathways in morphine-dependent rats are activated by opioid withdrawal precipitated outside the central nervous system. Withdrawal precipitated with a peripherally acting quaternary opioid antagonist (naloxone methiodide) increased Fos expression but caused a more restricted pattern of neuronal activation than systemic withdrawal (precipitated with naloxone which enters the brain). There was no effect on locus coeruleus and significantly smaller increases in Fos neurons were produced in most other areas. However in the ventrolateral medulla (A1/C1 catecholamine neurons), nucleus of the solitary tract (A2/C2 catecholamine neurons), lateral parabrachial nucleus, supramamillary nucleus, bed nucleus of the stria terminalis, accumbens core and medial prefrontal cortex no differences in the withdrawal treatments were detected. We have shown that peripheral opioid withdrawal can affect central nervous system pathways. Crown Copyright 2001 Published by Elsevier Science Ltd. All rights reserved. Keywords: Morphine dependence; Substance withdrawal syndrome; Naloxone methiodide; Proto-oncogene proteins c-fos
1. Introduction Opioid withdrawal can be precipitated peripherally in rodents by injecting morphine-dependent animals with hydrophilic opioid antagonists that effectively do not cross the blood-brain barrier (Brown and Goldberg, 1985). This produces gut hypermotility, secretomotor stimulation (diarrhoea, lacrimation, rhinorhoea and salivation) and other visceral responses but virtually none of the physical signs that are precipitated when opioid antagonists are injected into the brain (Schulteis and Koob, 1996). ‘Peripheral withdrawal’ also does not have the aversive behavioural effects that are elicited by systemic withdrawal (precipitated with antagonists that enter the brain) such as reductions in spontaneous locomotion, suppression of operant responding and conditioned place aversions (Hand et al., 1988; Mucha, 1989).
* Corresponding author. Tel.: +612-9382-7934; fax: +612-93822722. E-mail address: [email protected] (P.B. Osborne).
Based on these whole animal studies it could be assumed that central nervous system pathways are not activated by peripheral opioid withdrawal. However it has been reported recently that peripheral withdrawal can activate the dorsal horn of the rat sacral spinal cord, causing an increase in neurons that express the inducible transcription factor c-fos (Rohde et al., 1997a). In the brain, systemic opioid withdrawal markedly increases cfos and ‘acute’ Fos-related antigens (FRAs; e.g. Fos B, FRA-1 and FRA-2)(Nestler and Aghajanian, 1997; Harlan and Garcia, 1998) but the extent to which these inducible transcription factors are affected by peripheral withdrawal has not been determined. In this study, we have addressed this question by using the hydrophilic quaternary opioid antagonist naloxone methiodide to precipitate withdrawal in morphine-dependent rats. We demonstrate that peripheral withdrawal significantly increases Fos-immunoreactive (Fos-IR) neurons in many brain areas, and the pattern of recruitment is distinct from that seen when opioid withdrawal is precipitated systemically.
0028-3908/01/$ - see front matter. Crown Copyright 2001 Published by Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 8 - 3 9 0 8 ( 0 1 ) 0 0 1 0 1 - 0
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2. Methods Adult male Wistar rats (350–500 g) were used in all of the experiments. Two types of pretreatment (vehicle or morphine) were combined with three types of withdrawal treatment (saline, naloxone methiodide and naloxone). This gave six experimental groups that included four control groups (vehicle/saline, n=4; vehicle/naloxone methiodide, n=4; vehicle/naloxone, n=7; and morphine/saline, n=4), a peripheral withdrawal group (morphine/naloxone methiodide, n=7) and a systemic withdrawal group (morphine/naloxone, n=8). The treatment schedules were administered to batches of four rats in no particular sequence. The pretreatment consisted of three subcutaneous injections (48 h apart) of a slow release vehicle ± morphine HCl (50 mg/kg, Sigma pharmaceuticals). The vehicle was an emulsion of 42.5% paraffin oil and 7.5% mannide manooleate (Sigma-Aldrich) in a NaCl solution (0.9%) containing 311 nM NaOH to which 25 mg/ml of morphine HCl was added. The emulsion was warmed to 35°C and 2 ml/kg was injected subcutaneously. Then 48 h after the final pretreatment injection, vehicle or morphine treated rats were administered