BRAIN RESEARCH

ELSEVIER Brain Research 717 (1996) 127-134

Research report

Distribution of c-Fos in guinea-pig brain following morphine withdrawal

L . A . C h a h l a , , , j . L e a h b, T. H e r d e g e n c, L . T r u e m a n b, A . M . L y n c h - F r a m e a

a Faculty of Medicine and Health Sciences, University of Newcastle, Newcastle, N.S.W. 2308, Australia b School of Science, Griffith University, Nathan 4111, Australia

c H Physiologisches lnstitut, Universitiit Heidelberg, D6900 Heidelberg, Germany

Accepted 27 December 1995

Abstract

The distribution of the immediate-early gene and transcription factor protein, c-Fos, was examined in the brains of guinea-pigs following treatment with morphine, naloxone or naltrexone, or the induction of morphine withdrawal by these opioid antagonists. Guinea-pigs were given subcutaneous injections of morphine sulphate or tartrate three times per day in increasing doses for three days (total dose 690 mg/kg as base). Control animals received saline injections. Naloxone hydrochloride (30 mg/kg), naltrexone hydrochloride (15 mg/kg) or saline was administered subcutaneously 1 h after the last dose of morphine or saline, and the animals killed 1.5 h later by perfusion-fixation under deep sodium pentobarbitone anaesthesia. In the animals that were treated with morphine and withdrawn with either naloxone or naltrexone, c-Fos was expressed in neurons in many brain areas, including the frontal and cingulate cortices, olfactory tubercles, ventral pallidum, nucleus accumbens, habenular, paraventricular thalamic nucleus, septal and arcuate nuclei, lateral and posterior hypothalamic areas, ventral tegmental area, central grey, dorsal raphe nucleus, locus coeruleus, raphe magnus, lateral paragigantocellular nucleus and solitary tract nucleus. In contrast, only low levels of c-Fos were found in brains of animals that had been treated for three days with morphine followed by saline, or with saline followed by naltrexone or naloxone. The widespread distribution of c-Fos induced by morphine withdrawal reflects the complexity of the accompanying behavioural and autonomic responses.

Keywords: c-Fos; Morphine; Naltrexone; Opioid; Withdrawal; Brain; Guinea-pig

1. Introduct ion

Morphine and other opioids are widely used clinically for their powerful analgesic action. In addition to analge- sia, morphine produces a number of striking pharmacologi- cal effects, the most notable of which include respiratory depression, reward, reinforcement and dependence. The latter effect is manifest as a withdrawal response on cessa- tion of drug administration or administration of an opioid antagonist. Although the dopaminergic pathways of the mesolimbic system have been strongly implicated in the rewarding and reinforcing effects of opioids [24], the rela- tionship of these pathways to those involved in be- havioural changes, dependence and analgesia is still not clear.

The immediate-early genes and transcription factors c-fos, c-jun and krox-24 are rapidly expressed in cells by many stimuli, and numerous studies have now successfully

* Corresponding author. Fax: (61) (49) 602088.

0006-8993/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PII S0006-8993(96)00041-8

used expression of their mRNAs (written here as, e.g., c-fos) or proteins (e.g., c-Fos) to map the distribution of central nervous system neurons activated by stimulation in vivo. In previous studies acute morphine treatment of rats was found to increase the expression of c-fos mRNA a n d / o r c-Fos in caudate putamen [9,20], nucleus accum- hens [20] and ventromedial hypothalamus [7], but both acute and chronic morphine treatment reduced c-fos in several other regions, including the locus coeruleus [15]. In the spinal cord a single dose of morphine suppressed c-Fos expression evoked by noxious pressure in arthritic rats [1]. In contrast, morphine withdrawal following chronic treat- ment with morphine produced marked increases in the levels of c-fos and c-Fos in the locus coeruleus, amygdala, ventral tegmental area, nucleus accumbens, neostriatum and cerebral cortex [15]. However, no systematic study of the effects of morphine, opioid antagonists or morphine withdrawal on the induction of c-fos mRNA or c-Fos protein expression throughout the brain has been reported. Such a study would add to knowledge regarding the neurons activated by opioid withdrawal.

