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Amygdalar vasopressin mRNA increases in acute cocaine withdrawal: Evidence for opioid receptor modulation
Neuroscience 134 (2005) 1391–1397
AMYGDALAR VASOPRESSIN mRNA INCREASES IN ACUTE COCAINE WITHDRAWAL: EVIDENCE FOR OPIOID RECEPTOR MODULATION Y. ZHOU,* J. T. BENDOR, V. YUFEROV, S. D. SCHLUSSMAN, A. HO AND M. J. KREEK
Key words: hypothalamic–pituitary–adrenal axis, proopiomelanocortin, hypothalamus, ACTH.
Laboratory of the Biology of Addictive Diseases, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA
In humans, stress has long been recognized as a major factor contributing to relapse to drug abuse in abstinent individuals. All types of stressors enhance vulnerability to drug abuse. Drugs of abuse themselves or withdrawal from such drugs acts as a stressor (see reviews by Kreek, 1992; Kreek and Koob, 1998). In the animal model, stress modulates the effects of drugs of abuse on the acquisition of drug self-administration behavior and reinstatement of drug self-administration (Shaham et al., 1996). For instance, increased corticotropin-releasing factor (CRF) neuronal activity in the central nucleus of the amygdala is involved in the stress-like or anxiogenic consequences of withdrawal that are common in phenomenology to many drugs of abuse (Weiss et al., 2001; Zhou et al., 2003b). Recent evidence suggests that increased vasopressinergic neuronal activity in the amygdala or hypothalamus represents an important step in the neurobiology of stressrelated behaviors. For example: (1) acute stress increases extracellular levels of arginine vasopressin (AVP) in the rat amygdala or hypothalamus (Ebner et al., 2002; Wigger et al., 2004); and (2) activation of V1 receptors by AVP may underlie anxiogenic and depressive behaviors in the rat (Liebsch et al., 1996; Griebel et al., 2002; Wigger et al., 2004). Stress-related anxiety and depression are major psychiatric consequences of chronic drug abuse and withdrawal in drug addiction (Kreek and Koob, 1998). Of possible relevance, AVP gene expression in extrahypothalamic brain regions, including the medial amygdala, is regulated by testosterone (Szot and Dorsa, 1994). Tolerance to the stimulatory effects of cocaine on the hypothalamic–pituitary–adrenal (HPA) axis develops after chronic “binge” cocaine; this blunting of HPA axis activity is associated with a cocaine-induced reduction of CRF mRNA levels in the hypothalamus (Zhou et al., 1996). During short-term (2 days) cocaine withdrawal, there is increased HPA activity. In contrast to acute (3 h) “binge” cocaine, cocaine withdrawal does not lead to an increase in CRF mRNA levels in the hypothalamus (Zhou et al., 2003a,b). Hypothalamic AVP potentiates CRF-induced adrenocorticotropin hormone (ACTH) secretion from the anterior pituitary (Antoni, 1993). Acute stress results in increased CRF mRNA expression in the paraventricular nucleus (PVN), whereas after chronic stress a preferential activation of AVP gene expression in the PVN is observed (Aguilera, 1994). It remains unclear whether the stimulatory effects of acute cocaine withdrawal on the HPA axis
Abstract—In humans, stress is recognized as a major factor contributing to relapse to drug abuse in abstinent individuals; drugs of abuse themselves or withdrawal from such drugs act as stressors. In the animals, evidence suggests that centrally released arginine vasopressin in both amygdala and hypothalamus plays an important role in stressrelated anxiogenic behaviors. The stress responsive hypothalamic–pituitary–adrenal axis is under tonic inhibition via endogenous opioids, and cocaine withdrawal stimulates hypothalamic–pituitary–adrenal activity. The present studies were undertaken to determine whether: (1) 14-day (chronic) “binge” pattern cocaine administration (45 mg/kg/day) or its withdrawal for 3 h (acute), 1 day (subacute) or 10 days (chronic) alters arginine vasopressin mRNA levels in amygdala or hypothalamus; (2) the opioid receptor antagonist naloxone (1 mg/kg) alters arginine vasopressin mRNA or hypothalamic–pituitary–adrenal hormonal responses in acute cocaine withdrawal; and (3) there are associated changes of mu opioid receptor or proopiomelanocortin mRNA levels. In amygdala, arginine vasopressin mRNA levels were unchanged after chronic “binge” cocaine, but were increased during acute cocaine withdrawal. Naloxone completely blocked this increase. Neither chronic cocaine nor its acute withdrawal altered amygdalar mu opioid receptor mRNA levels. The increase in amygdalar arginine vasopressin mRNA levels was still observed after subacute withdrawal, but not after chronic withdrawal. Although hypothalamic–pituitary– adrenal tolerance developed with chronic “binge” cocaine, there were modestly elevated plasma adrenocorticotropin hormone levels during acute withdrawal. While naloxone produced modest adrenocorticotropin hormone elevations in cocaine-naïve rats, naloxone failed to elicit an adrenocorticotropin hormone response in cocaine-withdrawn rats. In hypothalamus, neither chronic cocaine nor acute withdrawal altered arginine vasopressin, proopiomelanocortin or mu opioid receptor mRNA levels. These results show that: (1) opioid receptors mediate increased amygdalar arginine vasopressin gene expression during acute cocaine withdrawal, and (2) cocaine withdrawal renders the hypothalamic–pituitary–adrenal axis insensitive to naloxone. Our findings suggest a potential role for amygdalar arginine vasopressin in the aversive consequences of early cocaine withdrawal. © 2005 IBRO. Published by Elsevier Ltd. All rights reserved. *Corresponding author. Tel: ⫹1-212-327-8248; fax: ⫹1-212-327-8574. E-mail address: [email protected] (Y. Zhou). Abbreviations: ACTH, adrenocorticotropin hormone; ANOVA, analysis of variance; AVP, arginine vasopressin; CRF, corticotropin-releasing factor; HPA, hypothalamic–pituitary–adrenal; MOP-r, mu opioid receptor; NIL/PL, neurointermediate lobe/posterior lobe of the pituitary; POMC, proopiomelanocortin; PVN, paraventricular nucleus.
0306-4522/05$30.00⫹0.00 © 2005 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2005.05.032
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are associated with AVP mRNA changes in the hypothalamus. Mu opioid receptor (MOP-r) radioligand binding density is increased in the rat amygdala after chronic “binge” cocaine (Unterwald et al., 1992). The stress responsive HPA axis is under tonic inhibition via endogenous opioid systems, including proopiomelanocortin (POMC)-derived beta-endorphin and MOP-r in the hypothalamus (Nikolarakis et al., 1987; Kreek et al., 2002). Although acute cocaine withdrawal has been reported to increase beta-endorphin release in the rat nucleus accumbens (Roth-Deri et al., 2003), it remains unclear whether there are functional alterations of the endogenous opioid systems within the amygdala or hypothalamus after chronic “binge” cocaine or its acute withdrawal. The present studies were undertaken to determine whether: (1) chronic (14 days) “binge” pattern cocaine administration (45 mg/kg/day) or its withdrawal for 3 h (acute), 1 day (subacute) or 10 days (chronic) alters AVP gene expression in the amygdala or hypothalamus, as measured by AVP mRNA levels; (2) the opioid receptorselective antagonist naloxone (1 mg/kg) alters AVP mRNA or HPA hormonal responses to acute cocaine withdrawal; and (3) there are associated changes of POMC, MOP-r or V1b receptor mRNA levels in the amygdala, hypothalamus or anterior pituitary.