a withdrawal injection of normal saline, naloxone methiodide (5 mg/kg s.c.; Research Biochemicals Inc.) or naloxone (5 mg/kg s.c., SigmaAldrich). Withdrawal injections were administered to each rat in a batch of four at 30 min intervals from 10 a.m. onwards. The animals were withdrawn in their individual home cage and then over a 15 min period were monitored for the following opioid withdrawal signs: diarrhoea, wet dog shakes, teeth chattering, ptosis, and rearing or digging. About 2 h after precipitating withdrawal, rats were deeply anaesthetised (sodium pentobarbitone 50 mg/kg i.p.) and transcardially perfused with 2% sodium nitrite solution followed by 4% formaldehyde in 0.1 M phosphate buffer (pH 7.4). The brains were then removed postfixed and processed for immunohistochemistry using a dual immunoperoxidase technique described previously (Smith and Day, 1993). Brains taken from concurrently treated batches of rats were processed simultaneously. A 1:4 series of forebrain sections were processed for Fos (1:100,000 for 48 h; rabbit polyclonal antibody, Santa Cruz Biotechnology) and a 1:5 series of midbrain and hindbrain sections were processed for Fos and tyrosine-hydroxylase (1:25,000; mouse polyclonal antibody, Incstar). Unilateral counts of Fos-IR neurons were made in coronal sections spaced at 160 µm intervals in cortex, forebrain and hypothalamus, and at 250 µm intervals in midbrain, pons and medulla. Equality of the six group means in each structure was compared using a one-way analysis of variance. Unplanned comparisons among pairs of group means were made using the Tukey HSD test for unequal sample sizes, or using the Games–Howell procedure where the
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raw and transformed data failed Levine’s test for homogeneity of variance.
3. Results 3.1. Behaviour Rats were monitored over a period of 15 min for somatic and behavioural signs of opioid withdrawal. In morphine-dependent rats both naloxone and naloxone methiodide injections (5 mg/kg s.c.) caused diarrhoea but only naloxone injections caused wet dog shakes, teeth chattering, ptosis, rearing, digging, and escape behaviours. Injections of naloxone, naloxone methiodide or saline did not cause withdrawal signs in vehicletreated rats, nor were they observed in morphine-dependent rats injected with saline. 3.2. Fos expression The antibody used was specific for the Fos protein and does not cross react with FRAs. Fos-IR neurons were counted in sixteen brain structures in which the number of Fos-IR neurons was increased by systemic opioid withdrawal with naloxone (Fig. 1). The mean number of Fos-IR neurons counted in each structure for the six treatment groups are presented in Table 1. An overall difference in the means of the six treatment groups was detected in each structure (One-way ANOVA, P ⬍0.001). Relatively low numbers of Fos-IR neurons were induced by the four control treatments (i.e. vehicle/saline, vehicle/naloxone, vehicle/naloxone methiodide, and morphine/saline) and in most structures there was no significant difference in the means (Table 1). However, in the central nucleus of the amygdala, Fos-IR neurons in the vehicle/naloxone group were increased relative to all other control groups (Tukey HSD, P⬍0.05). Differences amongst some control means were also detected in the parvocellular hypothalamic paraventricular nucleus (vehicle/naloxone ⬎ vehicle/saline, morphine/saline); medial prefrontal cortex (morphine/saline ⬎ vehicle/saline) and supramamillary nucleus (vehicle/naloxone, vehicle/naloxone methiodide ⬎ vehicle/saline). However, these results were ambiguous as there were overlaps in the sets of control means that were not significantly different. Peripheral withdrawal (morphine/naloxone methiodide) increased Fos-IR neurons in comparison to each of the four control groups in all of the structures except the locus coeruleus (morphine/naloxone methiodide = vehicle/saline, vehicle/naloxone, vehicle/naloxone methiodide, morphine/saline; Games–Howell, P⬎0.05) and the ventrolateral periaqueductal gray (PAG) (morphine/naloxone methiodide = morphine/saline Tukey HSD, P⬎0.05; morphine/naloxone methiodide ⬎
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Fig. 1. Fos-immunoreactivity in the lateral parabrachial nucleus (LPB) and locus coeruleus (LC) in morphine-dependent rats injected with saline, naloxone (systemic withdrawal) or naloxone methiodide (peripheral withdrawal). Calibration bars = 400 µm.
