Epipregnanolone and a novel synthetic neuroactive steroid reduce alcohol self-administration in rats.

This study was designed to compare the effects of several neuroactive steroids with varying patterns of modulation of gamma-aminobutyric acid (GABA)(A) and NMDA receptors on operant self-administration of ethanol or water. Once stable responding for 10% (w/v) ethanol was achieved, separate test sessions were conducted in which male Wistar rats were allowed to self-administer ethanol or water following pre-treatment with vehicle or one of the following neuroactive steroids: (3beta,5beta)-3-hydroxypregnan-20-one (epipregnanolone; 5, 10, 20 mg/kg; n=12), (3alpha,5beta)-20-oxo-pregnane-3-carboxylic acid (PCA; 10, 20, 30 mg/kg n=10), (3alpha,5beta)-3-hydroxypregnan-20-one hemisuccinate (pregnanolone hemisuccinate; 5, 10, 20 mg/kg; n=12) and (3alpha,5alpha)-3-hydroxyandrostan-17-one hemisuccinate (androsterone hemisuccinate; 5, 10, 20 mg/kg; n=11). The effect of the 3beta-epimer of PCA, (3beta,5beta)-20-oxo-pregnane-3-carboxylic acid (10, 20, 30 mg/kg; n=9), on ethanol self-administration was also examined. The compounds were administered using a Latin-square design 45 min prior to the weekly test sessions. The effects of the 30 mg/kg dose of the steroidal hemisuccinates on ethanol intake were also examined 5 min after administration of these drugs. Both epipregnanolone and PCA attenuated ethanol self-administration. However, neither of the hemisuccinate compounds significantly altered this behavior. The steroidal hemisuccinates (30 mg/kg; n=7) were also tested 5 min before behavior testing and had no effect on ethanol intake 5 min after administration. The 3beta-epimer of PCA also failed to alter ethanol intake. None of the test compounds altered water intake. In electrophysiological studies, the effects of PCA and androsterone hemisuccinate on evoked GABA(A) receptor-mediated inhibitory postsynaptic currents (GABA(A)-IPSCs) was examined in brain slices of the amygdala. PCA had a stimulatory effect at concentrations of 5 and 25 muM. Androsterone hemisuccinate had no agonist activity. The ability of epipregnanolone and PCA to alter ethanol intake appears to be related to different inhibitory actions of these compounds on either GABA(A) or NMDA receptors, respectively. Thus, dual modulation of these systems by selected neuroactive steroids may offer potential for modifying the reinforcing effects of alcohol.

Serotonin1B receptors in the ventral tegmental area modulate cocaine-induced increases in nucleus accumbens dopamine levels.

Previous work has demonstrated that peripheral serotonin(1B) (5-HT(1B)) receptor agonist administration facilitates the behavioral and neurochemical effects of cocaine. This study used dual probe microdialysis to investigate whether activation of serotonin(1B) (5-HT(1B)) receptors in the ventral tegmental area (VTA) alters the ability of peripherally administered cocaine to elevate dopamine (DA) levels in the ipsilateral nucleus accumbens (NAcc) of drug-naive Wistar rats. Intra-VTA administration of the selective 5-HT(1B) agonist 1,4-dihydro-3-(1,2,3,6-tetrahydro-4-pyridinyl)-5H-pyrrolo [3,2-b]pyridin-5-one dihydrochloride (CP 93,129) by reverse dialysis produced a dose-dependent (30 and 100 microM) potentiation of cocaine-induced (10 mg/kg i.p.) increases in NAcc DA efflux and concurrent cocaine-induced decreases in VTA GABA efflux. There was no effect of either local CP 93,129 or peripheral cocaine on VTA glutamate efflux. Intra-VTA administration of the 5-HT(1A/7) receptor agonist 8-hydroxy-2-dipropylaminotetralin (8-OH-DPAT; 100 microM) did not alter cocaine-induced alterations in NAcc DA or VTA GABA, suggesting that the effects of CP 93,129 were not mediated through 5-HT(1A) receptors. Moreover, the effects of intra-VTA CP 93,129 (100 microM) on both cocaine-induced increases in NAcc DA levels and cocaine-induced decreases in VTA GABA levels were reversed by coadministration of the selective 5-HT(1B) receptor antagonist 3-[3-(dimethylamine)propyl]-4-hydroxy-N-[4-(4-pyridinyl] phenyl] benzamide dihydrochloride (GR 55562; 300 microM). In the absence of cocaine, intra-VTA CP 93,139 produced an increase in NAcc DA and decrease in VTA GABA levels. However, intra-VTA GR 55562 alone had no effect on any of our neurochemical measures. These findings indicate that activation of VTA 5-HT(1B) receptors potentiates cocaine-induced increases in NAcc DA levels by enhancing the ability of cocaine to decrease VTA GABA efflux.