Male Fischer rats (190 –220 g, Charles River Laboratories, Kingston, NY, USA) were housed individually in a stress-minimized facility with free access to food and water. Animals were adapted to a standard 12-h light/dark cycle (lights on from 10:30 h to 22:30 h) for 7 days, and then received i.p. injections of cocaine (3⫻15 mg/kg) or equal volumes of saline for 14 days in their home cages following the “binge” pattern regimen: three times daily at 1 h intervals (11:00, 12:00 and 13:00 h) (Branch et al., 1992). On day 14, an i.p injection of naloxone (1 mg/kg) or saline was administered 30 min after the last “binge” cocaine or saline injection. The naloxone dose chosen was based on a pilot study, in which a single 1 mg/kg dose was observed to moderately increase ACTH levels in cocaine naïve rats (unpublished data). In experiment 1 chronic cocaine, rats were killed by decapitation after a brief exposure to CO2 at 14:00 h on day 14, 1 h after the last “binge” saline or cocaine injection. The 1-hour time point was considered as chronic cocaine as extracellular dopamine levels are elevated in the striatum 1 h after the last cocaine on day 13 of chronic “binge” cocaine (Maisonneuve et al., 1995). In experiment 2 acute (3 h) cocaine withdrawal, rats were killed at 16:00 h on day 14, 3 h after the last “binge” cocaine injection. The 3-hour period following the last cocaine was considered as acute cocaine withdrawal, a time in which extracellular dopamine levels in the striatum are lower than the normal levels: 3 h after the last cocaine on day 13 of chronic “binge” cocaine (Maisonneuve et al., 1995). For both experiments, animals were grouped into four groups of eight animals each for treatment: (1) Saline: “binge” saline injections for 14 days with one saline injection 30 min after
saline control cocaine
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AVP mRNA (pg/µg RNA)
EXPERIMENTAL PROCEDURES
hypothalamus
Fig. 1. Effects of chronic (14-day) “binge” cocaine administration and acute (3-hour) withdrawal from chronic cocaine with or without 1 mg/kg opioid antagonist naloxone (NXN) on AVP mRNA levels in the amygdala or hypothalamus. Data shown in graphs are treatment group mean⫹S.E.M. Significant differences are indicated: * P⬍0.05, Cocaine withdrawal vs. Saline control; # P⬍0.05, NXN⫹Cocaine withdrawal vs. Cocaine withdrawal; ⫹ P⬍0.05, two-way ANOVA with a significant main effect for NXN.
Y. Zhou et al. / Neuroscience 134 (2005) 1391–1397 the last “binge” saline; (2) Cocaine: “binge” cocaine injections for 14 days with one saline injection 30 min after the last “binge” cocaine; (3) Naloxone: “binge” saline injections for 14 days with one naloxone injection (1 mg/kg) 30 min after the last “binge” saline; and (4) Naloxone⫹Cocaine: “binge” cocaine injections for 14 days with one naloxone injection (1 mg/kg) 30 min after the last “binge” cocaine. In experiment 3 subacute (1 day) cocaine withdrawal, rats were killed at 14:00 h on day 15, 1 day after the last “binge” saline or cocaine injection. In experiment 4 chronic (10 days) cocaine withdrawal, rats were killed at 14:00 h on day 24, 10 days after the last “binge” saline or cocaine injection. For both experiments, animals were grouped into two groups of eight animals for each treatment: (1) Saline: “binge” saline injections for 14 days; and (2) Cocaine: “binge” cocaine injections for 14 days. The amygdala, hypothalamus, anterior pituitary and neurointermediate lobe/posterior lobe of the pituitary (NIL/PL) were dissected on ice, homogenized in guanidinium thiocyanate buffer and extracted with acidic phenol and chloroform. The solution hybridization ribonuclease protection–trichloroacetic acid precipitation protocol has been described in detail in earlier reports (Branch et al., 1992; Zhou et al., 1996). A 502 bp fragment from the rat AVP cDNA and a 1201 bp fragment from the rat V1b cDNA were cloned into the polylinker region of pCR II (Invitrogen, Carlsbad CA, USA). A 538 bp fragment from the rat POMC cDNA and a 2100 bp fragment from the rat MOP-r cDNA were cloned into the polylinker region of pSP64 (Promega, Madison, WI, USA) in both the sense and antisense orientations. To determine the total picograms of each mRNA in each extract, the amounts calculated from the standard curves were multiplied by 1.2 for AVP, 2.41 for V1bR, 2.04 for POMC or 5.71 for MOP-r to correct for the difference in length between the sense transcript and the full-length mRNA. At the time of kill, blood from each rat was collected, and plasma was separated. Corticosterone and testosterone levels were assayed using a rat corticosterone or testosterone 125I kit from ICN Biomedicals (Costa Mesa, CA, USA). ACTH immunore-
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activity levels were assayed from unextracted plasma by using a kit from Nichols Institute (San Juan Capistrano, CA, USA). All ACTH, corticosterone or testosterone values were determined in duplicate in a single assay. In experiments 1 and 2, a two-way analysis of variance (ANOVA) (Chronic Treatment: saline, cocaine; Antagonist Condition: saline, naloxone) followed by Newman-Keuls post Shoc tests, or a student’s t-test where appropriate was carried out for each tissue to evaluate the statistical significance of differences among treatment groups. In experiments 3 and 4, a two-way ANOVA (Chronic Treatment: saline, cocaine; Withdrawal Condition: 1-day, 10-day) followed by Newman-Keuls post hoc tests or a planned comparison where appropriate was carried out. To determine correlation between measured variables, linear regression analysis was performed. The accepted level of significance for all tests was P⬍0.05. We followed the Principles of Laboratory Animal Care (NIH publication no 86-23, 1996), and procedures were approved by the Rockefeller University Animal Care and Use Committee. During all procedures of experiments, the number of animals and their suffering by treatments were minimized.
RESULTS Effects of chronic (14-day) “binge” cocaine and acute (3-hour) cocaine withdrawal with naloxone (1 mg/kg) on AVP mRNA levels in the amygdala and hypothalamus Amygdala. In the chronic cocaine experiment, neither chronic “binge” cocaine administration nor naloxone challenge altered AVP mRNA levels (Fig. 1). In the cocaine withdrawal experiment, two-way ANOVA showed a significant Cocaine⫻Naloxone interaction (F(1,27)⫽5.49, P⬍ 0.05) (Fig. 1). Acute withdrawal led to a significant increase
Table 1. Effects of chronic (14-day) “binge” cocaine administration (A) and acute (3-h) withdrawal from chronic cocaine (B) with or without 1 mg/kg opioid antagonist naloxone on mRNA levels (pg/g total RNA) of AVP V1b receptor (V1b), POMC or MOP-r in the amygdala, hypothalamus, anterior pituitary or NIL/PL, and on plasma testosterone levels (ng/ml) A
Amygdala Hypothalamus Anterior pituitary NIL/PL Plasma
MOP-r POMC MOP-r V1b POMC POMC Testosterone
Saline control
Chronic cocaine
Naloxone
Naloxone⫹Chronic cocaine
0.94⫾0.07 7.7⫾1.2 1.3⫾0.04 0.59⫾0.04 184⫾18 4314⫾394 2.6⫾0.29
0.97⫾0.05 8.2⫾1.8 1.3⫾0.10 0.70⫾0.05* 198⫾33 4249⫾308 2.1⫾0.83
0.97⫾0.06 6.2⫾0.59 1.2⫾0.07 0.58⫾0.04 186⫾21 4555⫾204 3.7⫾0.59
0.94⫾0.12 6.5⫾0.80 1.2⫾0.09 0.73⫾0.02* 183⫾15 4175⫾312 2.2⫾0.38
Saline control
Cocaine withdrawal
Naloxone
Naloxone⫹Cocaine withdrawal
1.08⫾0.06 6.0⫾1.3 1.2⫾0.08 0.64⫾0.04 178⫾14 4359⫾400 0.51⫾0.06
1.05⫾0.14 4.9⫾0.88 1.4⫾0.06 0.69⫾0.03 183⫾8.5 4190⫾311 2.8⫾0.74**
0.97⫾0.09 5.2⫾0.83 1.2⫾0.14 0.66⫾0.03 207⫾19 4033⫾371 0.59⫾0.13
1.10⫾0.14 5.8⫾1.08 1.3⫾0.12 0.67⫾0.04 188⫾17 4257⫾375 4.6⫾0.87**#
B
Amygdala Hypothalamus Anterior pituitary NIL/PL Plasma
MOP-r POMC MOP-r V1b POMC POMC Testosterone
Data shown in tables are treatment group mean⫾SEM. Significant differences are indicated: * P⬍0.05, Chronic cocaine or Naloxone⫹Chronic cocaine vs. Saline control; ** P⬍0.001, Cocaine withdrawal or Naloxone⫹Cocaine withdrawal vs. Saline control; # P⬍0.05, Naloxone⫹Cocaine withdrawal vs. Cocaine withdrawal.
