Elevation of guinea pig brain preprodynorphin mRNA expression and hypothalamic-pituitary-adrenal axis activity by "binge" pattern cocaine administration.

The endogenous opioid system and the hypothalamic-pituitary-adrenal (HPA) axis have been implicated in many of the neurobiological effects of cocaine. Previous studies in our laboratory showed that "binge" pattern cocaine administration increases preprodynorphin (ppDyn) mRNA levels in the caudate putamen and circulating levels of corticosterone in the rat. The present study extended these findings to guinea pigs, a species known to have a kappa opioid receptor profile similar to that of humans. Male guinea pigs were treated with: (a) "binge" pattern cocaine for 7 days (subchronic) (3 x 15 mg/kg/day, hourly, intraperitoneal); (b) "binge" pattern saline for 5 days followed by "binge" pattern cocaine for 2 days (subacute); or (c) "binge" pattern saline for 7 days. Thirty minutes after the final injection, levels of ppDyn mRNA were quantitated in the nucleus accumbens, caudate putamen, frontal cortex, amygdala, hippocampus, and hypothalamus using a solution hybridization RNase protection assay. Regional distribution of ppDyn mRNA levels in the guinea pig brain was similar to that found in rat, with highest levels in the nucleus accumbens and caudate putamen. In the caudate putamen, ppDyn mRNA was significantly increased following either 2 days (38% increase) or 7 days (32% increase) of "binge" pattern cocaine administration as compared to saline-treated controls. No significant changes in ppDyn mRNA levels were found in any other brain region. Both subacute and subchronic "binge" cocaine administration significantly elevated plasma levels of adrenocorticotropin hormone (ACTH) and cortisol. However, the ACTH and cortisol increases were significantly blunted following 7 days of "binge" cocaine administration as compared to 2 days of drug treatment, reflecting the development of HPA tolerance or adaptation to repeated cocaine administration. Thus, the ppDyn mRNA and HPA responses to cocaine in guinea pigs are similar to those observed in rats.     

Functional and anatomical localization of mu opioid receptors in the striatum, amygdala, and extended amygdala of the nonhuman primate

The subregional distribution of mu opioid receptors and corresponding G-protein activation were examined in the striatum, amygdala, and extended amygdala of cynomolgus monkeys. The topography of mu binding sites was defined using autoradiography with [(3)H]DAMGO, a selective mu ligand. In adjacent sections, the distribution of receptor-activated G proteins was identified with DAMGO-stimulated guanylyl 5'(gamma-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding. Within the striatum, the distribution of [(3)H]DAMGO binding sites was characterized by a distinct dorsal-ventral gradient with a higher concentration of binding sites at more rostral levels of the striatum. [(3)H]DAMGO binding was further distinguished by the presence of patch-like aggregations within the caudate, as well as smaller areas of very dense receptor binding sites, previously identified in human striatum as neurochemically unique domains of the accumbens and putamen (NUDAPs). The amygdala contained the highest concentration of [(3)H]DAMGO binding sites measured in this study, with the densest levels of binding noted within the basal, accessory basal, paralaminar, and medial nuclei. In the striatum and amygdala, the distribution of DAMGO-stimulated G-protein activation largely corresponded with the distribution of mu binding sites. The central and medial nuclei of the amygdala, however, were notable exceptions. Whereas the concentration of [(3)H]DAMGO binding sites in the central nucleus of the amygdala was very low, the concentration of DAMGO-stimulated G-protein activation in this nucleus, as measured with [(35)S]GTPgammaS binding, was relatively high compared to other portions of the amygdala containing much higher concentrations of [(3)H]DAMGO binding sites. The converse was true in the medial nucleus, where high concentrations of binding sites were associated with lower levels of DAMGO-stimulated G-protein activation. Finally, [(3)H]DAMGO and [(35)S]GTPgammaS binding within the amygdala, particularly the medial nucleus, formed a continuum with the substantia innominata and bed nucleus of the stria terminalis, supporting the concept of the extended amygdala in primates.     

Chronic heroin self-administration desensitizes mu opioid receptor-activated G-proteins in specific regions of rat brain.