128 L.A. Chahl et al./Brain Research 717 (1996) 127 134

In the present study the distributions of c-Fos-labelled cells following treatment with morphine, naloxone, nal- trexone, and the induction of withdrawal by naloxone and naltrexone following chronic morphine treatment, were investigated in guinea-pig brain. Guinea-pigs were chosen because, like humans and unlike rats, they are clearly sedated by morphine [3], and they exhibit a marked loco- motor response on induction of withdrawal with an opioid antagonist [4,6,18]. Furthermore, guinea-pig brain has a proportion of opioid receptor subtypes which resembles that found in human brain [21].

2. Materials and methods

2.1. Treatment of animals

Adult guinea-pigs of either sex, 410-650 g, were made tolerant-dependent on morphine by administration of sub- cutaneous (s.c.) injections of morphine sulfate, 30 mg/ml , or tartrate, 120 rag/1.5 ml, at higher doses, at 8-hourly intervals in increasing doses for 3 days (total dose 690 m g / k g of morphine as base) according to the protocol described by Chahl [5]. On the first day guinea-pigs re- ceived doses of 10, 20, 40 mg/kg , on the second day, 40, 80, 100 mg/kg , and on the third day, 100, 100, 100 mg/kg . On the morning of the fourth day they were given a final dose of morphine, 100 mg/kg . Naloxone hydro- chloride (30 mg /kg ) (n = 3) or naltrexone hydrochloride (15 m g / k g ) (n = 4) was given s.c. I h later to induce withdrawal. Three control guinea-pigs were treated with morphine as described above but were given saline instead of naloxone or naltrexone. Other animals were given saline injections instead of morphine, and then saline (n = 2), naloxone (n = 2) or naltrexone (n = 2). The behaviour of the animals was observed for 90 rain, but was not quanti- fied for the present study. Animals were given sodium pentobarbitone, 80 mg/kg , intraperitoneally, 90 rain after the opioid antagonist or saline injection, and fixed by transcardial perfusion with warm heparinized phosphate- buffered saline (PBS) followed by 4% paraformaldehyde in phosphate buffer. The brains were removed, post-fixed overnight at 4°C and placed into 30% sucrose fixative solution for cryoprotection.

2.2. Localization of c-Fos

Coronal sections, 50 /xm, were cut on a cryostat. Free floating sections were washed in PBS, incubated for 20 min in methanol containing 0.3% hydrogen peroxide, washed in PBS and incubated for 1 h at room temperature in PBST (PBS containing 0.3% Triton X-100) containing 5% normal goat serum (blocking solution). They were then incubated in one of three different rabbit c-Fos antisera, viz. Fos 689/5 (diluted 1:15,000, obtained from Dr. Bravo, Squibb-Bristol Myers, Princeton) [19], c-Fos(4) (diluted

1:6000, from Santa Cruz, sc-52), or c-Fos (diluted 1:20,000, obtained from S. Hunt) [17], in blocking solution for 48 h at 4°C. Some sections were incubated in Jun and Krox-24 antisera [19]. Control sections were incubated in normal rabbit antiserum diluted in blocking solution. Sections were then washed three times in PBST and incubated for 1 h at room temperature in biotinylated anti-rabbit antibody diluted in blocking solution. After washing again, the sections were incubated for 2 h in avidin-biotin-peroxidase complex (Vectastain), washed and stained with 3,3'-di- aminobenzidine tetrahydrochloride 0.2 m g / m l solution in PBS containing 0.01% hydrogen peroxide, 0.01% nickel ammonium sulfate and 0.01% cobalt chloride.

Briefly, the characteristics of the c-Fos antisera were as follows. Fos 689/5, raised against the complete c-Fos molecule, showed a strong reaction with c-Fos, no reaction with Fos B and only a weak reaction with the Fos-related antigen, Fra-1 [19]. The c-Fos antibody obtained from Hunt was raised against the peptide sequence common to all Fos proteins and thus reacted with all members of the Fos protein family [17]. The commercially available anti- body c-Fos(4) from Santa Cruz, was highly selective for c-Fos and would not recognize other Fos proteins or Fos-related antigens.