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saline control cocaine
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Fig. 2. Effects of chronic (14-day) “binge” cocaine administration and acute (3-hour) withdrawal from chronic cocaine with or without 1 mg/kg opioid antagonist naloxone (NXN) on plasma ACTH and corticosterone levels. Data shown in graphs are treatment group mean⫹S.E.M. Significant differences are indicated: * P⬍0.05 or ** P⬍0.01 vs. Saline control.
in AVP mRNA levels (Newman-Keuls post hoc test, P⬍0.05). When the rats were treated with naloxone during acute withdrawal, the AVP mRNA increase was significantly attenuated (P⬍0.05).
“binge” cocaine resulted in a slight (20%), but significant increase in V1b receptor mRNA levels (P⬍0.05). Neither cocaine withdrawal alone nor with naloxone challenge had an effect on V1b receptor mRNA levels (Table 1B).
Hypothalamus. In the chronic cocaine experiment, chronic “binge” cocaine had no effect on AVP mRNA levels. However, two-way ANOVA showed a significant main effect for Naloxone: a 40% decrease in AVP mRNA levels by 1 mg/kg naloxone administration (F(1,27)⫽7.44, P⬍ 0.05) (Fig. 1). AVP mRNA levels were unaltered by either acute withdrawal alone or with naloxone.
POMC mRNA. POMC mRNA levels in the anterior pituitary and NIL/PL were unaltered by either drug treatment in either experiment (Table 1A and 1B). Effects of chronic “binge” cocaine and acute cocaine withdrawal with naloxone on plasma ACTH, corticosterone and testosterone levels
Effects of chronic “binge” cocaine and acute cocaine withdrawal with naloxone on V1b receptor and POMC mRNA levels in the anterior pituitary and NIL/PL
ACTH. In the chronic cocaine experiment, plasma ACTH levels were unchanged by either drug treatment (Fig. 2). In the cocaine withdrawal experiment, mean plasma ACTH levels were elevated in the Cocaine group (140% Saline), Naloxone⫹Cocaine group (180% Saline), and Naloxone group (170% Saline) (Fig. 2). Two-way ANOVA, however, just failed to show a significant main effect for either treatment (P⫽0.07). Since earlier studies have shown elevated ACTH levels after naloxone challenge [see review by Kreek et al. (2002)] or cocaine withdrawal (Zhou et al., 2003a), Student’s t-test was carried out and showed a significant difference between Saline and each of the other three groups (P⬍0.05).
V1b receptor mRNA. In the chronic cocaine experiment, two-way ANOVA showed a significant main effect for Cocaine (F(1,25)⫽11.3, P⬍0.01) (Table 1A). Chronic
Corticosterone. In the chronic cocaine experiment, two-way ANOVA showed a significant main effect for Cocaine (F(1,25)⫽9.69, P⬍0.005) (Fig. 2). Chronic “binge”
Effects of chronic “binge” cocaine and acute cocaine withdrawal with naloxone on POMC and MOP-r mRNA levels in the amygdala and hypothalamus Neither POMC nor MOP-r mRNA levels in the amygdala or hypothalamus were altered by either drug treatment in either experiment (Table 1A and 1B).
Y. Zhou et al. / Neuroscience 134 (2005) 1391–1397
AVP mRNA (pg/ µ g RNA)
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Fig. 3. Effects of subacute (1-day) or chronic (10-day) withdrawal from chronic (14-day) “binge” cocaine administration on AVP mRNA levels in the amygdala. Data shown in graphs are treatment group mean⫹S.E.M. Significant differences are indicated: * P⬍0.05 vs. Saline control.
cocaine resulted in a significant increase in plasma corticosterone levels (P⬍0.05). Neither cocaine withdrawal alone nor with naloxone had an effect on plasma corticosterone levels. Testosterone. In the chronic cocaine experiment, plasma testosterone levels were unaltered by either drug treatment (Table 1A). In the cocaine withdrawal experiment, two-way ANOVA showed a significant main effect for Cocaine (F(1,27)⫽35.5, P⬍0.0001). Plasma testosterone levels were elevated by acute cocaine withdrawal (P⬍0.05). When the cocaine withdrawn-rats were challenged with naloxone (1 mg/kg), the testosterone increase was significantly enhanced (P⬍0.05) (Table 1B). There was no correlation between amygdalar AVP mRNA and plasma testosterone levels in the rat after acute cocaine withdrawal. Effects of subacute (1-day) or chronic (10-day) withdrawal from chronic (14-day) “binge” cocaine on AVP mRNA levels in the amygdala After subacute cocaine withdrawal, the AVP mRNA levels were increased in the amygdala (Fig. 3). Two-way ANOVA, however, just failed to show a significant main effect for treatments. Since the acute withdrawal study showed elevated AVP mRNA levels in the amygdala, a planned comparison was carried out and showed a significant difference between Saline and 1-day Withdrawal (F(1,24)⫽4.98, P⬍0.05). However, after chronic withdrawal the AVP mRNA levels were no longer elevated in the amygdala (Fig. 3).
DISCUSSION We found that AVP mRNA levels in the amygdala were not altered after chronic “binge” cocaine. However, acute cocaine withdrawal was associated with increased amygdalar AVP mRNA levels, nearly three-fold that of the control level (Fig. 1). Also, this effect occurred at acute withdrawal stage (3 h), persisted for 1 day into subacute cocaine withdrawal, but not for 10 days of chronic withdrawal (Fig. 3). This novel finding parallels our recent studies demonstrating an increased AVP mRNA level in the amygdala during acute opiate withdrawal, but not after chronic opiate administration (Zhou et al., 2004). We further found that a moderate dose of naloxone (1 mg/kg) was able to completely block the AVP mRNA increase, suggesting that the stimulation of amygdalar AVP gene expression by acute cocaine withdrawal is mediated by opioid receptors. This observed interaction between the opioidergic and AVPergic systems may be fundamental to drug withdrawal stress-induced aversive consequences, like anxiogenesis. Earlier work has demonstrated an increased MOP-r binding density in the amygdala of the rat after 14-day chronic “binge” cocaine (Unterwald et al., 1992) and in the amygdala of cocaine-dependent men (Zubieta et al., 1996), suggesting involvement of amygdalar MOP-r functional alterations in cocaine dependence. In the amygdala, however, MOP-r mRNA levels were not affected by either chronic “binge” cocaine or its acute withdrawal (Table 1). Further time-course studies are necessary before alternate explanations, such as indirect
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amygdalar activation due to extraamygdalar naloxone action (e.g. in the nucleus accumbens), can be ruled out. There are relatively few studies on the effects of chronic cocaine or cocaine withdrawal on the brain vasopressinergic systems in the rat. As early as 1992, it was shown that subchronic cocaine administration for 5 days resulted in decreased AVP content in the hypothalamus and amygdala as measured by radioimmunoassay, which may suggest increased release and/or subsequent degradation (Sarnyai et al., 1992). AVP mRNA expression in the medial nucleus of the amygdala is under direct positive regulation by testosterone (Szot and Dorsa, 1994; Albeck et al., 1997). Although both amygdalar AVP mRNA and plasma testosterone levels were elevated during acute cocaine withdrawal, we found no correlation between these two parallel changes. Therefore, the observed effects of cocaine withdrawal on amygdalar AVP mRNA levels may not be directly related to plasma testosterone. The second aim of our study was to examine the effect of acute cocaine withdrawal on hypothalamic AVP gene expression, which is potentially involved in determining HPA activation during cocaine withdrawal. In the present study, 3 h after chronic “binge” cocaine we found similar ACTH elevations as found after 1-day withdrawal (Zhou et al., 2003a). Administration of 1 mg/kg naloxone caused a moderate, but significant, increase in ACTH levels (Fig. 2), consistent with a tonic inhibition of the HPA axis by endogenous opioids. Of interest, the same dose of naloxone did not further