In previous studies from our laboratory, chronic noncontingent morphine administration decreased mu opioid receptor-activated G-proteins in specific brainstem nuclei. In the present study, mu opioid receptor binding and receptor-activated G-proteins were examined after chronic heroin self-administration. Rats were trained to self-administer intravenous heroin for up to 39 d, achieving heroin intake up to 366 mg. kg(-1). d(-1). mu opioid-stimulated [(35)S]GTPgammaS and [(3)H]naloxone autoradiography were performed in adjacent brain sections. Agonist-stimulated [(35)S]GTPgammaS autoradiography also examined other G-protein-coupled receptors, including delta opioid, ORL-1, GABA(B), adenosine A(1), cannabinoid, and 5-HT(1A). In brains from heroin self-administering rats, decreased mu opioid-stimulated [(35)S]GTPgammaS binding was observed in periaqueductal gray, locus coeruleus, lateral parabrachial nucleus, and commissural nucleus tractus solitarius, as previously observed in chronic morphine-treated animals. In addition, decreased mu opioid-stimulated [(35)S]GTPgammaS binding was found in thalamus and amygdala after heroin self-administration. Despite this decrease in mu-activated G-proteins, [(3)H]naloxone binding demonstrated increased mu opioid receptor binding in several brain regions after heroin self-administration, and there was a significant decrease in mu receptor G-protein efficiency as expressed as a ratio between agonist-activated G-proteins and mu receptor binding. No effects on agonist-stimulated [(35)S]GTPgammaS binding were found for any other receptor examined. The effect of chronic heroin self-administration to decrease mu-stimulated [(35)S]GTPgammaS binding varied between regions and was highest in brainstem and lowest in the cortex and striatum. These results not only provide potential neuronal mechanisms that may contribute to opioid tolerance and dependence, but also may explain why various chronic effects of opioids develop to different degrees.     

The frequency of cocaine administration impacts cocaine-induced receptor alterations

The present study investigated the impact of dosing schedule on cocaine-induced receptor alterations. Rats were injected with 30 mg/kg per day of cocaine given either as a single injection or in two equally divided doses for 14 days. The effects of these two dosing regimens were compared with our previous findings following administration of cocaine three times daily at 1-h intervals. Using receptor autoradiography, twice daily injections of cocaine produced an upregulation of mu opioid receptors in the rostral nucleus accumbens, rostral caudate putamen, and layer I of the rostral cingulate cortex, whereas single daily injections resulted in a significant increase in the nucleus accumbens only. Only small insignificant increases in kappa opioid receptor densities were found following either once or twice daily cocaine injections, whereas three daily injections produced an increase in kappa receptor density in the cingulate cortex, nucleus accumbens, and caudate putamen. Increased dopamine D1 receptor binding was found in the nucleus accumbens and olfactory tubercle following twice daily cocaine injections, but not after single daily injections of the same total daily dose. These results demonstrate that the same total daily dose of cocaine administered in multiple small injections produces a greater effect on receptor regulation than a single larger injection. This suggests that the interval between cocaine injections is an important variable when studying the effects of cocaine on neurochemistry.     

Persistent upregulation of mu-opioid, but not adenosine, receptors in brains of long-term withdrawn escalating dose "binge" cocaine-treated rats

There is evidence showing that the opioid and adenosine systems play an important role in cocaine addiction; fewer studies have examined their roles in cocaine withdrawal. To determine whether cocaine and/or chronic withdrawal from cocaine alters the specific components of the opioid and adenosine systems, we carried out quantitative autoradiographic mapping of mu-opioid, A1 and A2A adenosine receptors in the brains of rats treated with an escalating dose "binge" cocaine administration paradigm and of rats chronically withdrawn from cocaine. Male Fischer rats were injected with saline or cocaine (15 x 3 mg/kg/day for 4 days, 20 x 3 mg/kg/day for 4 days, 25 x 3 mg/kg/day for 4 days and 30 x 3 mg/kg/day for 2 days) at 1-h intervals for 14 days. Similarly treated rats were withdrawn from that paradigm for 14 days. A significant increase in [(3)H]DAMGO binding to mu-receptors was detected in the frontal and cingulate cortex, as well as in the caudate putamen, of long-term withdrawn rats after an escalating dose "binge" cocaine administration paradigm and in chronic cocaine-treated rats. No significant cocaine-induced change was found in A1 or A2A receptor binding in any region analyzed. These results reconfirm that mu-opioid (MOP) receptors undergo upregulation in response to chronic escalating dose "binge" cocaine administration. This upregulation was shown for the first time to persist at least 14 days into withdrawal after escalating "binge" cocaine.     