Table 1 Mean (±SEM) number of Fos-IR neurons within different brain structures (values are total counts from evenly spaced sections through each structure identified using the Atlas of Paxinos and Watson) Vehicle treated
Morphine dependent
Structurea (no. of sections counted)
Saline (n=4)
Naloxone methiodide (n=4)
Naloxone (n=7) Saline (n=4)
MPC (3) ACB, shell (5) ACB, core (5) BST, laterodorsal (4) BST, ventromedial (4) BST, ventrolateral (2) CEA (5) PVN, magnocellular (2) PVN, parvocellular (2) SUM (3) VTA (7) PAG, ventrolateral (4) LPB (4) A6 (LC) (5) A2/C2 (NTS) (17) A1/C1 (VLM) (17)
13±5.2b 20±5 15±2.4 5±1.8 49±12.3 34±6.2 11±3d 3±0.4 3±0.9e 2±0.6*f 5±0.8 35±11.6 35±6.7 0h 7±0.6 9±0.9
11±3.9 21±6 11±3.7 27±6.1 38±9.4 29±3.4 27±10.8d 3±0.3 5±1.6 6±0.3f 9±0.6 31±5.2 33±4.3 0h 9±0.9 7±0.9
8±1.3 34±4.3 19±3 19±6 52±13.5 19±5.1 167±30.1*d 5±0.5 9±0.7*e 5±0.6f 5±0.9 38±5.4 36±4.4 1±0.5h 10±1.1 9±0.5
31±3.9*b 41±18.4 32±16.2 7.1±3.8 16±6.5 10±0.6 20±11d 4±1.4 2±0.7e 5±1.1 7.8±1 50±17g 56±7.3 0h 6±1 7±0.6
Naloxone methiodide (n=7)b
Naloxone (n=8)
131±12.2 251±19.3 149±11.9 80±8.3 202±15.8 128±10.7 487±28.2 35±4.5 200±22.3 42±5.2 24±2.8 104±15 (6)nsg 294±32.8 12±4.6 (6)nsh 52±5.4 59±7.7
156±9.3 525±19.4*c 173±18.4 115±17.6 270±21.7 192±20.3 1182±86.8*c 136±9.5*c 481±37.4*c 57±2.9 90±8.3*c 277±34 (7)*c 280±18.3 149±40.2 (7)*c 107±21 90±13.2
a MPC, medial prefrontal cortex; ACB, nucleus accumbens; BST, bed nucleus of the stria terminalis; CEA, central nucleus of the amygdala; PVN, paraventricular hypothalamic nucleus; SUM, supermammillary nucleus; VTA, ventral tegmental area; PAG, periaqueductal gray; LC, locus coeruleus; NTS, nucleus of the solitary tract; VLM, ventrolateral medulla. b P⬍0.05 vs. vehicle/saline. c P⬍0.05 vs. morphine/naloxone methiodide. d P⬍0.05 vs. vehicle/saline, vehicle/naloxone methiodide, morphine/saline. e P⬍0.05 vs. vehicle/saline, morphine/saline. f P⬍0.05 vs. vehicle/naloxone methiodide, vehicle/naloxone. g P⬎0.05 vs. morphine/naloxone (P⬍0.05 vs. vehicle/saline, vehicle/naloxone methiodide, vehicle/naloxone). h P⬎0.05 vs. vehicle/saline, vehicle/naloxone methiodide, vehicle/naloxone, morphine/saline.
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vehicle/saline, vehicle/naloxone, vehicle/naloxone methiodide; Tukey HSD, P⬍0.05). Systemic withdrawal (morphine/naloxone) increased Fos-IR neurons in comparison to the four control groups in all of the structures. Fig. 2 illustrates the differences between the control treatments and the effects of systemic and peripheral withdrawal by plotting the means of the pooled control groups together with the means of the morphine/naloxone methiodide and morphine/naloxone groups. The increase in Fos-IR neurons caused by peripheral withdrawal relative to systemic withdrawal is illustrated in Fig. 3 by plotting the normalised difference between the morphine/naloxone and vehicle/naloxone means, and the morphine/naloxone methiodide and vehicle/naloxone methiodide means. The data are ranked in order of the structure least affected by peripheral withdrawal, the locus coeruleus, to the structure most affected, the lateral parabrachial nucleus. No difference was detected between the means of the systemically and peripherally
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Fig. 3. Systemic withdrawal (open circles) and peripheral withdrawal (closed circles) represented as the differences between the antagonist/morphine and antagonist/vehicle treatments. The data has been normalised using the mean of the morphine/naloxone group in each structure and ranked according to the magnitude of the peripheral withdrawal effect. Bars are the SE of the difference between the means.