increase plasma ACTH levels when the animals were undergoing acute cocaine withdrawal (Fig. 2). Taken together, our results indicate HPA hyposensitivity to the MOP-r preferring antagonist naloxone during acute cocaine withdrawal. Both AVP- and CRF-producing parvocellular PVN cells express MOP-r and activities of these cells are under tonic inhibition by endogenous opioids, including beta-endorphin (Nikolarakis et al., 1987). Our observation of naloxone-insensitive HPA responsivity suggests that alterations in the hypothalamic beta-endorphin/ MOP-r system may contribute to this HPA hyporesponsivity after chronic cocaine. POMC mRNA levels in the hypothalamus were not affected by either chronic “binge” cocaine or its acute withdrawal, nor were hypothalamic MOP-r mRNA levels (Table 1). Thus, reduction of betaendorphin release or MOP-r signaling activity by chronic cocaine or its withdrawal does not necessarily affect their gene expression at the two time points examined. Neither hypothalamic CRF nor anterior pituitary receptor CRF type 1 receptor mRNA levels are altered during short-term (1 day) withdrawal from chronic “binge” cocaine (Zhou et al., 2003b). We hypothesized that increased HPA activity during acute cocaine withdrawal is mediated by alterations in the hypothalamic–pituitary AVP system. However, neither hypothalamic AVP nor anterior pituitary V1b receptor mRNA levels were affected by 3-hour acute cocaine withdrawal (Table 1). Although measurement of AVP mRNA levels in whole hypothalamic RNA extracts may be confounded by potentially differential responses of AVP mRNA levels in parvocellular and magnocellular cells,
a study using the same assays found increased AVP mRNA levels in the hypothalamus in response to acute injection stress (Zhou et al., 2004). The present results argue that the stimulatory effects of cocaine withdrawal on HPA activity may be associated with POMC-derived peptide-releasing factors other than AVP or CRF. Stimulation of the HPA axis is often observed after acute stress, such as acute cocaine administration. This activation of the HPA axis activity is associated with a stress-induced increase in plasma ACTH levels, and rapidly followed by a concomitant increase in plasma corticosterone levels (e.g. Rivier and Vale, 1987; Zhou et al., 1996). After chronic “binge” cocaine administration, however, there is a blunting of ACTH response with a still significant increase in corticosterone levels. Such dissociation of ACTH and corticosterone responses after chronic “binge” cocaine were observed before (Zhou et al., 1996, 2003a) and replicated here. It is possible that the increase in corticosterone is driven by factors other than ACTH (i.e. sympathetic nervous system or direct effect of CRF on the adrenal gland). Another potential explanation for such dissociation after chronic cocaine is that, similar to many cases of chronic stress, the adrenal gland becomes hypersensitive to ACTH (e.g. Sarnyai et al., 1998). In contrast to chronic cocaine, however, immediate cocaine withdrawal led to a significant increase in ACTH levels with a lack of corticosterone response, suggesting that the adrenal gland becomes less responsive to ACTH during this 3-hour immediate withdrawal. Although we did not have later time points, the immediate withdrawal-induced blunting of corticosterone responses seems short-lived. In fact, there is a parallel increase in both plasma ACTH and corticosterone levels during 1– 4 days of short-term withdrawal from chronic cocaine, as we reported before (Zhou et al., 2003a). Our results indicate that there is an uncoupling of ACTH and corticosterone during immediate withdrawal from chronic cocaine administration.
CONCLUSION The present studies determined the effects of chronic “binge” cocaine and its acute withdrawal on stress responsive AVP gene expression. We found that acute cocaine withdrawal resulted in a significant increase in amygdalar AVP mRNA levels. Of interest, amygdalar AVP gene expression stimulated by acute cocaine withdrawal was completely blocked by a single, moderate dose of naloxone, indicating an opioid receptor activation-mediated mechanism. In line with this finding, we have recently found an increase in amygdalar AVP mRNA levels in the rat during acute heroin withdrawal (Zhou et al., 2004), suggesting that there is enhanced vasopressinergic neuronal activity in the amygdala in the aversive and anxiogenic consequences of drug withdrawal. In contrast to the amygdala, we found no effect of either chronic cocaine administration or its acute withdrawal on AVP mRNA levels in the hypothalamus. In this study, we also showed differential responses of HPA and amygdalar stress systems to naloxone. Our finding of HPA hyposensitivity to naloxone after
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acute cocaine withdrawal indicates a decrease in opioidergic tone within the hypothalamus during acute cocaine withdrawal, and is consistent with studies in recently abstinent human cocaine addicts (Schluger et al., 2001). Acknowledgments—The authors would like to thank Dr. G. Aguilera for providing the V1b receptor cDNAs; Dr. G. Uhl for the MOP-r cDNA; Dr. J. Roberts for the POMC cDNA; and Drs. T. Nilsen and P. Maroney for the 18S DNA. The work was supported by NIDA Research Center Grant DA-P60-05130 and DA-00049 (M.J.K.), and by NIH Training Grant GM07524 (J.T.B.).
REFERENCES Aguilera G (1994) Regulation of pituitary ACTH secretion during chronic stress. Front Neuroendocrinol 15:321–350. Albeck DS, McKittrick CR, Blanchard DC, Blanchard RJ, Nikulina J, McEwen BS, Sakai RR (1997) Chronic social stress alters levels of corticotropin-releasing factor and arginine vasopressin mRNA in rat brain. J Neurosci 17:4895– 4903. Antoni FA (1993) Vasopressinergic control of pituitary adrenocorticotropin secretion comes of age. Front Neuroendocrinol 14:76 –122. Branch A, Unterwald E, Lee S, Kreek MJ (1992) Quantitation of preproenkephalin mRNA levels in brain regions from male Fischer rats following chronic cocaine treatment using a recently developed solution hybridization assay. Mol Brain Res 14:231–238. Ebner K, Wotjak C, Landgraf R, Engelmann M (2002) Forced swimming triggers vasopressin release within the amygdala to modulate stress-coping strategies in rats. Eur J Neurosci 15:384 –388. Griebel G, Simiand J, Gal CS, Wagnon J, Pascal M, Scatton B, Maffrand J, Soubrie P (2002) Anxiolytic- and antidepressant-like effects of the non-peptide vasopressin V1b receptor antagonist, SSR149415, suggest an innovative approach for the treatment of stress-related disorders. Proc Natl Acad Sci U S A 99:6370 – 6375. Kreek MJ (1992) Rationale for maintenance pharmacotherapy of opiate dependence. In: Addictive states (O’Brien CP, Jaffe JH, eds), pp. 205–230. New York: Raven Press. Kreek MJ, Koob GF (1998) Drug dependence: stress and dysregulation of brain reward pathways. Drug Alcohol Depend 51:23– 47. Kreek MJ, Borg L, Zhou Y, Schluger J (2002) Relationships between endocrine functions and substance abuse syndromes: Heroin and related short-acting opiates in addiction contrasted with methadone and other long-acting opioid agonists used in pharmacotherapy of addiction. In: Hormones, brain and behavior (Pfaff D, ed), pp781– 830. San Diego: Academic Press. Liebsch G, Wotjak CT, Landgraf R, Engelmann M (1996) Septal vasopressin modulates anxiety-related behavior in rats. Neurosci Lett 217:101–104. Maisonneuve IM, Ho A, Kreek MJ (1995) Chronic administration of a cocaine “binge” alters basal extracellular levels in male rats: an in vivo microdialysis study. J Pharmacol Exp Ther 272:652– 657. Nikolarakis KE, Almeida OFX, Herz A (1987) Feedback inhibition of opioid peptide release in the hypothalamus of the rat. Neuroscience 23:143–148. Rivier C, Vale W (1987) Cocaine stimulates adrenocorticotropin (ACTH) secretion through a corticotrophin-releasing factor (CRF)mediated mechanism. Brain Res 422:403– 406.