Comparison of G-protein activation in the brain by mu-, delta-, and kappa-opioid receptor agonists in mu-opioid receptor knockout mice

Mice lacking the mu-opioid receptor gene have been developed by a gene knockout procedure. In this study, the activity of opioid receptor coupled G-proteins was examined to investigate whether there is a change in the extent of coupling for mu, delta-, and kappa-opioid receptors in mu-opioid receptor knockout mice. Selective agonists of mu- (DAMGO), delta- (DPDPE), and kappa- (U-69,593) opioid receptors stimulated [(35)S]GTPgammaS binding in the caudate putamen and cortex of wild-type mice. In contrast, only U-69,593 stimulated [(35)S]GTPgammaS binding in these regions of mu-opioid receptor knockout mice. These results confirmed the absence of G-protein activation by a mu-opioid receptor agonist in mu-opioid receptor knockout mice, and demonstrated that coupling of the kappa-opioid receptor to G-proteins is preserved in these mice. However, G-protein activation by the delta-opioid receptor agonist, DPDPE, was reduced in the mu-opioid receptor knockout mice, at least in the brain regions studied using autoradiography.     

κ-opioid agonist stimulated regional distribution of [S]GTPγs binding in butorphanol continuously infused rat

Butorphanol is a mixed agonist/antagonist opioid analgesic agent, which exerts its effects mainly by interaction with the kappa-opioid receptor. Opioid receptors are coupled to G proteins of G(i)/G(o) family, and recently a decrease in micro-opioid activation of G proteins has been reported in specific brainstem nuclei after chronic morphine administration. The influence of centrally administered butorphanol on agonist-stimulated G protein coupling was examined in the rat brain, using in situ guanylyl-5'-O-(gamma-[(35)S]thio)-triphosphate (GTPgammaS) binding autoradiography. Rats were treated with butorphanol (26 nmol/microl/h) by intracerebroventricular infusion via osmotic minipumps for 3 days. The distribution of [(35)S]GTPgammaS binding in the brain 7 h after the termination of butorphanol infusion was measured in the presence or absence of the selective kappa-opioid agonist, U-50,488. This agonist significantly increased [(35)S]GTPgammaS binding in the parietal cortex, caudate putamen, thalamus, and central gray of control rats, but not in those regions of the butorphanol-infused animals. These results suggest that chronic administration of butorphanol developed tolerance and abolished U-50,488 activation of G proteins in these brain areas.     

kappa-opioid agonist stimulated regional distribution of [(35)S]GTPgammas binding in butorphanol continuously infused rat

Butorphanol is a mixed agonist/antagonist opioid analgesic agent, which exerts its effects mainly by interaction with the kappa-opioid receptor. Opioid receptors are coupled to G proteins of G(i)/G(o) family, and recently a decrease in micro-opioid activation of G proteins has been reported in specific brainstem nuclei after chronic morphine administration. The influence of centrally administered butorphanol on agonist-stimulated G protein coupling was examined in the rat brain, using in situ guanylyl-5'-O-(gamma-[(35)S]thio)-triphosphate (GTPgammaS) binding autoradiography. Rats were treated with butorphanol (26 nmol/microl/h) by intracerebroventricular infusion via osmotic minipumps for 3 days. The distribution of [(35)S]GTPgammaS binding in the brain 7 h after the termination of butorphanol infusion was measured in the presence or absence of the selective kappa-opioid agonist, U-50,488. This agonist significantly increased [(35)S]GTPgammaS binding in the parietal cortex, caudate putamen, thalamus, and central gray of control rats, but not in those regions of the butorphanol-infused animals. These results suggest that chronic administration of butorphanol developed tolerance and abolished U-50,488 activation of G proteins in these brain areas.     