The role of serotonin(2) receptors in mediating cocaine-induced convulsions.

Previous research in our laboratory suggests that serotonin (5-HT) neurotransmission mediates the expression of cocaine-induced convulsions. The role of 5-HT in mediating this toxic effect of cocaine appears to be due to activation of 5-HT(2) receptors, because cocaine-induced convulsions are blocked by the 5-HT(2) antagonists cinanserin, ketanserin, and pirenperone. The present study utilized a number of compounds that display a high affinity for 5-HT(2) receptors to further examine the role of these sites in mediating this toxic effect of cocaine. Cocaine-induced convulsions were observed following pretreatment with various doses of the following 5-HT(2) antagonists: mianserin, metergoline, MDL 11939, and methiothepin. In addition, 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl]piperazine (NAN 190) was tested to examine the influence of 5-HT(1) sites and the agonist compound 1-(3-triflurormethylphenyl)piperazine (TFMPP) was examined to further explore the role of 5-HT(1) and 5-HT(2) sites. Each 5-HT(2) antagonist attenuated cocaine-induced convulsions. Conversely, NAN 190 did not alter this toxic effect of cocaine. In addition, TFMPP significantly potentiated cocaine-induced convulsions. The results from this study support the hypothesis that 5-HT neurotransmission, acting primarily at 5-HT(2) receptors, plays an important role in mediating cocaine-induced convulsions.

Molecular serotonergic mechanisms appear to mediate genetic sensitivity to cocaine-induced convulsions.

Cocaine-induced convulsions appear to be mediated by serotonin (5-HT) neurotransmission, acting primarily at 5-HT(2) receptors. However, this effect of cocaine is attenuated by cocaine binding at sigma and muscarinic M(1) and M(2) sites. This study examined whether the aforementioned neural sites mediate the nearly two-fold difference in sensitivity to cocaine-induced convulsions across C57BL/6J (6J) and C57BL/6ByJ (6ByJ) mice. Experiment 1 compared 5-HT transporter densities across several brain regions of 6J and 6ByJ mice and cocaine-induced convulsions following pretreatment with the 5-HT reuptake inhibitor fluoxetine. Experiment 2 compared 5-HT(2) receptor densities across these mice and cocaine-induced convulsions following pretreatment with the 5-HT(2) antagonist cinanserin. There were no differences in 5-HT transporter densities, however, fluoxetine produced a greater facilitation of cocaine-induced convulsions in 6ByJ relative to 6J mice, suggesting that sensitivity to convulsions is mediated postsynaptically. Indeed, 5-HT(2) density was higher in 6ByJ relative to 6J mice in the amygdaloid ridge, hypothalamus, and midbrain. In addition, cinanserin attenuated convulsions more potently in 6J relative to 6ByJ mice. There were no differences in the densities or affinities of 5-HT(1), muscarinic, or sigma receptors across these strains, suggesting that density of these latter sites does not mediate genetic sensitivity to cocaine-induced convulsions. Since 6ByJ mice are less sensitive to convulsions despite the fact that they have more 5-HT(2) receptors, we hypothesized that these mice may exhibit a weaker linkage of 5-HT(2) sites to their second-messenger system relative to 6J mice. However, in experiment 3 we demonstrated that 5-HT(2)-receptor mediated phosphoinositide hydrolysis was higher in 6ByJ relative to 6J mice in the same regions also displaying higher 5-HT(2) densities. This study suggests that 5-HT(2) receptors mediate genetic sensitivity to cocaine-induced convulsions, further supporting the role of these sites in mediating this toxic effect of cocaine.

Antidepressant drugs appear to enhance cocaine-induced toxicity.