Cells positive for c-Fos were plotted by camera lucida, using the adjacent neutral red stained sections as guides. No cells were stained in control sections. The results are presented semiquantitatively according to the density of c-Fos labelled cells in each area: + + + , for areas with almost all cells labelled, to + , for areas with only a sparse distribution of labelled cells.

3. Results

A semiquantitative representation of the distribution of c-Fos expression in brain regions of guinea-pigs that were treated for 3 days with morphine and withdrawn with naltrexone is shown in Table 1. The distributions of c-Fos in corresponding sections from the brains of control guinea-pigs treated with morphine for 3 days followed by saline, and of animals treated with saline for 3 days followed by naltrexone, are also shown in Table 1. Ani- mals given only saline injections showed virtually no c-Fos-labelled cells in any brain region (not shown). Jun and Krox-24 proteins could not be detected in guinea-pig brain following morphine withdrawal.

3.1. Expression of c-Fos induced by morphine

In guinea-pigs treated for 3 days with morphine and not withdrawn (Table 1), sparse c-Fos labelling was found in some of the same regions as in morphine withdrawn guinea-pigs (see below). These regions included the cingu- late, frontal, piriform, and retrosplenial cortical areas, ol- factory tubercle, median preoptic area, bed nucleus of stria

L.A. Chahl et al. /Bra in Research 717 (1996) 127-134 1 2 9

T a b l e 1

A r e a s t h a t s h o w e d c - F o s e x p r e s s i o n f o l l o w i n g m o r p h i n e , n a l t r e x o n e , a n d n a l t r e x o n e - i n d u c e d m o r p h i n e w i t h d r a w a l

B r a i n a r e a M o r p h i n e N a l t r e x o n e M o r p h i n e / N a l t r e x o n e

A 5 n o r a d r e n a l i n e c e l l s - - +

A H i a m y g d a l o h i p p o c a m p a l a r e a - + + +

A r c a r c u a t e n. - + + +

B S T b e d n. o f s t r i a t e r r n i n a l i s - + +

C G c e n t r a l g r e y - + + +

C G D c e n t r a l g r e y , d o r s a l - + +

C g c i n g u l a t e c o r t e x + + + +

C l i c a u d a l l i n e a r r a p h e n. - - + +

D R d o r s a l r a p h e n. + + + +

E n t e n t o r h i n a l c o r t e x - + +

E W E d i n g e r W e s t p h a l n. - + + +

F r f r o n t a l c o r t e x + + +

H D B n. o f h o r i z o n t a l l i m b o f d i a g o n a l b a n d - - + +

I C i n f e r i o r c o l l i c u l u s + + +

I L i n f r a l i m b i c c o r t e x - + + +

I P i n t e r p e d u n c u l a r n. - - +

L A l a t e r o a n t e r i o r h y p o t h a l a m i c n, - - + +

L C l o c u s c o e r u l e u s - - +

L D T g l a t e r a l d o r s a l t e g m e n t a l n. - - + +

L H l a t e r a l h y p o t h a l a m i c a r e a - - + +

L H b l a t e r a l h a b e n u l a r n. - + 4. 4.

L P B l a t e r a l p a r a b r a c h i a l n~ - - + 4.

L P G i l a t e r a l p a r a g i g a n t o c e l l u l a r n. - + +

L S I l a t e r a l s e p t a l n . , i n t e r m e d i a t e p a r t - - + + +

L S V l a t e r a l s e p t a l n . , v e n t r a l - + 4- 4.

M H b m e d i a l h a b e n u l a r n. - + + +

M G m e d i a l g e n i c u l a t e n. - - + +

M P O m e d i a l p r e o p t i c n. - 4. + +

M n R m e d i a n r a p h e n. - - +

M S m e d i a l s e p t a l n. - - + + +

M P A m e d i a l p r e o p t i c a r e a 4. - + + 4.

M V e m e d i a l v e s t i b u l a r n. - + 4.

O c o c c i p i t a l c o r t e x - - 4- 4.

P a p a r a v e n t r i c u l a r h y p o t h a l a m i c n. - - + 4.