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Roth-Deri I, Zangen A, Aleli M, Goelman RG, Pelled G, Nakash R, Gispan I, Green T, Shaham Y, Yadid G (2003) Effect of experimenter-delivered and self-administered cocaine on extracellular -endorphin levels in the nucleus accumbens. J Neurochem 84: 930 –938. Sarnyai Z, Vecsernyes M, Laczi F, Biro E, Szabo G, Kovacs GL (1992) Effects of cocaine on the contents of neurohypophyseal hormones in the plasma and in different brain structures in rats. Neuropeptides 23:27–31. Sarnyai Z, Dhabhar FS, McEwen BS, Kreek MJ (1998) Neuroendocrine-related effects of long-term, “binge” cocaine administration: Diminished individual differences in stress-induced corticosterone response. Neuroendocrinology 68:334 –344. Schluger JH, Borg L, Ho A, Kreek MJ (2001) Altered HPA axis responsivity to metyrapone testing in methadone maintained former heroin addicts with ongoing cocaine addiction. Neuropsychopharmacology 24:568 –575. Shaham Y, Rajabi H, Stewart J (1996) Relapse to heroin-seeking in rats under opioid maintenance: the effects of stress, heroin, priming and withdrawal. J Neurosci 16:1957–1963. Szot P, Dorsa DM (1994) Expression of cytoplasmic and nuclear vasopressin RNA following castration and testosterone replacement: evidence for transcriptional regulation. Mol Cell Neurosci 5:1–10. Unterwald EM, Horne-King J, Kreek MJ (1992) Chronic cocaine alters brain mu opioid receptors. Brain Res 584:314 –318. Weiss F, Ciccocioppo R, Parsons LH, Katner S, Liu X, Zorrilla EP, Valdez GR, Ben-Shahar O, Angeletti S, Richter RR (2001) Compulsive drug-seeking behavior and relapse: neuroadaptation, stress, and conditioning factors. Ann N Y Acad Sci 937:1–26. Wigger A, Sánchez MM, Mathys KC, Ebner K, Frank E, Liu D, Kresse A, Neumann ID, Holsboer F, Plotsky PM, Landgraf R (2004) Alterations in central neuropeptide expression, release, and receptor binding in rats bred for high anxiety: critical role of vasopressin. Neuropsychopharmacology 29:1–14. Zhou Y, Spangler R, LaForge KS, Maggos C, Ho A, Kreek MJ (1996) Corticotropin-releasing factor and CRF-R1 mRNAs in rat brain and pituitary during “binge” pattern cocaine administration and chronic withdrawal. J Pharmacol Exp Ther 279:351–358. Zhou Y, Spangler R, Schlussman SD, Ho A, Kreek MJ (2003a) Alterations in hypothalamic-pituitary-adrenal axis activity and in levels of proopiomelanocortin and corticotrophin-releasing hormone-receptor 1 mRNAs in the pituitary and hypothalamus of the rat during chronic “binge” cocaine and withdrawal. Brain Res 964:187–199. Zhou Y, Spangler R, Ho A, Kreek MJ (2003b) Increased CRH mRNA levels in the rat amygdale during short-term withdrawal from chronic “binge” cocaine. Mol Brain Res 114:73–79. Zhou Y, Choi J, Hofmann L, McCall T, Schlussman SD, Ho A, Kreek MJ (2004) Differential effects on stress responsive gene expression in rat hypothalamic-pituitary and amygdala by heroin challenge after chronic heroin withdrawal and acute stress. Program No. 265.5. 2004 Abstract Viewer and Itinerary Planner. Washington, DC: Society for Neuroscience, 2004. Zubieta JK, Gorelick DA, Stauffer R, Ravert HT, Dannals RF, Frost JJ (1996) Increased mu opioid receptor binding detected by PET in cocaine-dependent men is associated with cocaine craving. Nat Med 2:1225–1229.
(Accepted 16 May 2005)
AMYGDALAR VASOPRESSIN mRNA INCREASES IN ACUTE COCAINE WITHDRAWAL: EVIDENCE FOR OPIOID RECEPTOR MODULATION Y. ZHOU,* J. T. BENDOR, V. YUFEROV, S. D. SCHLUSSMAN, A. HO AND M. J. KREEK
Key words: hypothalamic–pituitary–adrenal axis, proopiomelanocortin, hypothalamus, ACTH.
Laboratory of the Biology of Addictive Diseases, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA
In humans, stress has long been recognized as a major factor contributing to relapse to drug abuse in abstinent individuals. All types of stressors enhance vulnerability to drug abuse. Drugs of abuse themselves or withdrawal from such drugs acts as a stressor (see reviews by Kreek, 1992; Kreek and Koob, 1998). In the animal model, stress modulates the effects of drugs of abuse on the acquisition of drug self-administration behavior and reinstatement of drug self-administration (Shaham et al., 1996). For instance, increased corticotropin-releasing factor (CRF) neuronal activity in the central nucleus of the amygdala is involved in the stress-like or anxiogenic consequences of withdrawal that are common in phenomenology to many drugs of abuse (Weiss et al., 2001; Zhou et al., 2003b). Recent evidence suggests that increased vasopressinergic neuronal activity in the amygdala or hypothalamus represents an important step in the neurobiology of stressrelated behaviors. For example: (1) acute stress increases extracellular levels of arginine vasopressin (AVP) in the rat amygdala or hypothalamus (Ebner et al., 2002; Wigger et al., 2004); and (2) activation of V1 receptors by AVP may underlie anxiogenic and depressive behaviors in the rat (Liebsch et al., 1996; Griebel et al., 2002; Wigger et al., 2004). Stress-related anxiety and depression are major psychiatric consequences of chronic drug abuse and withdrawal in drug addiction (Kreek and Koob, 1998). Of possible relevance, AVP gene expression in extrahypothalamic brain regions, including the medial amygdala, is regulated by testosterone (Szot and Dorsa, 1994). Tolerance to the stimulatory effects of cocaine on the hypothalamic–pituitary–adrenal (HPA) axis develops after chronic “binge” cocaine; this blunting of HPA axis activity is associated with a cocaine-induced reduction of CRF mRNA levels in the hypothalamus (Zhou et al., 1996). During short-term (2 days) cocaine withdrawal, there is increased HPA activity. In contrast to acute (3 h) “binge” cocaine, cocaine withdrawal does not lead to an increase in CRF mRNA levels in the hypothalamus (Zhou et al., 2003a,b). Hypothalamic AVP potentiates CRF-induced adrenocorticotropin hormone (ACTH) secretion from the anterior pituitary (Antoni, 1993). Acute stress results in increased CRF mRNA expression in the paraventricular nucleus (PVN), whereas after chronic stress a preferential activation of AVP gene expression in the PVN is observed (Aguilera, 1994). It remains unclear whether the stimulatory effects of acute cocaine withdrawal on the HPA axis
Abstract—In humans, stress is recognized as a major factor contributing to relapse to drug abuse in abstinent individuals; drugs of abuse themselves or withdrawal from such drugs act as stressors. In the animals, evidence suggests that centrally released arginine vasopressin in both amygdala and hypothalamus plays an important role in stressrelated anxiogenic behaviors. The stress responsive hypothalamic–pituitary–adrenal axis is under tonic inhibition via endogenous opioids, and cocaine withdrawal stimulates hypothalamic–pituitary–adrenal activity. The present studies were undertaken to determine whether: (1) 14-day (chronic) “binge” pattern cocaine administration (45 mg/kg/day) or its withdrawal for 3 h (acute), 1 day (subacute) or 10 days (chronic) alters arginine vasopressin mRNA levels in amygdala or hypothalamus; (2) the opioid receptor antagonist naloxone (1 mg/kg) alters arginine vasopressin mRNA or hypothalamic–pituitary–adrenal hormonal responses in acute cocaine withdrawal; and (3) there are associated changes of mu opioid receptor or proopiomelanocortin mRNA levels. In amygdala, arginine vasopressin mRNA levels were unchanged after chronic “binge” cocaine, but were increased during acute cocaine withdrawal. Naloxone completely blocked this increase. Neither chronic cocaine nor its acute withdrawal altered amygdalar mu opioid receptor mRNA levels. The increase in amygdalar arginine vasopressin mRNA levels was still observed after subacute withdrawal, but not after chronic withdrawal. Although hypothalamic–pituitary– adrenal tolerance developed with chronic “binge” cocaine, there were modestly elevated plasma adrenocorticotropin hormone levels during acute withdrawal. While naloxone produced modest adrenocorticotropin hormone elevations in cocaine-naïve rats, naloxone failed to elicit an adrenocorticotropin hormone response in cocaine-withdrawn rats. In hypothalamus, neither chronic cocaine nor acute withdrawal altered arginine vasopressin, proopiomelanocortin or mu opioid receptor mRNA levels. These results show that: (1) opioid receptors mediate increased amygdalar arginine vasopressin gene expression during acute cocaine withdrawal, and (2) cocaine withdrawal renders the hypothalamic–pituitary–adrenal axis insensitive to naloxone. Our findings suggest a potential role for amygdalar arginine vasopressin in the aversive consequences of early cocaine withdrawal. © 2005 IBRO. Published by Elsevier Ltd. All rights reserved. *Corresponding author. Tel: ⫹1-212-327-8248; fax: ⫹1-212-327-8574. E-mail address: [email protected] (Y. Zhou). Abbreviations: ACTH, adrenocorticotropin hormone; ANOVA, analysis of variance; AVP, arginine vasopressin; CRF, corticotropin-releasing factor; HPA, hypothalamic–pituitary–adrenal; MOP-r, mu opioid receptor; NIL/PL, neurointermediate lobe/posterior lobe of the pituitary; POMC, proopiomelanocortin; PVN, paraventricular nucleus.