Fig. 2. Mean number of Fos-IR neurons counted in the pooled control groups and in the peripheral withdrawal and systemic withdrawal groups. Asterisks indicate a significant difference between peripheral withdrawal and systemic withdrawal groups (P⬍0.05). Error bars are the SE of the means.
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withdrawn groups in several structures (Table 1; Fig. 2). These included medullary regions that relay and process peripheral sensory information such as the lateral parabrachial nucleus, A1/C1 catecholamine neurons in the ventrolateral medulla (VLM) and A2/C2 catecholamine neurons in the nucleus of the solitary tract (NTS) (Fig. 4). However there was also no difference in midbrain and forebrain structures that included the core of the accumbens, medial prefrontal cortex, supramammillary nucleus and the bed nucleus of the stria terminalis (BST).
Fig. 4. Fos-IR neurons in morphine-dependent rats injected with naloxone (left panels) or naloxone methiodide (right panels). Shown in the micrographs are transverse sections containing the nucleus accumbens (ACB), central nucleus of the amygdala (CEA), PVN, NTS and VLM. The lighter cytoplasmic staining in the NTS and VLM sections is tyrosine hydroxylase immunoreactivity, and in the CEA and PVN sections corresponds to immunoreactivity for corticotrophin releasing factor (calibration bars = 500 µm).
4. Discussion In this paper, we report that withdrawal precipitated outside the central nervous system in morphine-dependent rats increases Fos-IR neurons in many regions of the brain. We compared the effect of peripheral opioid withdrawal to systemic withdrawal precipitated with naloxone, which is an opioid antagonist that readily crosses the blood-brain barrier. No significant difference in the number of Fos-IR neurons induced by the two treatments was detected in: A1/C1 catecholamine neurons in VLM, A2/C2 catecholamine neurons in NTS, lateral parabrachial nucleus, supramamillary nucleus, BST, nucleus accumbens core, and medial prefrontal cortex. This suggests that most, if not all, of the induction of Fos was triggered by withdrawal activation of opioiddependent neurons or tissues located outside the central nervous system. If this is the case then induction of cfos mRNA and Fos protein reported in these regions by previous studies (Hayward et al., 1990; Stornetta et al., 1993; Baraban et al., 1995; Couceyro and Douglass, 1995; Chahl et al., 1996; Nye and Nestler, 1996; Georges et al., 2000) is unlikely to be involved in the centrally mediated aversive behavioral effects or somatic signs of withdrawal (reviewed by Schulteis and Koob, 1996). In contrast, no significant difference between the controls and peripheral withdrawal was detected in A6 catecholamine neurons in the locus coeruleus. Therefore in this structure induction of Fos could entirely be due to the withdrawal activation of opioid-dependent neurons located in the central nervous system. Both peripheral and central withdrawal mechanisms appeared to contribute to the induction of Fos in the remaining brain regions examined-PAG, ventral tegmental area, magnocellular and parvocellular divisions of the paraventricular nucleus of the hypothalamus (PVN), central nucleus of the amygdala, and nucleus accumbens shell. We used the hydrophilic opioid antagonist naloxone methiodide (also known as methylnaloxonium iodide) to precipitate peripheral withdrawal in morphine-dependent rats. As reported by previous studies, this caused diarrhoea but did not induce somatic withdrawal signs that are precipitated in dependent animals by intracerebral injections of opioid antagonists (Bozarth, 1994; Rohde et al., 1997a,b). Naloxone methiodide and certain other quaternary salts of opioid antagonists are hydrophilic and have an extremely restricted ability to cross the blood-brain barrier in rat (Brown and Goldberg, 1985). This has been effectively demonstrated by the failure of quaternary naloxone to reduce supraspinal and spinal antinociception induced by morphine, even when doses as high as 300 mg/kg i.p. are used (Russell et al., 1982; Milne et al., 1990). Conditioned place-preferences induced centrally by morphine are also not affected ˚ gmo et al., 1992), and place aversions induced by (A methyl naloxone administered into the cerebral ven-
