Estrogen alters baseline and inflammatory-induced cytokine levels independent from hypothalamic-pituitary-adrenal axis activity.

Although estrogen reduces inflammatory-mediated pain responses, the mechanisms behind its effects are unclear. This study investigated if estrogen modulates inflammatory signaling by reducing baseline or inflammation-induced cytokine levels in the injury-site, serum, dorsal root ganglia (DRG) and/or spinal cord. We further tested whether estrogen effects on cytokine levels are in part mediated through hypothalamic-pituitary-adrenal (HPA) axis activation. Lumbar DRG, spinal cord, serum, and hind paw tissue were analyzed for cytokine levels in 17ß-estradiol-(20%) or vehicle-(100% cholesterol) treated female rats following ovariectomy/sham adrenalectomy (OVX), adrenalectomy/sham ovariectomy (ADX) or ADX+OVX operation at baseline and post formalin injection. Formalin significantly increased pro-inflammatory interleukin (IL)-6 levels in the paw, as well as pro- and anti-inflammatory cytokine levels in the DRG, spinal cord and serum in comparison to naïve conditions. Estrogen replacement significantly increased anti-inflammatory IL-10 levels in the DRG. Centrally, estradiol significantly decreased pro-inflammatory tumor necrosis factor (TNF)-α and IL-1ß levels, as well as IL-10 levels, in the spinal cord in comparison to cholesterol treatment. At both sites, most estradiol modulatory effects occurred irrespective of pain or surgical condition. Estradiol alone had no influence on cytokine release in the paw or serum, indicating that estrogen effects were site-specific. Although cytokine levels were altered between surgical conditions at baseline and following formalin administration, ADX operation did not significantly reverse estradiol's modulation of cytokine levels. These results suggest that estrogen directly regulates cytokines independent of HPA axis activity in vivo, in part by reducing cytokine levels in the spinal cord.

Estradiol-induced antinociceptive responses on formalin-induced nociception are independent of COX and HPA activation.

Estrogen modulates pain perception but how it does so is not fully understood. The aim of this study was to determine if estradiol reduces nociceptive responses in part via hypothalamic-pituitary-adrenal (HPA) axis regulation of cyclooxygenase (COX)-1/COX-2 activity. The first study examined the effects of estradiol (20%) or vehicle with concurrent injection nonsteroidal antiinflammatory drugs (NSAIDs) on formalin-induced nociceptive responding (flinching) in ovariectomized (OVX) rats. The drugs were ibuprofen (COX-1 and COX-2 inhibitor), SC560 (COX-1 inhibitor), or NS398 (COX-2 inhibitor). In a second study, estradiol's effects on formalin-induced nociception were tested in adrenalectomized (ADX), OVX, and ADX+OVX rats. Serum levels of prostaglandins (PG) PGE(2) and corticosterone were measured. Estradiol significantly decreased nociceptive responses in OVX rats with effects during both the first and the second phase of the formalin test. The nonsteroidal antiinflammatory drugs (NSAIDs) did not alter nociception at the doses used here. Adrenalectomy neither altered flinching responses in female rats nor reversed estradiol-induced antinociceptive responses. Estradiol alone had no effect on corticosterone (CORT) or prostaglandin levels after the formalin test, dissociating the effects of estradiol on behavior and these serum markers. Ibuprofen and NS398 significantly reduced PGE2 levels. CORT was not decreased by OVX surgery or by estradiol below that of ADX. Only IBU significantly increased corticosterone levels. Taken together, our results suggest that estradiol-induced antinociception in female rats is independent of COX activity and HPA axis activation.

Progesterone attenuates cocaine-induced responses.

In this review, we summarize literature focused on how progesterone alters cocaine-induced psychomotor, reinforcement, and physiological responses. Clinical studies suggest that progesterone attenuates the subjective effects of cocaine. Similarly, preclinical studies have demonstrated that cocaine-induced reward and psychomotor responses are attenuated after progesterone administration. In rats progesterone also reduces the reinforcement effects of cocaine attenuates acquisition, escalation, reinstatement of cocaine self-administration, and cocaine-seeking behaviors. Progesterone also counteracts the facilitatory effects of estrogen on cocaine self-administration and psychomotor activation. These findings suggest that progesterone has a potential in clinical applications as a treatment for cocaine addiction. Constantly changing progesterone serum levels in female humans and rats affect the female's reinforcement responses to cocaine and may in part contribute to the known sex differences in cocaine responses.

Cocaine-induced sex differences in D1 dopamine receptor mRNA levels after acute cocaine administration.