Specific activation of the mu opioid receptor (MOR) by endomorphin 1 and endomorphin 2

The recently discovered endomorphin 1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin 2 (Tyr-Pro-Phe-Phe-NH2) were investigated with respect to their direct receptor-binding properties, and to their ability to activate G proteins and to inhibit adenylyl cyclase in both cellular and animal models. Both tetrapeptides activated G proteins and inhibited adenylyl cyclase activity in membrane preparations from cells stably expressing the mu opioid receptor, an effect reversed by the mu receptor antagonist CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2), but they had no influence on cells stably expressing the delta opioid receptor. To further establish the selectivity of these peptides for the mu opioid receptor, brain preparations of mice lacking the mu opioid receptor gene were used to study their binding and signalling properties. Endomorphin 2, tritiated by a dehalotritiation method resulting in a specific radioactivity of 1.98 TBq/mmol (53.4 Ci/mmol), labelled the brain membranes of wild-type mice with a Kd value of 1.77 nM and a Bmax of 63.33 fmol/mg protein. In membranes of mice lacking the mu receptor gene, no binding was observed, and both endomorphins failed to stimulate [35S]guanosine-5'-O-(3-thio)triphosphate ([35S]GTPgammaS) binding and to inhibit adenylyl cyclase. These data show that endomorphins are capable of activating G proteins and inhibiting adenylyl cyclase activity, and all these effects are mediated by the mu opioid receptors.     

Effect of chronic "binge cocaine" on basal levels and cocaine-induced increases of dopamine in the caudate putamen and nucleus accumbens of C57BL/6J and 129/J mice

In vivo microdialysis was used to measure the effect of chronic "binge" pattern cocaine administration on basal and cocaine-induced dopamine levels in the caudate putamen and nucleus accumbens of C57BL/6J and 129/J mice. Mice were implanted with a guide cannula in the caudate putamen or nucleus accumbens and after 4 days recovery, one group received "binge" pattern cocaine administration for 13 days (15 mg/kg x 3, i.p. at hourly intervals) while another group received saline in the same pattern. On the day before microdialysis, dialysis probes were lowered into the caudate putamen and nucleus accumbens. The next morning, after baseline dopamine collection, all animals received "binge" cocaine administration. Dialysates were collected every 20 min and dopamine content was determined by HPLC with electrochemical detection. In the basal condition, the mean level of dopamine in the dialysate from both brain regions of mice pretreated with "binge" pattern cocaine administration was significantly lower than that of the mice pretreated with saline administration. The absolute levels of dopamine achieved following "binge" pattern cocaine challenge were lower in the mice that had received chronic cocaine administration. However, when expressed as percent increase over baseline, the dopamine response to cocaine in the nucleus accumbens was significantly higher in mice that received chronic than in mice that received acute cocaine administration. Chronic cocaine administration led to a lowering of both basal dopamine and the absolute levels of cocaine-induced increases of dopamine in the two brain regions, but enhanced the percent increases over the baseline in response to cocaine in the nucleus accumbens of both mouse strains.     

Opioid peptide receptor studies. 12. Buprenorphine is a potent and selective mu/kappa antagonist in the [35S]-GTP-gamma-S functional binding assay.

We utilized the [(35)S]-GTP-gamma-S functional binding assay to determine the selectivity of opioid receptor agonists in guinea pig caudate membranes. The study focused on two opioid agonists used for treating opioid-dependent patients: methadone and buprenorphine. Selective antagonists were used to generate agonist-selective conditions: TIPP + nor-BNI to measure mu receptors, CTAP + nor-BNI to measure gamma receptors and TIPP + CTAP to measure kappa receptors. The assay was first validated with opioid agonists of known subtype specificity (DAMGO for mu, SNC80 for delta, and U69, 593 for kappa receptors). Methadone-stimulated [(35)S]-GTP-gamma-S binding was mu-specific and less potent and efficacious than etorphine (K(d) = 1,537 nM vs. K(d) = 7.8 nM). Buprenorphine failed to stimulate [(35)S]-GTP-gamma-S binding but inhibited agonist-stimulated [(35)S]-GTP-gamma-S binding. The antagonist-K(i) values (nM) of buprenorphine at mu, delta, and kappa receptors were 0.088 nM, 1.15 nM, and 0.072 nM, respectively. The antagonist-K(i) values (nM) of naloxone at mu, delta, and kappa receptors were 1.39 nM, 25.0 nM, and 11.4 nM, respectively. Autoradiographic studies showed that buprenorphine failed to stimulate [(35)S]-GTP-gamma-S binding in caudate-level rat brain sections but blocked DAMGO-stimulated [(35)S]-GTP-gamma-S binding. In cells expressing the cloned rat mu receptor, buprenorphine was a partial agonist and potent mu antagonist. Administration of buprenorphine to rats produced a long-lasting (>24 h) decrease in mu and kappa2 receptor binding and attenuated mu-stimulated [(35)S]-GTP-gamma-S binding. Viewed collectively, these data indicate that, in this assay system, buprenorphine is a potent mu and gamma receptor antagonist. The clinical implications remain to be elucidated. Synapse 34:83-94, 1999. Published 1999 Wiley-Liss, Inc.     