It has been shown that cocaine-induced convulsions and lethality appear to be mediated by serotonin and dopamine neurotransmission, respectively. However, many antidepressants considered for treatment of cocaine addiction target these monoamine systems and may thus amplify these toxic effects during relapse. In this study, the authors assessed whether pretreatment with antidepressants influences cocaine-induced toxicity in mice as well as the potency of these medications at cocaine-binding sites previously shown to be associated with cocaine toxicity. Overall, selective serotonin reuptake inhibitors (SSRIs) facilitated cocaine-induced convulsions but not lethality. Dopamine uptake inhibition facilitated cocaine-induced lethality, but not convulsion. The SSRI sertraline enhanced neither convulsions nor lethality and may be unique due to its high affinity for sigma receptors. These results have important implications for safe and effective addiction treatments.

Serotonin(2C) receptors appear to mediate genetic sensitivity to cocaine-induced convulsions.

RATIONALE: C57BL/6ByJ (6ByJ) and C57BL/6J (6J) mice differ in their sensitivity to cocaine-induced convulsions, with CD(50) values being 100 and 70 mg/kg, respectively. This genetic sensitivity to cocaine-induced convulsions is probably related to 5-HT(2) receptors, since the density of these sites and the concentration of 5-HT(2) antagonists required to block cocaine-induced convulsions is lower in 6J mice relative to 6ByJ mice. OBJECTIVE: Although 5-HT(2) receptors appear to play a role in mediating genetic sensitivity to cocaine-induced convulsions, the role of 5-HT(2) receptor subtypes in this effect of cocaine has not been examined. METHODS: The present study compared the effects of the preferential 5-HT(2C) agonists m-chlorophenylpiperazine (mCPP) and 6-chloro-2-(1-piperazinyl)pyrazine (MK212) on cocaine-induced convulsions in 6ByJ and 6J mice. General activity was also measured following pretreatment with mCPP and MK212. RESULTS: Both mCPP and MK212 potentiated cocaine-induced convulsions and the effect of these agonists was more robust in 6ByJ mice relative to 6J mice. CONCLUSION: The findings from this study support previous research suggesting that 5-HT(2) receptors play a role in mediating cocaine-induced convulsions, and extend previous research by suggesting that the 5-HT(2C) receptor subtype mediates cocaine-induced convulsions and genetic sensitivity to this toxic effect of cocaine.

Behavioral effects of psychomotor stimulant infusions into amygdaloid nuclei.

The role of amygdaloid nuclei in locomotion, stereotypy, and conditioned place preference (CPP) produced by psychomotor stimulants was examined. Five 2-day conditioning trials were conducted over 10 consecutive days. Rats received bilateral intracranial infusions of saline, cocaine (25-100 micrograms/side), or amphetamine (0.31-20 micrograms/side) into the ventricles (ICV), basolateral amygdala (BlA), or central amygdala (CeA) and were confined to a compartment. On alternating days, rats received sham infusions and were confined to a different compartment. Locomotion was measured daily, stereotypy was measured on trials 1 and 5, and CPP was measured 24 h after conditioning. ICV infusions of cocaine or amphetamine produced locomotion, rearing, and CPP. Intra-BlA and intra-CeA infusions of the highest dose of cocaine produced locomotion. In contrast, intra-CeA infusions of amphetamine potently produced locomotion and CPP. Intra-BlA infusions of amphetamine, however, did not produce any behavioral changes. These results suggest that the CeA, but not the BlA, is involved in initiating reward and locomotion produced by amphetamine.

Effects of SCH-23390 on dopamine D1 receptor occupancy and locomotion produced by intraaccumbens cocaine infusion.