INTRODUCTION: Although it is known that female rats have a more robust behavioral response to acute cocaine administration than male rats, the neurobiological mechanisms underlying these differences remain unclear. The purpose of the present study was to determine if there are sex differences in cocaine's regulation of dopamine D1 and D2 receptor mRNA levels. METHODS: Male and female Fischer rats received acute cocaine (20 mg/kg, intraperitoneal) or saline. Ambulatory activity was recorded one hour post drug treatment. Rats were then sacrificed either 1 or 24 hours post drug treatment and D1/D2 DA receptor mRNA levels were measured via solution hybridization assay. RESULTS: Cocaine-induced ambulatory activity was greater in female than male rats. There were no sex differences in baseline levels of D1 and D2 receptor mRNA in the caudate putamen (CPu) or the nucleus accumbens (NAc). Cocaine administration reduced levels of D1 mRNA in the NAc only in male rats. CONCLUSION: Our findings suggest that the regulation of striatal D1 mRNA levels after acute cocaine administration is a sexually dimorphic process. We also hypothesize that the D1 receptor may be an important substrate in the regulation of sex differences in cocaine-induced locomotor activity.

Acute and chronic estradiol replacements differentially alter corticosterone and COX-mediated responses to an inflammatory stimulus in female rats.

INTRODUCTION: This study aimed to determine whether the previously reported differential effects of estradiol on inflammation-induced behavioral responses are in part explained through differential activation of the corticosterone-cyclooxygenase (CORT-COX) regulatory pathway. METHODS: Prostaglandin E2 (PGE2), COX, and CORT levels were analyzed before and after a formalin administration (1% vs. 5%, representing different intensities of inflammatory stimuli). RESULTS: In vehicle-treated rats, chronic estradiol administration increased corticosterone, and decreased COX and PGE2. After acute estradiol administration, although corticosterone serum levels were increased, COX protein levels were unchanged. In rats treated with formalin, PGE2 serum levels were higher in rats administered 5% formalin than vehicle- and 1%-treated rats. Significant correlations were observed between PGE2 serum levels, CORT serum levels, and COX protein levels. CONCLUSIONS: Our results suggest that the administration of exogenous estradiol may mediate inflammatory responses by regulating the levels of PGE2 and/or CORT release, thereby mediating the nociceptive response to an inflammatory stimulus.

Endogenous gonadal hormones regulate females' behavioral responses to formalin through prostaglandin E2 release.

INTRODUCTION: This study aimed to determine if endogenous gonadal hormones affect the intracellular mechanisms in the spinal cord that control inflammatory pain responses. METHODS: We analyzed behavioral responses to, and changes in, serum levels of prostaglandin E2, estradiol, progesterone, and corticosterone after administration of 5% formalin in intact and ovariectomized (OVX) female rats. RESULTS: OVX females displayed significantly more flinching than did intact females during Phase I, and after formalin administration their corticosterone levels were significantly lower. No differences were seen across COX-1 and COX-2 protein expression in the spinal cord of either naive or formalin-treated rats. However, subsequent to formalin a main effect of gonadectomy was seen in prostaglandin E2 levels; OVX animals had significantly lower prostaglandin E2 levels than intact animals. CONCLUSIONS: These results indicate that in female rats nociceptive responses to formalin are regulated through the levels of prostaglandin E2, an important mediator in inflammation, whereas protein levels of COX-1 and COX-2 play a more limited role.

Sex differences in dopamine D2-like receptor-mediated G-protein activation in the medial prefrontal cortex after cocaine.

INTRODUCTION: Sexually dimorphic behavioral responses to cocaine have been linked to a difference in activation of dopamine receptors. Our study was conducted to determine whether dopamine D2-like receptor-activated G-protein contributes to sex differences in response to cocaine in the medial prefrontal cortex (mPFC). METHOD: In vitro functional autoradiography was performed using dopamine receptor D2 agonist (quinpirole, 100 microM) to stimulate [35S]GTPgammaS binding in brain tissue sections from male and female Fischer rats treated with saline (1 mL/kg) or cocaine (20 mg/kg; i.p.). RESULTS: Overall, quinpirole increased G-protein activation in the caudate-putamen, nucleus accumbens, and frontal cortex in both sexes. Although saline-treated male rats had higher [35S]GTPyS binding in the mPFC than their female counterparts, cocaine-treated females had higher [35S]GTPgammaS binding in the mPFC than cocaine-treated males. CONCLUSIONS: These data suggest that both intrinsic and activational effects of dopamine D2-like receptor-mediated G-protein activation in the mPFC may contribute to the differences between males and females in their response to acute cocaine administration.

Progesterone does not affect cocaine-induced conditioned place preference or locomotor activity in male rats.

INTRODUCTION: The present study aimed to determine if, as occurs in female rats, progesterone attenuates cocaine-induced reward and psychomotor responses in male rats. METHODS: The role of progesterone in the acquisition and/or expression of cocaine-induced conditioned place preference (CPP) and locomotor responses of intact male rats was studied. For chronic progesterone treatment, rats received Silastic capsules with either progesterone (100%) or vehicle 1 week prior to conditioning. For acute progesterone treatment, rats received subcutaneous injections of progesterone (500 microg) or vehicle (sesame oil) 4 hours before intraperitoneal injections of saline or cocaine administration (20 mg/kg) on conditioning days (acquisition phase-formation of reward associations) or before testing (expression phase-recall of reward associations). RESULTS: Both progesterone-treatment paradigms produced equivalent progesterone serum levels. Progesterone administered chronically or acutely during the acquisition and expression phases of cocaine conditioning did not block cocaine-induced CPP. Nor did progesterone affect ambulatory or rearing behaviors after cocaine administration. CONCLUSION: These results suggest that, unlike the findings with female rats (in which similar treatment paradigms inhibited the formation and recall of cocaine-induced CPP), progesterone plays a limited role in the cocaine-induced reward or psychomotor responses of male rats.