P a r p a r i e t a l c o r t e x - - +

P e p e r i v e n t r i c u l a r h y p o t b a l a m i c n. - + 4- + +

P F p a r a f a s c i c u l a r t h a l a m i c n. - - +

P H p o s t e r i o r h y p o t h a l a m i c a r e a - + + +

P i t p i r i f o r m c o r t e x + + + 4- + +

P n p o n t i n e n. - - +

P P T p o s t e r i o r p r e t e c t a l n. - - + +

P V A p a r a v e n t r i c u l a r t h a l a m i c n . , a n t e r i o r - 4. + + +

P V G p e r i v e n t r i c u l a r g r e y - - + +

P V P p a r a v e n t r i c u l a r t h a l a m i c n . , p o s t e r i o r - - 4- 4. +

R r e d n. - - + +

R M g r a p h e m a g n u s n. - + +

R P n r a p h e p o n t i s n. - - +

R R F r e t r o r u b r a l f i e l d - - + +

R S r e t r o s p l e n i a l c o r t e x + + + +

S s u b i c u l u m - 4, +

S C s u p e r i o r c o l l i c u l u s - - +

S C h s u p r a c h i a s m a t i c n. + - 4. + +

S H y s e p t o h y p o t h a l a m i c n. - + +

S N s u b s t a n t i a n i g r a - - + +

S o l n. o f s o l i t a r y t r a c t - - +

S p 5 s p i n a l t r i g e m i n a l n. - - +

S t H y s t r i o h y p o t h a l a m i c n. - - + + +

T u o l f a c t o r y t u b e r c l e 4. - + + +

V D B n. o f v e r t i c a l l i m b o f d i a g o n a l b a n d - - + +

V M H v e n t r o m e d i a l h y p o t h a l a m i c n. - - 4. +

V P v e n t r a l p a l l i d u m - - + + +

V T A v e n t r a l t e g m e n t a l a r e a + - 4. +

130 L.A. Chahl et al./Brain Research 717 (1996) 127 134

A central grey, inferior colliculus, dorsal raphe, raphe mag- nus and lateral paragigantocellular nuclei. However, the extent of the distribution of c-Fos expressed in these areas in saline-treated animals given naltrexone was much less than in animals withdrawn from morphine (Table 1). Fur- thermore, the distribution of c-Fos in cortical areas induced by naltrexone in saline-treated animals differed from that produced by chronic morphine treatment (Fig. 1).

3.3. Expression q[ c-Fos resulting from naltrexone-induced morphine withdrawal

B

C

Fig. 1. Diagrammatic representation of coronal sections of guinea-pig forebrain showing distribution of c-Fos-positive neurons induced by naltrexone-induced morphine withdrawal (A), by chronic morphine treat- ment (B), and by naltrexone following chronic saline treatment (C). For description see text and abbreviations in Table 1.

terminalis, suprachiasmatic nucleus, inferior colliculus, and dorsal raphe nucleus. The only area with marked c-Fos expression in these guinea-pigs was the rostral region of the piriform cortex (Fig. 1).

3.2. Expression o[ c-Fos induced by naltrexone

Sparse c-Fos was also found in the brains of guinea-pigs treated for 3 days with saline injections and then given naltrexone (Table 1). Several of these areas were also affected by naltrexone-induced withdrawal in morphine treated guinea-pigs; for example, the frontal, cingulate, retrosplenial, piriform and entorhinal cortical areas, bed nucleus of stria terminalis, septal, habenular and hypotha- lamic nuclei, amygdalohippocampal area, substantia nigra,

All guinea-pigs treated chronically with morphine ex- hibited a marked withdrawal response upon injection of naloxone or naltrexone. This was characterized by intense locomotor activity interspersed with bouts of digging, rear- ing, chewing, face-washing and grooming. None of these behaviours, or any other change in behaviour was ever observed in any animal given saline alone, morphine fol- lowed by saline, or saline injections followed by naloxone or naltrexone. These behaviours were identical in nature to those observed in guinea-pigs given a single dose of morphine followed by naloxone [18], although the re- sponses following 3 days' treatment with morphine were both more marked and longer lasting (70-90 min for animals given naltrexone, 40-60 min for animals given naloxone) than those observed in guinea-pigs given a single dose of morphine and withdrawn with naloxone (15-20 min). Naloxone hydrochloride, 30 mg/kg , was used in the first experiments to induce withdrawal. This dose was higher than that used in previous experiments (15 mg/kg) , to induce withdrawal following a single dose of morphine [18]. However, in the present study, the higher dose of naloxone was found to be necessary to induce a long lasting withdrawal response, presumably because naloxone has a short half-life. Qualitatively similar results were obtained using naloxone hydrochloride, 30 mg/kg , and naltrexone hydrochloride, 15 mg/kg , to induce with- drawal, but the response was longer lasting with naltrexone and thus it was used in all subsequent experiments.