0306-4522/05$30.00⫹0.00 © 2005 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2005.05.032
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are associated with AVP mRNA changes in the hypothalamus. Mu opioid receptor (MOP-r) radioligand binding density is increased in the rat amygdala after chronic “binge” cocaine (Unterwald et al., 1992). The stress responsive HPA axis is under tonic inhibition via endogenous opioid systems, including proopiomelanocortin (POMC)-derived beta-endorphin and MOP-r in the hypothalamus (Nikolarakis et al., 1987; Kreek et al., 2002). Although acute cocaine withdrawal has been reported to increase beta-endorphin release in the rat nucleus accumbens (Roth-Deri et al., 2003), it remains unclear whether there are functional alterations of the endogenous opioid systems within the amygdala or hypothalamus after chronic “binge” cocaine or its acute withdrawal. The present studies were undertaken to determine whether: (1) chronic (14 days) “binge” pattern cocaine administration (45 mg/kg/day) or its withdrawal for 3 h (acute), 1 day (subacute) or 10 days (chronic) alters AVP gene expression in the amygdala or hypothalamus, as measured by AVP mRNA levels; (2) the opioid receptorselective antagonist naloxone (1 mg/kg) alters AVP mRNA or HPA hormonal responses to acute cocaine withdrawal; and (3) there are associated changes of POMC, MOP-r or V1b receptor mRNA levels in the amygdala, hypothalamus or anterior pituitary.
Male Fischer rats (190 –220 g, Charles River Laboratories, Kingston, NY, USA) were housed individually in a stress-minimized facility with free access to food and water. Animals were adapted to a standard 12-h light/dark cycle (lights on from 10:30 h to 22:30 h) for 7 days, and then received i.p. injections of cocaine (3⫻15 mg/kg) or equal volumes of saline for 14 days in their home cages following the “binge” pattern regimen: three times daily at 1 h intervals (11:00, 12:00 and 13:00 h) (Branch et al., 1992). On day 14, an i.p injection of naloxone (1 mg/kg) or saline was administered 30 min after the last “binge” cocaine or saline injection. The naloxone dose chosen was based on a pilot study, in which a single 1 mg/kg dose was observed to moderately increase ACTH levels in cocaine naïve rats (unpublished data). In experiment 1 chronic cocaine, rats were killed by decapitation after a brief exposure to CO2 at 14:00 h on day 14, 1 h after the last “binge” saline or cocaine injection. The 1-hour time point was considered as chronic cocaine as extracellular dopamine levels are elevated in the striatum 1 h after the last cocaine on day 13 of chronic “binge” cocaine (Maisonneuve et al., 1995). In experiment 2 acute (3 h) cocaine withdrawal, rats were killed at 16:00 h on day 14, 3 h after the last “binge” cocaine injection. The 3-hour period following the last cocaine was considered as acute cocaine withdrawal, a time in which extracellular dopamine levels in the striatum are lower than the normal levels: 3 h after the last cocaine on day 13 of chronic “binge” cocaine (Maisonneuve et al., 1995). For both experiments, animals were grouped into four groups of eight animals each for treatment: (1) Saline: “binge” saline injections for 14 days with one saline injection 30 min after
saline control cocaine
10
saline control+NXN cocaine+NXN
8 6
10 8
+
6
4
4
2 1.0 0.8 0.6 0.4 0.2 0.0
2 1.0 0.8 0.6 0.4 0.2 0.0
*
amygdala
al w ith dr aw
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w ith dr
ch ro n
aw al
#
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AVP mRNA (pg/µg RNA)
EXPERIMENTAL PROCEDURES
hypothalamus
Fig. 1. Effects of chronic (14-day) “binge” cocaine administration and acute (3-hour) withdrawal from chronic cocaine with or without 1 mg/kg opioid antagonist naloxone (NXN) on AVP mRNA levels in the amygdala or hypothalamus. Data shown in graphs are treatment group mean⫹S.E.M. Significant differences are indicated: * P⬍0.05, Cocaine withdrawal vs. Saline control; # P⬍0.05, NXN⫹Cocaine withdrawal vs. Cocaine withdrawal; ⫹ P⬍0.05, two-way ANOVA with a significant main effect for NXN.
Y. Zhou et al. / Neuroscience 134 (2005) 1391–1397 the last “binge” saline; (2) Cocaine: “binge” cocaine injections for 14 days with one saline injection 30 min after the last “binge” cocaine; (3) Naloxone: “binge” saline injections for 14 days with one naloxone injection (1 mg/kg) 30 min after the last “binge” saline; and (4) Naloxone⫹Cocaine: “binge” cocaine injections for 14 days with one naloxone injection (1 mg/kg) 30 min after the last “binge” cocaine. In experiment 3 subacute (1 day) cocaine withdrawal, rats were killed at 14:00 h on day 15, 1 day after the last “binge” saline or cocaine injection. In experiment 4 chronic (10 days) cocaine withdrawal, rats were killed at 14:00 h on day 24, 10 days after the last “binge” saline or cocaine injection. For both experiments, animals were grouped into two groups of eight animals for each treatment: (1) Saline: “binge” saline injections for 14 days; and (2) Cocaine: “binge” cocaine injections for 14 days. The amygdala, hypothalamus, anterior pituitary and neurointermediate lobe/posterior lobe of the pituitary (NIL/PL) were dissected on ice, homogenized in guanidinium thiocyanate buffer and extracted with acidic phenol and chloroform. The solution hybridization ribonuclease protection–trichloroacetic acid precipitation protocol has been described in detail in earlier reports (Branch et al., 1992; Zhou et al., 1996). A 502 bp fragment from the rat AVP cDNA and a 1201 bp fragment from the rat V1b cDNA were cloned into the polylinker region of pCR II (Invitrogen, Carlsbad CA, USA). A 538 bp fragment from the rat POMC cDNA and a 2100 bp fragment from the rat MOP-r cDNA were cloned into the polylinker region of pSP64 (Promega, Madison, WI, USA) in both the sense and antisense orientations. To determine the total picograms of each mRNA in each extract, the amounts calculated from the standard curves were multiplied by 1.2 for AVP, 2.41 for V1bR, 2.04 for POMC or 5.71 for MOP-r to correct for the difference in length between the sense transcript and the full-length mRNA. At the time of kill, blood from each rat was collected, and plasma was separated. Corticosterone and testosterone levels were assayed using a rat corticosterone or testosterone 125I kit from ICN Biomedicals (Costa Mesa, CA, USA). ACTH immunore-
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activity levels were assayed from unextracted plasma by using a kit from Nichols Institute (San Juan Capistrano, CA, USA). All ACTH, corticosterone or testosterone values were determined in duplicate in a single assay. In experiments 1 and 2, a two-way analysis of variance (ANOVA) (Chronic Treatment: saline, cocaine; Antagonist Condition: saline, naloxone) followed by Newman-Keuls post Shoc tests, or a student’s t-test where appropriate was carried out for each tissue to evaluate the statistical significance of differences among treatment groups. In experiments 3 and 4, a two-way ANOVA (Chronic Treatment: saline, cocaine; Withdrawal Condition: 1-day, 10-day) followed by Newman-Keuls post hoc tests or a planned comparison where appropriate was carried out. To determine correlation between measured variables, linear regression analysis was performed. The accepted level of significance for all tests was P⬍0.05. We followed the Principles of Laboratory Animal Care (NIH publication no 86-23, 1996), and procedures were approved by the Rockefeller University Animal Care and Use Committee. During all procedures of experiments, the number of animals and their suffering by treatments were minimized.