Kappa- and delta-opioid receptor functional activities are increased in the caudate putamen of cannabinoid CB1 receptor knockout mice

The purpose of this study was to examine the functional interaction between endogenous opioid and cannabinoid receptor systems in the caudate putamen and nucleus accumbens. We therefore examined by autoradiography the functional activity and density of micro-, kappa- and delta-opioid receptors in both brain regions of cannabinoid CB1 receptor knockout mice. Functional activity was estimated by measuring agonist-stimulated [35S]GTPgammaS binding. Results showed that deletion of the CB1 cannabinoid receptor markedly increased kappa-opioid (50%) and delta-opioid (42%) receptor activities whereas no differences were found in micro-opioid receptor in the caudate putamen. In contrast, binding autoradiography showed a similar density of micro-, kappa- and delta-opioid receptors between mutant and wild-type mice. No differences were found in densities or activities of micro-, kappa- and delta-opioid receptors between mutant and wild-type mice in the nucleus accumbens. Taken together, our results revealed that deletion of CB1 cannabinoid receptors produced a pronounced increase in the activity of kappa- and delta-opioid receptors in the caudate putamen. This endogenous interaction between opioid and cannabinoid receptors may be relevant to further understand a variety of neuroadaptative processes involving the participation of opioid receptors, such as motor behaviour, emotional responses and drug dependence.     

Neuroanatomical localization ofκ1and κ2 opioid receptors in rat and guinea pig brain

The neuroanatomical localization of kappa opioid receptors in rat and guinea pig brain was determined by quantitative in vitro receptor autoradiography. Our study shows striking differences in kappa 1 and kappa 2 receptor distributions both between species and within each species. In the rat brain, kappa 1 sites (labeled by [3H]U-69,593) are of low density and confined to a small number of structures. These include the claustrum, endopiriform nucleus, caudate putamen, nucleus accumbens, midline nuclear group of the thalamus, superficial grey layer of the superior colliculus, and central grey. kappa 2 sites (labeled by [3H]ethylketocyclazocine or [3H]bremazocine under conditions in which mu, delta, and kappa 1 binding was suppressed) are more widely distributed throughout all levels of rat brain. kappa 2 sites occur at high density in the caudate putamen, nucleus accumbens, amygdala, thalamus, and interpeduncular nuclei. In guinea pig brain, kappa 1 sites predominate and are of high density in layers I and VI of the neocortex, claustrum, endopiriform nucleus, caudate putamen, nucleus accumbens, and molecular layer of the cerebellum. As in rat brain, kappa 2 sites in guinea pig are more uniformly and widely distributed throughout the brain than are kappa 1 sites. The highest density of kappa 2 sites is in the dorsal parabrachial nucleus, interpeduncular nuclei, mammillary nuclei, and posterior thalamic nuclei. Results from this study demonstrate important interspecies differences in the distribution of kappa 1 and kappa 2 opioid receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective heterologous regulation of 5-HT1A receptor-stimulated 35S GTPgammaS binding in the anterior cingulate cortex as a result of 5-HT2 receptor activation