It is seen in Table 1 that morphine withdrawal induced widespread c-Fos expression in the guinea-pig brain, in marked contrast to the effects of either morphine or nal- trexone alone which each produced much less c-Fos ex- pression. Morphine withdrawal induced c-Fos in several cortical areas, viz. the frontal, cingulate, retrosplenial, piriform and entorhinal regions, c-Fos was also found in the olfactory tubercle, ventral pallidum, caudate-putamen, globus pallidus, amygdala, nucleus accumbens, diagonal band, bed nucleus of stria terminalis, septal, preoptic, habenular and hypothalamic nuclei, paraventricular and parafascicular thalamic nuclei, central grey, substantia ni- gra, ventral tegmental area (Fig. 2), red nucleus and supe- rior colliculus. Surprisingly, there were few c-Fos-labelled cells in the hippocampus (Fig. 3), but several cells in the subiculum expressed c-Fos. More caudally, morphine

L.A. Chahl et a l . /Brain Research 717 (1996) 127-134 131

m,

Fig. 2. Photomicrographs showing neurons positive for c-Fos in the rostral ventral tegmental area of guinea-pig brain following naltrexone-in- duced morphine withdrawal. Bar = 200 /xm (upper) and 80 /xm (lower).

i - ' 4 :

?~ "

withdrawal induced c-Fos in the dorsal raphe, raphe mag- nus, lateral dorsal tegmental nuclei, inferior colliculus, locus coeruleus, lateral paragigantocellular nucleus and the nucleus of the solitary tract.

Qualitatively similar results were obtained with all three c-Fos antibodies, although some differences in the number of c-Fos-positive cells were found. In Fig. 4 it can be seen that the c-Fos(4) antibody from Santa Cruz detected a slightly higher number of cells in adjacent sections from the same guinea-pig than the c-Fos antibody from S. Hunt.

4. Discussion

Chronic morphine treatment induced a small expression of c-Fos in several, mainly cortical, areas of the guinea-pig brain. It is not known whether the effect of morphine on the expression of c-Fos, which was presumably induced by

k . /

ii!iii!:ii~iii!!iiii~i!;~il

Fig. 3. Photomicrographs of guinea-pig cortex (upper) and hippocampus (middle and lower) from the same section, showing relative lack of neurons positive for c-Fos following naltrexone-induced morphine with- drawal in the hippocampus. Bar = 200 /xm (middle) and 80 /xm (upper and lower).

132 L.A. Chahl et al. / Brain Research 717 (1996) 127-134

A Locus cocmleus

• " ...!:.:-...:. ..'!~':,.':'.. ::. :. . :i:?;......'

B

" .~b .. '~: "

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C .. [ Ventral PAG \ ~ ....

;:"i: . ~~!~" • .. .. ~,::... .:..... . .: .,. . '.';~.~' ..' .:. -..:...

':'. • , ' " . ' : . . , . : . A . . ' . " • , " ~ , ' ~ " g ' - . ~ . . ~ . ' " " , . ' " • ~ " . . " • . • " ' . 4 ~ : : : , , ' . " . , ~ . . " . " ~ . ' ~ " " ' • " •

• ' " • "'.' "'> " . ! ' "~,~ '. ' " z " ' :

Fig. 4. Camera lucida drawings showing the distributions of c-Fos- posit ive neurons in locus coeruleus (A), habenular and paraventricular thalamic area (B), and ventral periaqueductal gray (PAG) (C) in two

adjacent sections from a guinea-pig brain following naltrexone-induced morphine withdrawal. For each brain level the drawing on the left shows c-Fos neurons detected by the c-Fos antibody used by Hunt [17], and the drawing on the right shows neurons detected by the Santa Cruz c-Fos(4)

antibody.