RESULTS Effects of chronic (14-day) “binge” cocaine and acute (3-hour) cocaine withdrawal with naloxone (1 mg/kg) on AVP mRNA levels in the amygdala and hypothalamus Amygdala. In the chronic cocaine experiment, neither chronic “binge” cocaine administration nor naloxone challenge altered AVP mRNA levels (Fig. 1). In the cocaine withdrawal experiment, two-way ANOVA showed a significant Cocaine⫻Naloxone interaction (F(1,27)⫽5.49, P⬍ 0.05) (Fig. 1). Acute withdrawal led to a significant increase
Table 1. Effects of chronic (14-day) “binge” cocaine administration (A) and acute (3-h) withdrawal from chronic cocaine (B) with or without 1 mg/kg opioid antagonist naloxone on mRNA levels (pg/g total RNA) of AVP V1b receptor (V1b), POMC or MOP-r in the amygdala, hypothalamus, anterior pituitary or NIL/PL, and on plasma testosterone levels (ng/ml) A
Amygdala Hypothalamus Anterior pituitary NIL/PL Plasma
MOP-r POMC MOP-r V1b POMC POMC Testosterone
Saline control
Chronic cocaine
Naloxone
Naloxone⫹Chronic cocaine
0.94⫾0.07 7.7⫾1.2 1.3⫾0.04 0.59⫾0.04 184⫾18 4314⫾394 2.6⫾0.29
0.97⫾0.05 8.2⫾1.8 1.3⫾0.10 0.70⫾0.05* 198⫾33 4249⫾308 2.1⫾0.83
0.97⫾0.06 6.2⫾0.59 1.2⫾0.07 0.58⫾0.04 186⫾21 4555⫾204 3.7⫾0.59
0.94⫾0.12 6.5⫾0.80 1.2⫾0.09 0.73⫾0.02* 183⫾15 4175⫾312 2.2⫾0.38
Saline control
Cocaine withdrawal
Naloxone
Naloxone⫹Cocaine withdrawal
1.08⫾0.06 6.0⫾1.3 1.2⫾0.08 0.64⫾0.04 178⫾14 4359⫾400 0.51⫾0.06
1.05⫾0.14 4.9⫾0.88 1.4⫾0.06 0.69⫾0.03 183⫾8.5 4190⫾311 2.8⫾0.74**
0.97⫾0.09 5.2⫾0.83 1.2⫾0.14 0.66⫾0.03 207⫾19 4033⫾371 0.59⫾0.13
1.10⫾0.14 5.8⫾1.08 1.3⫾0.12 0.67⫾0.04 188⫾17 4257⫾375 4.6⫾0.87**#
B
Amygdala Hypothalamus Anterior pituitary NIL/PL Plasma
MOP-r POMC MOP-r V1b POMC POMC Testosterone
Data shown in tables are treatment group mean⫾SEM. Significant differences are indicated: * P⬍0.05, Chronic cocaine or Naloxone⫹Chronic cocaine vs. Saline control; ** P⬍0.001, Cocaine withdrawal or Naloxone⫹Cocaine withdrawal vs. Saline control; # P⬍0.05, Naloxone⫹Cocaine withdrawal vs. Cocaine withdrawal.
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saline control cocaine
250
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*
*
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ACTH, pg/ml
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Corticosterone, ng/ml
**
200
250
corticosterone
Fig. 2. Effects of chronic (14-day) “binge” cocaine administration and acute (3-hour) withdrawal from chronic cocaine with or without 1 mg/kg opioid antagonist naloxone (NXN) on plasma ACTH and corticosterone levels. Data shown in graphs are treatment group mean⫹S.E.M. Significant differences are indicated: * P⬍0.05 or ** P⬍0.01 vs. Saline control.
in AVP mRNA levels (Newman-Keuls post hoc test, P⬍0.05). When the rats were treated with naloxone during acute withdrawal, the AVP mRNA increase was significantly attenuated (P⬍0.05).
“binge” cocaine resulted in a slight (20%), but significant increase in V1b receptor mRNA levels (P⬍0.05). Neither cocaine withdrawal alone nor with naloxone challenge had an effect on V1b receptor mRNA levels (Table 1B).
Hypothalamus. In the chronic cocaine experiment, chronic “binge” cocaine had no effect on AVP mRNA levels. However, two-way ANOVA showed a significant main effect for Naloxone: a 40% decrease in AVP mRNA levels by 1 mg/kg naloxone administration (F(1,27)⫽7.44, P⬍ 0.05) (Fig. 1). AVP mRNA levels were unaltered by either acute withdrawal alone or with naloxone.
POMC mRNA. POMC mRNA levels in the anterior pituitary and NIL/PL were unaltered by either drug treatment in either experiment (Table 1A and 1B). Effects of chronic “binge” cocaine and acute cocaine withdrawal with naloxone on plasma ACTH, corticosterone and testosterone levels
Effects of chronic “binge” cocaine and acute cocaine withdrawal with naloxone on V1b receptor and POMC mRNA levels in the anterior pituitary and NIL/PL
ACTH. In the chronic cocaine experiment, plasma ACTH levels were unchanged by either drug treatment (Fig. 2). In the cocaine withdrawal experiment, mean plasma ACTH levels were elevated in the Cocaine group (140% Saline), Naloxone⫹Cocaine group (180% Saline), and Naloxone group (170% Saline) (Fig. 2). Two-way ANOVA, however, just failed to show a significant main effect for either treatment (P⫽0.07). Since earlier studies have shown elevated ACTH levels after naloxone challenge [see review by Kreek et al. (2002)] or cocaine withdrawal (Zhou et al., 2003a), Student’s t-test was carried out and showed a significant difference between Saline and each of the other three groups (P⬍0.05).
V1b receptor mRNA. In the chronic cocaine experiment, two-way ANOVA showed a significant main effect for Cocaine (F(1,25)⫽11.3, P⬍0.01) (Table 1A). Chronic
Corticosterone. In the chronic cocaine experiment, two-way ANOVA showed a significant main effect for Cocaine (F(1,25)⫽9.69, P⬍0.005) (Fig. 2). Chronic “binge”
Effects of chronic “binge” cocaine and acute cocaine withdrawal with naloxone on POMC and MOP-r mRNA levels in the amygdala and hypothalamus Neither POMC nor MOP-r mRNA levels in the amygdala or hypothalamus were altered by either drug treatment in either experiment (Table 1A and 1B).
Y. Zhou et al. / Neuroscience 134 (2005) 1391–1397
AVP mRNA (pg/ µ g RNA)
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saline control cocaine
0.75
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da y 10
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da y
w ith dr aw al
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Fig. 3. Effects of subacute (1-day) or chronic (10-day) withdrawal from chronic (14-day) “binge” cocaine administration on AVP mRNA levels in the amygdala. Data shown in graphs are treatment group mean⫹S.E.M. Significant differences are indicated: * P⬍0.05 vs. Saline control.
cocaine resulted in a significant increase in plasma corticosterone levels (P⬍0.05). Neither cocaine withdrawal alone nor with naloxone had an effect on plasma corticosterone levels. Testosterone. In the chronic cocaine experiment, plasma testosterone levels were unaltered by either drug treatment (Table 1A). In the cocaine withdrawal experiment, two-way ANOVA showed a significant main effect for Cocaine (F(1,27)⫽35.5, P⬍0.0001). Plasma testosterone levels were elevated by acute cocaine withdrawal (P⬍0.05). When the cocaine withdrawn-rats were challenged with naloxone (1 mg/kg), the testosterone increase was significantly enhanced (P⬍0.05) (Table 1B). There was no correlation between amygdalar AVP mRNA and plasma testosterone levels in the rat after acute cocaine withdrawal. Effects of subacute (1-day) or chronic (10-day) withdrawal from chronic (14-day) “binge” cocaine on AVP mRNA levels in the amygdala After subacute cocaine withdrawal, the AVP mRNA levels were increased in the amygdala (Fig. 3). Two-way ANOVA, however, just failed to show a significant main effect for treatments. Since the acute withdrawal study showed elevated AVP mRNA levels in the amygdala, a planned comparison was carried out and showed a significant difference between Saline and 1-day Withdrawal (F(1,24)⫽4.98, P⬍0.05). However, after chronic withdrawal the AVP mRNA levels were no longer elevated in the amygdala (Fig. 3).