Previous studies have shown that administration of the 5-HT(2) receptor agonist DOI to rats results in the heterologous desensitization of 5-HT(1A) receptor-mediated behavioral and neuroendocrine responses [Neuropsychopharmacology 19 (1998) 354; J. Neurosci. 21 (2001) 7919]. We hypothesized that the basis for these changes in 5-HT(1A) receptor function may involve changes in the capacity of the 5-HT(1A) receptor to activate G proteins. We examined the effect of chronic administration of DOI on the regulation of 5-HT(1A) receptor function at the level of receptor-G protein interaction using quantitative autoradiography of [(35)S]GTPgammaS binding stimulated by the 5-HT(1A) receptor agonist (+/-)8-OH-DPAT (1 microM). Repeated administration of DOI (1 mg/kg, s.c. once daily for 8 days) resulted in a marked down-regulation in 5-HT(2A) binding sites, as labeled by the antagonist radioligand [(3)H]ketanserin, throughout the cerebral cortex. Chronic DOI treatment also resulted in a significant and selective attenuation of 5-HT(1A) receptor-stimulated [(35)S]GTPgammaS binding in the anterior cingulate cortex (vehicle-treated: 74+/-7.7% above basal; DOI-treated: 43+/-4.6% above basal). Interestingly, 5-HT(1A) receptor-stimulated [(35)S]GTPgammaS binding was not altered in the dorsal or median raphe, or in the limbic structures and other cortical regions examined. The decrease in 5-HT(1A) receptor-stimulated [(35)S]GTPgammaS binding in anterior cingulate cortex was not due to a decrease in 5-HT(1A) receptor number, indicating that the capacity of the 5-HT(1A) receptor to activate G proteins is attenuated in this cortical area following repeated DOI treatment. The heterologous regulation of 5-HT(1A) receptor function by chronic 5-HT(2) receptor activation in the anterior cingulate cortex raises interesting questions as to how the regulatory interaction between these serotonin receptor subtypes influences cognition, memory and emotion.     

Autoradiographic differentiation of mu, delta, and kappa opioid receptors in the rat forebrain and midbrain

While there is an abundance of pharmacological and biochemical evidence to suggest the existence of multiple opioid receptors, their precise localization within the brain is unclear. To help clarify this issue, the present study examined the distributions of the mu, delta, and kappa opioid receptor subtypes in the rat forebrain and midbrain using in vitro autoradiography. Mu and delta receptors were labeled with the selective ligands 3H-DAGO (Tyr- D-Ala-Gly-MePhe-Gly-ol), and 3H-DPDPE (D-Pen2, D-Pen5-enkephalin), respectively, while the kappa receptors were labeled with 3H-(-)bremazocine in the presence of unlabeled DAGO and DPDPE. Based on previous findings in our laboratory, the labeling conditions were such that each ligand selectively occupied approximately 75% of each of the opioid sites. The results demonstrated that all 3 opioid receptor subtypes were differentially distributed in the rat brain. Mu binding was dense in anterior cingulate cortex, neocortex, amygdala, hippocampus, ventral dentate gyrus, presubiculum, nucleus accumbens, caudate putamen, thalamus, habenula, interpeduncular nucleus, pars compacta of the substantia nigra, superior and inferior colliculi, and raphe nuclei. In contrast, delta binding was restricted to only a few brain areas, including anterior cingulate cortex, neocortex, amygdala, olfactory tubercle, nucleus accumbens, and caudate putamen. Kappa binding, while not as widespread as observed with mu binding, was densely distributed in the amygdala, olfactory tubercle, nucleus accumbens, caudate putamen, medial preoptic area, hypothalamus, median eminence, periventricular thalamus, and interpeduncular nucleus. While all 3 opioid receptor subtypes could sometimes be localized within the same brain area, their precise distribution within the region often varied widely. For example, in the caudate putamen, mu binding had a patchy distribution, while delta and kappa sites were diffusely distributed, with delta sites being particularly dense ventrolaterally and kappa sites being concentrated ventromedially. These results support the existence of at least 3 distinct opioid receptors with possibly separate functional roles.     

Chronic cocaine alters brain mu opioid receptors

The possibility that dopamine may modulate the expression of opioid receptors was investigated by determining the effects of chronic cocaine administration on the density of μ opioid receptors. Quantitative in vitro autoradiography with the highly selective μ opioid ligand [³H]DAMGO was used to measure and localize changes in μ opioid receptors in the brains of rats administered cocaine or saline three times daily for 14 days. Significant increases in [³H]DAMGO binding were measured in areas of the cingulate cortex, nucleus accumbens, caudate putamen, and basolateral amygdaloid nucleus of the cocaine-treated animals. These results demonstrate that μ opioid receptors undergo upregulation in response to chronic cocaine exposure and suggest that dopamine activity can regulate the expression of μ opioid receptors.     