neuronal excitation, was produced in inhibitory neurons or excitatory neurons. Although the overall behavioural effect of morphine in the guinea-pig is inhibitory, the possibility remains that excitatory effects might occur in some brain pathways, either indirectly by disinhibition such as has been shown to occur in the hippocampus [26], or directly such as has been shown to occur in sensory neurons [12]. If such excitatory effects of morphine do occur in the central nervous system, they may be masked by inhibition of downstream neurons.

The present results are in general agreement with the previous observations that morphine induced c-Fos and/or c-fos mRNA in rat brain [7,9,20]. However, the effect of chronic morphine treatment of guinea-pigs on c-Fos ex- pression was less than that induced by acute administration of morphine (unpublished observations). This observation

is in agreement with that of Chang et al. [8] who showed loss of c-Fos responsiveness of rat brain regions normally responsive to acute treatment, following chronic treatment with morphine, ethanol or cocaine. Activation and subse- quent down-regulation of immediate-early genes was also seen in neuroblastoma cells in vitro following chronic morphine treatment. When these cells were treated daily with morphine each dose caused a transient expression of c-fos, with the amplitude of each expression decreasing over 7 days. Concomitantly, there was a persisting back- ground elevation in c-fos that increased over the 7 days [10]. In the rat nucleus accumbens, acute cocaine treatment caused the expression of c-fi)s, c-jun, fos B, jun B and krox-24 [16]. However, chronic cocaine treatment resulted in down-regulation of these immediate-early genes, an effect which could not be overcome by a further acute challenge with cocaine [16].

The effects of chronic morphine found in the present study would most likely have been affected by the devel- opment of tolerance to the effects of morphine, although suppression of c-fos expression might have played a role. The mechanisms that mediate down-regulation and long- term suppression of immediate-early gene responses on chronic drug treatment are not known. The protein, c-Fos, binds to DNA at AP-I sites and acts as a transcription factor, regulating the expression of other genes. It has been proposed that chronic drug treatments, through the induc- tion of novel Fos-related proteins, might alter the configu- ration of the AP-1 complex and thus alter its transcrip- tional activity and lead to changes in gene expression such that prolonged suppression of c-los transcription occurs [23]. Alternatively, c-Fos may inhibit production of a k i n a s e / p h o s p b a t a s e that specifically phospho- rylates/dephosphorylates proteins, including transcription factors that regulate c-Fos expression. Another possibility is that there is a long-term inhibition of membrane recep- tors, ion channels and second messenger systems necessary for stimulus-induced c-Fos expression.

Naltrexone and naloxone, given to animals not previ- ously treated with morphine, had slight effects in several areas of the central nervous system. However, morphine withdrawal produced a much more marked effect in these areas than either chronic morphine or the opioid antago- nists alone. Withdrawal also induced c-Fos expression in many other areas which have been previously implicated in rewarding, analgesic and dependence/withdrawal ef- fects of opioids [24,25], including the nucleus accumbens, ventral tegmental area, hypothalamic nuclei, striatum, sub- stantia nigra, central grey, locus coeruleus and raphe nu- clei. It is possible that c-Fos was induced in different cell populations in some of the same areas by morphine, opioid antagonists, and morphine withdrawal, but this cannot be determined from the present study,

The pattern of c-Fos expression induced by withdrawal from chronic morphine reflects the wide distribution of endogenous opioid peptides and opioid receptors [21].

L.A. Chahl et al. / Brain Research 717 (1996) 127-134 133

Such an extensive expression of c-Fos following with- drawal in animals chronically treated with morphine would be expected in light of the major behavioural and auto- nomic changes that occur with morphine withdrawal, and the wide distribution of opioid receptors in the central nervous system. In agreement with the findings of Hay- ward et al. [15] who examined c- fos distribution in rat brain following morphine withdrawal using immuno- blotting, Northern blotting and in situ hybridization, c-Fos was found in the locus coeruleus, amygdala, ventral tegmental area, nucleus accumbens, neostriatum and cere- bral cortex of the guinea-pig brain. A notable finding in both the present study and that of Hayward et al. [15] was the relative lack of induction of c-Fos in the hippocampus. This lack of induction of c-Fos in the hippocampus follow- ing morphine withdrawal is worthy of further investiga- tion, since c-Fos is readily induced in the hippocampus by many other stimuli.