DISCUSSION We found that AVP mRNA levels in the amygdala were not altered after chronic “binge” cocaine. However, acute cocaine withdrawal was associated with increased amygdalar AVP mRNA levels, nearly three-fold that of the control level (Fig. 1). Also, this effect occurred at acute withdrawal stage (3 h), persisted for 1 day into subacute cocaine withdrawal, but not for 10 days of chronic withdrawal (Fig. 3). This novel finding parallels our recent studies demonstrating an increased AVP mRNA level in the amygdala during acute opiate withdrawal, but not after chronic opiate administration (Zhou et al., 2004). We further found that a moderate dose of naloxone (1 mg/kg) was able to completely block the AVP mRNA increase, suggesting that the stimulation of amygdalar AVP gene expression by acute cocaine withdrawal is mediated by opioid receptors. This observed interaction between the opioidergic and AVPergic systems may be fundamental to drug withdrawal stress-induced aversive consequences, like anxiogenesis. Earlier work has demonstrated an increased MOP-r binding density in the amygdala of the rat after 14-day chronic “binge” cocaine (Unterwald et al., 1992) and in the amygdala of cocaine-dependent men (Zubieta et al., 1996), suggesting involvement of amygdalar MOP-r functional alterations in cocaine dependence. In the amygdala, however, MOP-r mRNA levels were not affected by either chronic “binge” cocaine or its acute withdrawal (Table 1). Further time-course studies are necessary before alternate explanations, such as indirect
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amygdalar activation due to extraamygdalar naloxone action (e.g. in the nucleus accumbens), can be ruled out. There are relatively few studies on the effects of chronic cocaine or cocaine withdrawal on the brain vasopressinergic systems in the rat. As early as 1992, it was shown that subchronic cocaine administration for 5 days resulted in decreased AVP content in the hypothalamus and amygdala as measured by radioimmunoassay, which may suggest increased release and/or subsequent degradation (Sarnyai et al., 1992). AVP mRNA expression in the medial nucleus of the amygdala is under direct positive regulation by testosterone (Szot and Dorsa, 1994; Albeck et al., 1997). Although both amygdalar AVP mRNA and plasma testosterone levels were elevated during acute cocaine withdrawal, we found no correlation between these two parallel changes. Therefore, the observed effects of cocaine withdrawal on amygdalar AVP mRNA levels may not be directly related to plasma testosterone. The second aim of our study was to examine the effect of acute cocaine withdrawal on hypothalamic AVP gene expression, which is potentially involved in determining HPA activation during cocaine withdrawal. In the present study, 3 h after chronic “binge” cocaine we found similar ACTH elevations as found after 1-day withdrawal (Zhou et al., 2003a). Administration of 1 mg/kg naloxone caused a moderate, but significant, increase in ACTH levels (Fig. 2), consistent with a tonic inhibition of the HPA axis by endogenous opioids. Of interest, the same dose of naloxone did not further increase plasma ACTH levels when the animals were undergoing acute cocaine withdrawal (Fig. 2). Taken together, our results indicate HPA hyposensitivity to the MOP-r preferring antagonist naloxone during acute cocaine withdrawal. Both AVP- and CRF-producing parvocellular PVN cells express MOP-r and activities of these cells are under tonic inhibition by endogenous opioids, including beta-endorphin (Nikolarakis et al., 1987). Our observation of naloxone-insensitive HPA responsivity suggests that alterations in the hypothalamic beta-endorphin/ MOP-r system may contribute to this HPA hyporesponsivity after chronic cocaine. POMC mRNA levels in the hypothalamus were not affected by either chronic “binge” cocaine or its acute withdrawal, nor were hypothalamic MOP-r mRNA levels (Table 1). Thus, reduction of betaendorphin release or MOP-r signaling activity by chronic cocaine or its withdrawal does not necessarily affect their gene expression at the two time points examined. Neither hypothalamic CRF nor anterior pituitary receptor CRF type 1 receptor mRNA levels are altered during short-term (1 day) withdrawal from chronic “binge” cocaine (Zhou et al., 2003b). We hypothesized that increased HPA activity during acute cocaine withdrawal is mediated by alterations in the hypothalamic–pituitary AVP system. However, neither hypothalamic AVP nor anterior pituitary V1b receptor mRNA levels were affected by 3-hour acute cocaine withdrawal (Table 1). Although measurement of AVP mRNA levels in whole hypothalamic RNA extracts may be confounded by potentially differential responses of AVP mRNA levels in parvocellular and magnocellular cells,
a study using the same assays found increased AVP mRNA levels in the hypothalamus in response to acute injection stress (Zhou et al., 2004). The present results argue that the stimulatory effects of cocaine withdrawal on HPA activity may be associated with POMC-derived peptide-releasing factors other than AVP or CRF. Stimulation of the HPA axis is often observed after acute stress, such as acute cocaine administration. This activation of the HPA axis activity is associated with a stress-induced increase in plasma ACTH levels, and rapidly followed by a concomitant increase in plasma corticosterone levels (e.g. Rivier and Vale, 1987; Zhou et al., 1996). After chronic “binge” cocaine administration, however, there is a blunting of ACTH response with a still significant increase in corticosterone levels. Such dissociation of ACTH and corticosterone responses after chronic “binge” cocaine were observed before (Zhou et al., 1996, 2003a) and replicated here. It is possible that the increase in corticosterone is driven by factors other than ACTH (i.e. sympathetic nervous system or direct effect of CRF on the adrenal gland). Another potential explanation for such dissociation after chronic cocaine is that, similar to many cases of chronic stress, the adrenal gland becomes hypersensitive to ACTH (e.g. Sarnyai et al., 1998). In contrast to chronic cocaine, however, immediate cocaine withdrawal led to a significant increase in ACTH levels with a lack of corticosterone response, suggesting that the adrenal gland becomes less responsive to ACTH during this 3-hour immediate withdrawal. Although we did not have later time points, the immediate withdrawal-induced blunting of corticosterone responses seems short-lived. In fact, there is a parallel increase in both plasma ACTH and corticosterone levels during 1– 4 days of short-term withdrawal from chronic cocaine, as we reported before (Zhou et al., 2003a). Our results indicate that there is an uncoupling of ACTH and corticosterone during immediate withdrawal from chronic cocaine administration.
CONCLUSION The present studies determined the effects of chronic “binge” cocaine and its acute withdrawal on stress responsive AVP gene expression. We found that acute cocaine withdrawal resulted in a significant increase in amygdalar AVP mRNA levels. Of interest, amygdalar AVP gene expression stimulated by acute cocaine withdrawal was completely blocked by a single, moderate dose of naloxone, indicating an opioid receptor activation-mediated mechanism. In line with this finding, we have recently found an increase in amygdalar AVP mRNA levels in the rat during acute heroin withdrawal (Zhou et al., 2004), suggesting that there is enhanced vasopressinergic neuronal activity in the amygdala in the aversive and anxiogenic consequences of drug withdrawal. In contrast to the amygdala, we found no effect of either chronic cocaine administration or its acute withdrawal on AVP mRNA levels in the hypothalamus. In this study, we also showed differential responses of HPA and amygdalar stress systems to naloxone. Our finding of HPA hyposensitivity to naloxone after
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acute cocaine withdrawal indicates a decrease in opioidergic tone within the hypothalamus during acute cocaine withdrawal, and is consistent with studies in recently abstinent human cocaine addicts (Schluger et al., 2001). Acknowledgments—The authors would like to thank Dr. G. Aguilera for providing the V1b receptor cDNAs; Dr. G. Uhl for the MOP-r cDNA; Dr. J. Roberts for the POMC cDNA; and Drs. T. Nilsen and P. Maroney for the 18S DNA. The work was supported by NIDA Research Center Grant DA-P60-05130 and DA-00049 (M.J.K.), and by NIH Training Grant GM07524 (J.T.B.).
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(Accepted 16 May 2005)