This study examined the effects of both systemic and intraaccumbens administration of SCH-23390 in rats on dopamine D1 receptor occupancy and on locomotor activity produced by intraaccumbens infusion of cocaine. In experiment 1, rats received SCH-23390 (0-1 mg/kg, i.p.) 15 minutes prior to intraaccumbens infusion of cocaine (0 or 100 microg/side). In experiment 2, rats received coinfusion of SCH-23390 (0-1 microg/side) and cocaine (0 or 100 microg/side) into the nucleus accumbens (NAc). After behavioral testing, receptors occupied by SCH-23390 were quantified by injecting animals with their respective dose of SCH-23390, followed by a systemic injection of the irreversible antagonist N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). Receptors occupied by SCH-23390, and therefore protected from EEDQ-induced inactivation, were quantified from autoradiograms of sections labeled with 3H-SCH-23390. Systemic administration of SCH-23390 dose-dependently (0.1-1.0 mg/kg) reversed cocaine-induced locomotion and occupied 72-100% of D1-like receptors in the anterior NAc. D1 receptor occupancy following systemic administration of SCH-23390 was evident as an inverted U-shaped, dose-dependent change, with the greatest occupancy observed at the intermediate dose of 0.3 mg/kg. Intraaccumbens infusion of SCH-23390 did not alter cocaine-induced locomotor activity despite occupying 40-60% of D1-like receptors in the anterior NAc core and shell. The findings that systemic, but not intraaccumbens, administration of SCH-23390 potently reversed locomotion produced by intraaccumbens cocaine infusion suggest that stimulation of D1 receptors in regions other than the NAc is involved in locomotion produced by intraaccumbens infusion of cocaine, and that stimulation of D1 receptors in the NAc is not necessary for this behavior.

Time-dependent changes in cocaine-seeking behavior and extracellular dopamine levels in the amygdala during cocaine withdrawal.

Cocaine and cocaine-associated cues elicit craving in addicts and reinstate cocaine-seeking behavior in rats. Craving and cocaine-seeking behavior may be mediated by withdrawal-induced changes in dopamine (DA) neurotransmission in the amygdala. To examine whether there are concomittant changes in cocaine-seeking behavior and extracellular DA levels during withdrawal, experimental rats were trained to self-administer cocaine (0.75 mg/kg i.v.). After 14 daily 3-hour training sessions, animals underwent either a 1-day, 1-week, or 1-month withdrawal period. Extracellular DA levels were assessed during baseline, extinction, cue reinstatement, and cocaine (15 mg/kg i.p.) reinstatement of cocaine-seeking behavior (i.e., defined as the difference in nonreinforced lever presses on an active minus inactive lever). Cocaine-seeking behavior became more intense during the course of cocaine withdrawal. Additionally, basal and cocaine-induced extracellular DA levels were enhanced after the 1-month withdrawal period. We suggest that the former may reflect a persistent elevation in tonic extracellular DA levels in the amygdala, whereas the latter may reflect a persistent elevation in phasic extracellular DA levels.

Dopamine overflow in the nucleus accumbens during extinction and reinstatement of cocaine self-administration behavior.

Both cocaine and cocaine-associated stimuli can reinstate extinguished self-administration behavior in animals. It has been suggested that reinstatement of drug-seeking behavior may be mediated by enhanced dopamine (DA) neurotransmission. To examine this hypothesis, DA overflow was measured in the nucleus accumbens (NAc) of rats during both extinction and cocaine-induced reinstatement of self-administration behavior. Rats were either allowed to self-administer cocaine for 3 hours daily for 14 days, or they received yoked administration of saline. A stimulus light above the lever was illuminated during drug delivery. Baseline DA overflow was measured in the NAc, using in vivo microdialysis 7 to 8 days after the last self-administration session. The rats were then placed into the operant chambers and allowed to respond in extinction for 90 minutes, during which responses resulted in presentation of the stimulus light. The rats then received a cocaine injection that reinstated self-administration behavior. Contrary to our hypothesis, cocaine-experienced animals exhibited less DA overflow in the NAc relative to controls during both extinction and reinstatement.

Differential effects of intra-accumbens sulpiride on cocaine-induced locomotion and conditioned place preference.

The effects of intra-accumbens sulpiride on conditioned place preference and locomotion produced by i.v. cocaine were investigated. Every other day during conditioning, rats received infusions of sulpiride (0-0.4 microgram) into the nucleus accumbens (NAc) or caudate-putamen. Fifteen min later, they were placed into a distinct compartment and injected with saline or cocaine (4.2 mg/kg, i.v.). On the alternate days, rats received sham intracranial injections and were placed into a different compartment. Locomotion and stereotypies were assessed after the first and last injection, and conditioned place preference was assessed 24 hr after the last conditioning day. After behavioral testing, receptors occupied by sulpiride were quantified by injecting rats intracranially with their respective dose of sulpiride, followed by a systemic injection of the irreversible antagonist N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). Receptors protected from EEDQ induced inactivation by sulpiride were revealed on autoradiograms of sections labeled with 3H-sulpiride. Sulpiride did not alter cocaine-conditioned place preference or cocaine-induced stereotypies. However, the two lowest doses of intra-accumbens sulpiride attenuated cocaine-induced locomotion and occupied > 42% of the sulpiride binding sites in the NAc, and the highest dose completely reversed cocaine-induced locomotion and occupied > 96% of the sulpiride binding sites in the NAc. Intracaudate sulpiride also attenuated cocaine-induced locomotion without occupying a significant number of binding sites in the NAc. These findings suggest that D2-like receptors in the NAc and anterior medial caudate-putamen are involved in cocaine-induced locomotion, but not cocaine-conditioned place preference.