In the present study c-Fos was found in a number of brain regions including the dorsal raphe, periaqueductal grey and lateral paragigantocellular nucleus, where c-Fos was not found in the study of Hayward et al. [15]. It is possible that these differences represent species differences resulting from the different distributions of opioid recep- tors, since the high dose of morphine used in chronic treatment might act not only on /x-opioid receptors but also on 8- and K-receptors, and guinea-pigs have a greater number of K-receptors than rats [21]. However, it is no- table that Chieng et al. [11] also found increased c-Fos in the ventrolateral periaqueductal grey in rats following mor- phine withdrawal. Activation of neurons in the periaque- ductal grey would be expected, since this area is consid- ered to play a major role in the autonomic and behavioural responses to opioid withdrawal [2].

It is unlikely that the present results were confounded by the presence of Fos B or Fos-related antigens, because two of the three antisera used have been reported to be highly specific for c-Fos. Moreover, at the time of killing the animals (90 min after induction of withdrawal), c-Fos would have been the major antigen present, the Fos-related antigens being induced later than c-Fos itself [22]. Differ- ences in affinity of the antibodies might have been respon- sible for the slightly different numbers of c-Fos positive neurons seen with different antisera. Surprisingly, expres- sion of other immediate-early gene proteins, Fos B, c-Jun, Jun B or Krox-24, was not detected in the guinea-pig brain following morphine withdrawal, although c- jun was found in rat brain following morphine withdrawal [15].

It has been shown using regional cerebral glucose uti- lization, measured by [~4C]2-deoxyglucose autoradiog- raphy (2-DG), that morphine withdrawal produced dose- dependent increases in glucose utilization, particularly in the diagonal band, medial and lateral preoptic areas, globus pallidus, paraventricular hypothalamus, lateral hypothala- mus, nucleus accumbens, medial and lateral septum, cen- tral nucleus of amygdala, medial and lateral mammillary

nuclei, anteroventral thalamus, lateral habenular, ventral tegmental area, interpeduncular nucleus and dorsal and median raphe [13]. Other regions such as cortical areas, medial geniculate nucleus and red nucleus did not show morphine dose-dependent changes in 2-DG uptake. Studies on the time-dependent nature of regional cerebral glucose utilization responses indicated that the earliest effects dur- ing naloxone-induced morphine withdrawal occurred in the lateral septum and lateral habenular [14], areas where c-Fos was found in the present study. Furthermore, it was shown that activation of /~-opioid receptors was sufficient to produce both the behavioural changes and the pattern of regional cerebral glucose utilization induced by morphine [25]. Thus, regional cerebral glucose utilization has been shown to be a sensitive and specific indicator of the brain regions involved in morphine withdrawal [25]. Although dose- and time-dependence were not investigated in the present study, the results are in overall agreement with those using glucose utilization, and have thus established that c-Fos is a sensitive indicator of morphine withdrawal, not only at the regional level, but also at the level of individual neurons.

The genes for the precursor proteins for the enkephalins, dynorphins, somatostatin, and nerve growth factor, all contain AP-1 sites in their promoter regions, and thus their expression could be changed by the c-Fos induced by opioid withdrawal, c-Fos, and other transcription factors, are also likely to bind to the promoters of other genes whose proteins produce long-lasting or even permanent alterations in the functioning of the neurons in which they are expressed. Some of these proteins could change the responses of neurons to subsequent exposures to opioids or their antagonists, and mediate the phenomena of tolerance and dependence. Thus it may be that long after chronic morphine treatment and antagonist-induced withdrawal, the responses of the neurons to further applications of morphine and /or its antagonists would be altered. Such a possibility warrants further investigation.

Acknowledgements

This study was supported by the National Health and Medical Research Council of Australia.

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