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tricles are not induced by subcutaneous injections (Hand et al., 1988). In morphine-dependent rats, peripheral injections of quaternary opioid antagonists cause virtually none of the physical signs that are precipitated by intracerebral antagonist injections (Maldonado et al., 1992) and do not induce aversive behavioral effects that are elicited by systemic withdrawal (Hand et al., 1988; Mucha, 1989; Koob et al., 1989). Intra-cerebral injection of opioid antagonists in morphine-dependent rats induce physical and behavioral signs which must be due to withdrawal activation of neural circuits within the brain. Structures that could be part of these circuits have been identified by precipitating withdrawal signs with localised antagonist microinjections. For example, somatic withdrawal signs can be elicited in morphine-dependent rats by microinjections of methylnaloxonium into the locus coeruleus, ventrolateral PAG and central nucleus of the amygdala (Maldonado et al., 1992). Similarly, in behavioural studies, i.c.v. injections of antagonists precipitate conditioned aversions (Hand et al., 1988; Mucha, 1989) and disruption of operant responding (Koob et al., 1989), whereas place aversions are formed when opioid antagonists are microinjected into the PAG, ventral tegmental area, amygdala, nucleus accumbens as well as the medial dorsal thalamus (Stinus et al., 1990). Our study has confirmed previous reports that systemic withdrawal increases Fos-IR neurons in most of these regions (reviewed by Harlan and Garcia, 1998). In contrast, peripheral withdrawal did not significantly increase Fos-IR neurons in the locus coeruleus, and caused a significantly smaller increase than systemic withdrawal in the ventrolateral PAG, central nucleus of the amygdala, ventral tegmental area, and the shell of the nucleus accumbens. Fos-IR neurons that are induced in these regions by systemic withdrawal and not peripheral withdrawal could therefore be part of the opioid-dependent central pathways that mediate physical or behavioural withdrawal signs. We propose that the pattern of Fos expression induced by peripheral withdrawal could occur as a consequence of visceral stimulation of supraspinal sensory pathways and subsequent activation of forebrain areas involved in emotional motor control. Although opioid withdrawal in the periphery has not been completely characterised, it is well established that the syndrome includes hypermotility of visceral organs such as the gut and bladder, and secretomotor effects such as diarrhoea, lacrimation and rhinorhoea (e.g. review by Schulteis and Koob, 1996). There are two main systems by which related visceral sensory information could reach the brain (reviewed by Craig, 1996; Saper, 1996). Sympathetic afferent information is carried by the visceral sensory fibres in the spinal nerves that terminate in the superficial layers of the spinal dorsal horn. From here secondary afferent neurons send projections to the intralaminar and ventro-
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posterior lateral thalamic nuclei, that also branch and supply visceral regulatory cell groups in the VLM, nucleus of the solitary tract and parabrachial nucleus. Parasympathetic afferent information is carried by visceral sensory fibres that enter the brain via the vagus, glossopharyngeal, facial and trigeminal cranial nerves and terminate in the nucleus of the solitary tract. Studies by Rohde et al. (1997a,b) report that peripheral opioid withdrawal increases Fos-IR neurons in the dorsal horn in sacral but not lumbar segments of the spinal cord, which was attributed to withdrawal activation of organs such as the colon and bladder. In the present study peripheral withdrawal elicited a corresponding increase of supraspinal Fos-IR neurons in the VLM and parabrachial nucleus, two of the sites most densely supplied with lamina I spinal cord projections (Craig, 1996), and in A2/C2 catecholamine neurons in the NTS. However peripheral withdrawal did not induce Fos in A6 catecholamine neurons in the locus coeruleus and a limited increase in the ventrolateral PAG, which also receive sympathetic and parasympathetic sensory information. We suggest this difference could be due to the nature of the sensory stimulus elicited by peripheral withdrawal. Although noxious visceral stimuli induce Fos in the ventrolateral PAG, locus coeruleus and parabrachial nucleus, Fos is also induced in the latter by non-noxious stimuli such as duodenal loading with glucose (Lanteri-Minet et al., 1993; Rodella et al., 1998; Wang et al., 1999). This is consistent with the lateral parabrachial nucleus functioning as a general visceral integration site that responds to non-noxious stimuli such as changes in thermoregulation, salivation and gastrointestinal motility (Reilly, 1999). We found that peripheral withdrawal could account for most of the induction of Fos-IR neurons caused by systemic withdrawal in the medial prefrontal cortex, accumbens core, BST, and at least part of the increase produced in the central nucleus of the amygdala. Within the context of addiction research, Fos induced by systemic opioid withdrawal in these structures is typically attributed to the expression of emotional and motivational aspects of opioid addiction (for example see reviews by Schulteis and Koob, 1996; Koob and Le Moal, 1997). However these regions also form functional networks that have a significant role in coordinating motor function in a behavioral context such as robust visceral activity that is caused by emotion and stress (McDonald et al., 1999). Within the complex formed by the orbital and medial prefrontal cortex (OMPFC), visceral/gustatory sensory information relayed by thalamus is directed to the gustatory and agranular insular cortex where it is processed and passed on to the medial prefrontal network, which in the rat encompasses the infralimbic and prelimbic cortices and acts as the primary source of visceromotor output from the OMPFC (Price, 1999). This anatomical arrangement is consistent