Cocaine alters GABA(B)-mediated G-protein activation in the ventral tegmental area of female rats: modulation by estrogen

In female rats, estrogen has been reported to enhance cocaine sensitization. Here we investigated the effect of estrogen and cocaine treatments on GABA(B)-stimulated [(35)S]GTPgammaS binding. Ovariectomized rats without (OVX) and with estrogen treatment (OVX-EB) were pretreated with saline or cocaine (15 mg/kg, i.p.) for 5 days and after 1 week of withdrawal challenged with cocaine. One hour after the final injection, animals were sacrificed, brains immediately frozen, and stored at -70 degrees C for subsequent cryosectioning. In vitro functional autoradiography was performed using baclofen (300 microM), a GABA(B) receptor agonist, to stimulate [(35)S]GTPgammaS binding in tissue sections at the level of the ventral tegmental area (VTA). OVX-EB rats showed lower levels of [(35)S]GTPgammaS binding in the VTA (-15%) and entorhinal cortex (EC) (-60%). The effect of cocaine on GABA(B)-mediated G-protein activation varied with the presence of estrogen. Repeated cocaine administration reduced [(35)S]GTPgammaS binding in the VTA and EC of OVX rats and increased it in OVX-EB. Thus, our data suggest that estrogen reduces GABA(B)-mediated G-protein activation in female rats. The results also show that estrogen strongly influences cocaine-induced alterations in GABA(B) function in the VTA and EC of female rats.     

Effect of acute binge cocaine on levels of extracellular dopamine in the caudate putamen and nucleus accumbens in male C57BL/6J and 129/J mice

Levels of dopamine, both basal and after binge-pattern cocaine administration, were measured in the caudate putamen and nucleus accumbens of C57BL/6J and 129/J mice by in vivo microdialysis. Six-week old males were surgically implanted with a CMA guide cannula into the caudate putamen or nucleus accumbens. After 4 days recovery, dialysis probes were lowered into the caudate putamen or the nucleus accumbens and mice were placed in individual microdialysis chambers. The next morning experiments were carried out on freely moving animals. Experimental animals received 1-day binge cocaine administration (15 mg/kgx3, i.p. at hourly intervals) while control animals received saline in the same pattern. Dialysates were collected every 20 min and dopamine content was determined by HPLC with electrochemical detection. Basal levels of dopamine in the dialysate of the caudate putamen were 4.2+/-0.2 nM in C57BL/6J mice and 5.0+/-0.3 nM in 129/J mice. In the nucleus accumbens, basal levels of dopamine were 0.65+/-0.04 nM in the C57BL/6J mice and 0.75+/-0.03 nM in 129/J mice, with no significant differences between strains in either region. Binge cocaine administration significantly increased mean dopamine levels in the caudate putamen in the C57BL/6J mice (with a 3-h mean of 6.80 nM) and in the 129J mice (9.94 nM). In this region, 129/J mice had significantly higher levels of cocaine-induced dopamine than did C57BL/6J mice. In the nucleus accumbens, administration of cocaine also significantly increased dopamine levels in both strains (1.32 nM in C57BL/6J and 1.43 nM in 129/J), but with no difference between strains.     

Effects of cocaine administration on receptor binding and subunits mRNA of GABA(A)-benzodiazepine receptor complexes

The effects of intermittent intraperitoneal (i.p.) administration of cocaine (20 mg/kg) on GABA(A)-benzodiazepine (BZD) receptors labeled by t-[(35)S]butylbicyclophosphorothionate (TBPS), and on several types of mRNA subunits were investigated in rat brain by in vitro quantitative receptor autoradiography and in situ hybridization. Phosphor screen imaging with high sensitivity and a wide linear range of response was utilized for imaging analysis. There was a significant decrease in the level of alpha 1, alpha 6, beta 2, beta 3, and gamma 2 subunits mRNA, with no alteration of [(35)S]TBPS binding in any regions in the brain of rats at 1 h following a single injection of cocaine. In chronically treated animals, the mean scores of stereotyped behavior were increased with the number of injections. The level of beta 3 subunit mRNA was decreased in the cortices and caudate putamen, at 24 h after a final injection of chronic administrations for 14 days. In the withdrawal from cocaine, the frontal cortex and hippocampal complexes showed a significant increase in [(35)S]TBPS binding and alpha1 and beta 3 subunit mRNA in the rats 1 week after a cessation of chronic administration of cocaine. These findings suggest that the disruption of GABA(A)-BZD receptor formation is closely involved in the development of cocaine-related behavioral disturbances. Further studies on the physiological functions on GABA(A)-BZD receptor complex will be necessary for an explanation of the precise mechanisms underlying the acute effects, development of hypersensitization, and withdrawal state of cocaine.