Short-Term Cocaine Treatment Causes Neuroadaptive Changes in G q and G 11 Proteins in Rats Undergoing Withdrawal

One of the characteristics of drug dependence is that a drug has to be administered repeatedly before withdrawal effects can be observed. We have previously shown that withdrawal after 14 days of cocaine treatment produces a supersensitivity of hypothalamic 5-hydroxytryptamine (serotonin) 2A (5-HT2A) receptors, which is accompanied by increases in the levels of G q and G 11 proteins. Unfortunately, the exact duration of cocaine treatment necessary to induce alterations in G protein levels during cocaine withdrawal is unknown. The present study investigated the minimum cocaine treatment period required to produce changes in protein levels of membraneand cytosol-associated G q and G 11 proteins in the hypothalamic paraventricular nucleus, amygdala, and frontal cortex. Rats were injected with cocaine (15 mg/kg i.p., b.i.d.) for 0, 1, 3, 5, and 7 days and tested after 2 days of withdrawal. The levels of G q and G 11 proteins were increased in the paraventricular nucleus and the amygdala but not in the frontal cortex. Although 1 and 3 days of cocaine treatment were sufficient to maximally elevate the protein levels of G 11 and G q proteins in the amygdala, 5 days of treatment were required to maximally increase the levels of G 11 and G q proteins in the paraventricular nucleus. The data suggest that the amygdala shows a faster neuroadaptation to the effects of cocaine than the hypothalamic paraventricular nucleus. These findings provide insight into the relative importance of individual components of 5-HT2A receptor signal transduction system in regulating the overall sensitivity of this signaling in cocaine-treated rats. Cocaine produces a variety of actions in neuronal function (Levy et al., 1994a). Cocaine binds with high affinity to neurotransmitter uptake sites on monoaminergic (serotonergic, dopaminergic, and noradrenergic) neurons in the brain and peripheral tissues, blocking the reuptake of serotonin, dopamine, and norepinephrine into the presynaptic neuron (Levy et al., 1994a). The influence of cocaine on serotonergic neurotransmission has received increasing attention in recent years. The cocaine-induced reuptake blockade leads to an increased concentration of 5-HT in the synapse and thus to stimulation of postsynaptic 5-HT receptors (Hanson et al., 1987). Furthermore, cocaine reduces the activity of 5-HT neurons in the dorsal raphe (Cunningham and Lakoski, 1988), presumably as a consequence of increased stimulation of somatodendritic 5-HT1A autoreceptors in the dorsal raphe nucleus (Pan et al., 1989). 5-HT2A and 5-HT2C receptors, which activate the dopamine mesoaccumbens pathway, play an important role in the behavioral effects of cocaine (McMahon et al., 2001). In addition, 5-HT2A receptors are expressed by neurons in the hypothalamic paraventricular nucleus (Zhang et al., 2002). Activation of 5-HT2A receptors in the hypothalamic paraventricular nucleus increases the secretion of ACTH, corticosterone, oxytocin, and prolactin (Van de Kar et al., 2001). Using neuroendocrine responses to the 5-HT2A/2C receptor agonist DOI, we found increased sensitivity of 5-HT2A receptors that stimulate the secretion of ACTH, corticosterone, and prolactin after 42-h withdrawal from repeated cocaine treatment (15 mg/kg i.p., twice a day for 7 days) (Levy et al., 1992). The mechanism of this cocaine-induced supersensitivity of 5-HT2A receptors in the paraventricular nucleus might be an This study was supported by United States Health Service Grant DA13669 (to L.D.V.d.K.), MH068612 (to N.A.M.), and DA07741 (to G.B). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.104.069807. ABBREVIATIONS: 5-HT, 5-hydroxytryptamine (serotonin); ACTH, adrenocorticotropic hormone; DOI, ( )-1-(2,5 dimethoxy-4-iodophenyl)-2amino-propane HCl; RGS, regulators of G protein signaling; IOD, integrated optical density; ANOVA, analysis of variance; AGS, activator of G protein signaling; MDL 100,907 ( )-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidinemethanol. 