Dose-dependent characterization of the rewarding and stimulant properties of cocaine following intraperitoneal and intravenous administration in rats.

Dose-dependent differences in the rewarding and stimulant properties of cocaine administered intravenously (IV) and intraperitoneally (IP) were compared. Six 2-day conditioning trials were conducted over consecutive days. Rats received cocaine and were placed into a compartment on one day of the trial, and were directly placed into a different compartment without drug on the other day. Rats were exposed to the compartments for either 20 or 40 min. The effects of cocaine on stimulant behaviors, including locomotion and stereotypies, were compared following the first and last injection. After conditioning, three tests were given with 1 rest day intervening each: (1) conditioned place preference (CPP) was measured as an increase in the amount of time animals spent in the injection compartment relative to the noninjection compartment when given access to both, (2) conditioned activity (CA) was measured as an increase in stimulant behaviors in cocaine-treated animals relative to saline controls following an injection of saline in the injection compartment and (3) context-independent sensitization was measured as an increase in stimulant behaviors following an injection of cocaine in the noninjection compartment relative to the animals' behavior following the first injection. Cocaine did not reliably produce sensitization of locomotion under any of the conditions examined. Cocaine produced sensitization of headbobbing that was more robust following IP administration than it was following IV administration. In both cases, sensitization of headbobbing involved a context-independent component. Cocaine produced CPP and CA with both routes of administration. CPP was established more readily with 40-min relative to 20-min exposures following IV administration, whereas CA was more prevalent with 20-min relative to 40-min exposures. This study provides a thorough characterization of the behavioral effects of cocaine administered IV and a new efficient method for assessing the effects of cocaine on conditioned and unconditioned behaviors following repeated administration.

Localization of dopamine receptor subtypes occupied by intra-accumbens antagonists that reverse cocaine-induced locomotion.

The purpose of this study was to examine whether blockade of either dopamine D1-like or D2-like receptors by selective antagonist administration into the nucleus accumbens (NAc) is sufficient to reverse cocaine-induced locomotion, and to develop a new technique that enables the population of receptors occupied by the antagonists to be quantified. Locomotor activity was assessed in rats that had received bilateral intra-accumbens injections of the D1-selective antagonist SCH-23390 (0-3.0 micrograms/0.5 microliters/side) or the D2/D3-selective antagonist sulpiride (0-1.0 micrograms/0.5 microliters/side), followed 15 min later by injections of saline or cocaine (15 mg/kg, i.p.). To assess receptor occupancy by the antagonists, 105 min prior to sacrifice the rats received intra-accumbens injections of the antagonist, followed 15 min later by an injection of the non-selective irreversible antagonist, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ; 10 mg/kg, i.p.). Receptors were labeled with [3H]SCH-23390 or [3H]sulpiride in sections containing the NAc, and the autoradiograms allowed quantitation of receptors occupied (i.e. protected from EEDQ) by the antagonist given in vivo. Only a dose of 3 micrograms/side SCH-23390 reversed cocaine-induced locomotion, whereas a dose of 0.5 microgram/side did not alter cocaine-induced locomotion despite occupying the same amount of [3H]SCH-23390 binding sites in the NAc. Intermediate doses of 0.1 and 0.3 microgram/side sulpiride reversed cocaine-induced locomotion, and also occupied the greatest number of [3H]sulpiride binding sites in the NAc. The results suggest that blockade of D2-like, but not D1-like, receptors in the NAc is sufficient to reverse cocaine-induced locomotion, and also demonstrate the importance of quantifying receptors occupied by drugs administered intracranially.