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with a report that Fos can be induced in the prefrontal cortex by noxious visceral stimulation (Traub et al., 1996). In the rat, infralimbic cortex functions as true visceral motor cortex — in addition to providing a major descending projection through the lateral hypothalamus it also innervates the VLM, NTS, parabrachial nucleus and the PAG (Saper, 1996). Direct contacts are made onto sympathetic preganglionic neurons in these regions and in the intermediolateral column of the thoracic spinal cord. The extended amygdala, which encompasses the BST and central nucleus of the amygdala, also receives descending projections from the medial prefrontal cortex as well as ascending projections from the parabrachial nucleus, VLM and NTS (McDonald et al., 1999). Systemic opioid withdrawal can induce Fos in A1 and A2 catecholamine neurons that project to the BST (Delfs et al., 2000). These catecholamine projections have been linked to the formation of conditioned place aversions to opioid withdrawal, as these are strongly attenuated when beta-adrenoceptor antagonists is microinjected into the BST. Our data suggests that Fos induction is not a reliable marker for identifying A1 and A2 neurons that are critical for opioid withdrawal-induced aversions. This is because the Fos induction in these neurons could mostly be attributed to peripheral withdrawal, which does not cause place aversions (Hand et al., 1988). Induction of Fos in VLM catecholamine neurons could indicate the activation of autonomic motor pathways. The adrenergic C1 neurons in the rostral VLM include pre-sympathetic bulbospinal neurons that project to the intermediolateral spinal cord and are activated during reflex stimulation of sympathetic outflow (Janig, 1988). The noradrenergic A1 neurons project to both caudal VLM and hypothalamus, and participate in autonomic control as well as activation of the hypothalamicpituitary axis. This interpretation is consistent with the limited ability of peripheral withdrawal to increase FosIR neurons in the ventrolateral PAG, stimulation of which causes a well-defined pattern of responses that include behavioral hyporeactive immobility, sympathoinhibition and anti-nociception (e.g. Keay et al., 1997). We found that injections of naloxone in morphine naı¨ve rats caused induction of Fos in the central nucleus of the amygdala. This has been reported previously and attributed to block of the effects of tonically released endogenous opioids (Carr et al., 1999; Gestreau et al., 2000). Although we did detect some other antagonist effects in morphine naı¨ve rats, these results were ambiguous due to overlaps in the sets of control means that were not significantly different. In the report by Gestreau et al. (2000) naloxone injections in morphine naı¨ve rats also induce Fos-IR neurons in the caudal, intermediate and rostral divisions of the NTS; area postrema and rostral VLM; Ko¨ lliker-Fuse nucleus and supramamillary nucleus. We were unable to detect these effects although a similar trend was apparent in the supramamillary
nucleus. However in our study only catecholamine neurons in the NTS and RV were counted, and the Ko¨ llikerFuse and area postrema were not analysed. Furthermore, to increase the sensitivity of their assay, Gestreau et al. (2000) habituated their animals to handling and saline injections over a 4 day period prior to administering the experimental injections used to induce Fos. In conclusion, our study has shown that peripheral withdrawal precipitated by antagonism of opioid receptors located outside the central nervous system can increase Fos-IR neurons in the brain. The induction of Fos was widespread and was observed in a variety of brain regions that have been implicated in motivational aspects of opioid addiction. Peripheral withdrawal mechanisms are thought not to contribute to the development of addiction, which is consistent with the absence of abuse liability reported for peripherally selective opioid agonists such as the antidiarrheal agent loperamide (Jaffe et al., 1980; Korey et al., 1980). If peripheral withdrawal can alter immediate-early gene expression in the brain then it could also contribute significantly to other molecular or cellular changes that are induced in the brain when opioid withdrawal is precipitated systemically. If so, then the motivational significance of these changes in the context of addiction warrants further investigation.
Acknowledgement Supported by NHMRC project grant 971126 (P.B.O.)
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