0022-3565/04/3111-349–355$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 311, No. 1 Copyright © 2004 by The American Society for Pharmacology and Experimental Therapeutics 69807/1169708 JPET 311:349–355, 2004 Printed in U.S.A. 349 at A PE T Jornals on M ay 8, 2017 jpet.asjournals.org D ow nladed from altered expression of proteins associated with the 5-HT2A receptor signaling pathway, including G q and G 11 proteins, and regulators of G protein signaling proteins, such as regulators of G protein signaling (RGS)4 and RGS7 proteins. Our previous study (Carrasco et al., 2003) showed that withdrawal (2 days) from chronic cocaine treatment (14 days, 15 mg/kg twice a day or using a binge protocol) produces a transient and region-specific increase in the levels of membrane-associated G q and G 11 proteins in the hypothalamic paraventricular nucleus and the amygdala, but not in the frontal cortex (Carrasco et al., 2003). Exposure to chronic cocaine does not produce changes in the levels of membraneor cytosol-associated 5-HT2A receptors, RGS4 or RGS7 proteins in frontal cortex, amygdala, or paraventricular nucleus (Carrasco et al., 2003). These results support the conclusion that withdrawal from chronic cocaine treatment increases 5-HT2A receptor function by altering postreceptor signal transduction mechanisms, rather than increasing 5-HT2A receptor density. In this article, we focused on the minimum number of days of cocaine treatment required to change the levels of 5-HT2A receptor signaling proteins in the hypothalamic paraventricular nucleus, amygdala, and frontal cortex. These regions were selected because of their prominent role in stress, anxiety, neuroendocrine function, and addiction (Carrasco and Van de Kar, 2003; Yun and Fields, 2003). The hypothalamic paraventricular nucleus plays a central role in mediating neuroendocrine responses to serotonergic activation (Bagdy, 1996). The hypothalamic paraventricular nucleus receives serotonergic projections from the raphe nuclei, which also send collaterals to other limbic structures, notably the amygdala (Liposits et al., 1987; Petrov et al., 1994). The amygdala is a limbic structure with interconnections to the cortex and the nucleus accumbens and plays a central role in the reinforcing effects of drugs of abuse (Yun and Fields, 2003). Various drugs of abuse require repeated administration before withdrawal effects can be observed (Levy et al., 1994a). Because the immediate effect of cocaine is to reduce serotonergic firing rate (Cunningham and Lakoski, 1988), there is a good likelihood that the reduction in 5-HT release in the hypothalamus will gradually lead to supersensitive postsynaptic 5-HT2A receptors. However, the minimum duration of cocaine exposure required to induce the adaptive mechanisms leading to an increase in the levels of G q and G 11 proteins in the hypothalamic paraventricular nucleus and amygdala during withdrawal from cocaine has not been determined. In the present study, we evaluated the minimum number of days of cocaine treatment that will produce an increase in the levels of G q and G 11 proteins in rats withdrawn from cocaine. Materials and Methods Animal Treatment. Male Sprague-Dawley rats (225–275 g) (Harlan, Indianapolis, IN) were housed two per cage in an environment controlled for lighting, temperature, and humidity. Food and water were available ad libitum. All procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory animals as approved by the Loyola University Institutional Animal Care and Use Committee. The rats were allowed to acclimate to their environment for at least 4 days before the start of the treatment period. Eight to 12 rats were randomly assigned to each group. Rats received injections of cocaine (15 mg/kg i.p., 8:30 AM and 3:30 PM) for 1, 3, 5, and 7 days. A control group of rats was injected with 0.9% saline (1 ml/kg i.p., 8:30 AM and 3:30 PM) for 7 days. All the rats were killed 2 days after the last injection. The brains were immediately removed, and the frontal cortex was dissected and frozen in liquid nitrogen. The remainder of the brain was frozen in dry ice and all tissues were stored at 80°C. Tissue Preparation. Rat brains were placed in a cryostat at 10°C, and coronal sections were cut to obtain a 700m-thick section containing the paraventricular nucleus and rostral amygdala and a 1200m-thick section containing the caudal amygdala. The paraventricular nucleus and amygdala were microdissected from these frozen sections with the aid of a dissecting stereomicroscope. Plasma membranes of frontal cortex, amygdala, and paraventricular nucleus of the hypothalamus were prepared as described previously (Carrasco et al., 2003). All procedures were conducted at 4°C. Briefly, the frontal cortex, hypothalamic paraventricular nucleus, and amygdala were homogenized in 50 mM Tris buffer (pH 7.4) containing 150 mM NaCl, 10% sucrose, and 0.5 mM phenylmethylsulfonyl fluoride, and additional protease inhibitors purchased as a cocktail [containing 4-(2-aminoethyl)benzenesulfonyl fluoride, pepstatin A, trans-epoxysuccinyl-L-leucyl-amido(4-guanidino)butane, bestatin, leupeptin, and aprotinin] from Sigma-Aldrich (St. Louis, MO; 1.5 l/30 mg of tissue). After centrifugation at 20,000g for 60 min, the supernatant was collected and stored at 80°C for further analyses of cytosolassociated protein levels. The pellets were collected and resuspended by sonication in a 20 mM Tris buffer (pH 8), containing 1 mM EDTA, 100 mM NaCl, 1% sodium cholate, and 1 mM dithiothreitol, plus the protease inhibitory cocktail (1.5 l of cocktail/30 mg of tissue). The resuspended pellets were incubated while shaking for 1 h at 4°C and then centrifuged at 100,000g for 60 min. The supernatant was collected for the Western blot analyses of membrane-associated protein levels. Protein concentration was measured using a bicinchoninic acid protein assay kit (Pierce Chemical, Rockford, IL). The membrane proteins were stored at 80°C for Western blot analyses. Western Blot Analysis. Samples containing 2 g (hypothalamic paraventricular nucleus), 3 g (amygdala), and 4 g (frontal cortex) of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis containing 0.1% SDS, 12.5% acrylamide/bisacrylamide (30:0.2), 4.6 M urea, and 275 mM Tris, pH 8.7. Gels were transferred electrophoretically by semi-dry blot to nitrocellulose membranes. After incubation with a blocking buffer (phosphatebuffered saline containing 0.2% casein and 0.1% Tween 20), the nitrocellulose membranes were probed overnight at 4°C with polyclonal antisera. Immunodetection was performed with either antiG 11 (1:500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), antiG q (1:500; Santa Cruz Biotechnology, Inc.), or anti-G z (1:6000; Santa Cruz Biotechnology, Inc.). The overnight incubation with G q, G 11, and G z antibodies was followed by incubation with peroxidase-labeled anti-rabbit antibody (1:20,000; 1 h at room temperature; Santa Cruz Biotechnology, Inc.). Finally, the membranes were incubated with the enhanced chemiluminescence substrate solution (Amersham Biosciences Inc., Piscataway, NJ) and then exposed to X-ray film (Eastman Kodak, Rochester, NY). Protein loading for each lane was verified using an anti-actin antibody (1:20,000; Santa Cruz Biotechnology, Inc.). Negative controls included either omission of primary antibody or addition of preimmune rabbit immunoglobulins. Film Analysis. Films were analyzed densitometrically using Scion Image software (Scion Corporation, Frederick, MD). The gray scale density readings were calibrated using a transmission stepwedge standard. The integrated optical density (IOD) of each band was calculated as the sum of the optical densities of all the pixels within the area of the band outlined. An adjacent area was used to calculate the background optical density of the film. The IOD for the film background was subtracted from the IOD for each band. Each sample was measured on three independent gels. All samples were standardized to controls and normalized to its respective actin level. Statistics. All data are expressed as the mean S.E.M., where n indicates the number of rats per group. A one-way analysis of vari350 Carrasco et al. at A PE T Jornals on M ay 8, 2017 jpet.asjournals.org D ow nladed from ance (ANOVA) was used to analyze the Western blot data, considering p 0.05 as statistically significant. Group means were compared by Newman-Keuls multiple range test (Steel and Torrie, 1960). GBSTAT software (Dynamic Microsystems, Inc., Silver Spring, MD) was used for all statistical analyses.