Basal and cocaine-induced sex differences in the DARPP-32-mediated signaling pathway.

RATIONALE: Behavioral and dopamine responses to cocaine are sexually dimorphic: Female rats exhibit higher levels of locomotor and reward-associated behaviors after cocaine administration and dopamine release than do males. Activation of the dopamine- and cAMP-regulated phosphoprotein of Mr 32 kDa (DARPP-32) intracellular cascade mediates responses to cocaine. OBJECTIVE: To examine the possibility that acute cocaine administration alters the DARPP-32 cascade in a sexually dimorphic pattern. MATERIALS AND METHODS: Male and female rats received either saline or cocaine (30 mg/kg). Protein levels of DARPP-32, phosphorylation of DARPP-32 at the Thr34 site (P-Thr34-DARPP-32), protein phosphatase 1 (PP-1), and protein phosphatase 2B (PP-2B) in nucleus accumbens were measured via Western blot analysis. RESULTS: Females had higher protein levels of DARPP-32, P-Thr34-DARPP-32, calcineurin A (CaN-A; catalytic subunit of PP-2B), and calcineurin B (CaN-B; regulatory subunit of PP-2B) than males 5 min after saline treatment. In females, CaN-A protein levels were also higher at 15 min and PP-1 protein levels were higher 30 min after saline administration than males. In male rats, cocaine significantly increased CaN-A protein levels at 30 min and CaN-B protein levels at 15 min. In females, cocaine administration significantly decreased protein levels of DARPP-32, P-Thr34-DARPP-32, and CaN-A at 45 min but increased PP-1 protein levels at 30 min. Overall, males had higher activation of the DARPP-32 pathway after cocaine administration than did females. CONCLUSION: These novel results show that basal and cocaine-induced sex differences in the DARPP-32/PP-1 cascade may be responsible for the sexual dimorphism in acute cocaine-induced behavioral responses.

Progesterone attenuates cocaine-induced conditioned place preference in female rats.

Progesterone replacement attenuates the intensity of cocaine-induced conditioned place preference (CPP) behaviors in female rats. The present study aimed to expand that finding by (i) determining the role of progesterone in the acquisition and/or expression of cocaine-induced CPP and (ii) determining if progesterone's effects might be meditated through learning and memory. To this end, female rats were administered progesterone during cocaine conditioning or object recognition tasks; rats received subcutaneous injections of progesterone (500 microg) or vehicle (sesame oil) 4 h before saline or cocaine (5 mg/kg) on conditioning days (acquisition phase) or before testing (expression phase or object recognition tasks). Progesterone treatment during both the acquisition and the expression phases of cocaine conditioning blocked cocaine-induced CPP. Progesterone affected neither the number of entrances and explorations in the CPP chambers nor the ambulatory and rearing behaviors. In the object recognition task (a non-spatial learning and memory task), progesterone treatment had no effect. However, in the object placement task (a spatial learning and memory task), progesterone treatment significantly impaired retention in hormone-treated rats as compared with control groups. These results suggest that progesterone treatment interferes with cocaine-induced reward associations, possibly through effects on spatial working memory consolidation The observed effects of acute progesterone treatment on cocaine-induced CPP may in part contribute reported menstrual effects and sex disparities in overall cocaine use and rates of relapse.

Progesterone and allopregnanolone are induced by cocaine in serum and brain tissues of male and female rats.

Acute and chronic-cocaine administration increase serum levels of progesterone in both male and female rats. This study aimed to determine whether progesterone and its bioactive metabolite allopregnanolone (ALLOP) are altered in the hippocampus and striatum (areas known to modulate cocaine-induced behavioral response) after acute cocaine administration. To this end, male and female rats were administered 20 mg/kg and 5 mg/kg of cocaine, respectively (doses that produce equivalent behavioral responses between the sexes). Thirty minutes after drug treatment, serum and brain were collected and later analyzed for progesterone and ALLOP levels using HPLC measurements. At these cocaine doses, no sex differences in the overall behavioral responses after drug treatment were observed. In saline-treated controls, female rats had overall higher levels of progesterone in the serum than did male rats. After cocaine administration, progesterone and ALLOP levels in serum, hippocampus, and striatum were increased at similar levels in both sexes. In the hippocampus, progesterone levels were increased in both males and females, but ALLOP levels were increased only in females.

Coadministration of estrogen and progesterone differentially affects locomotor responses to cocaine in rats.

Fluctuations in ovarian hormones throughout the estrous cycle may underlie sex differences in behavior. In this study, estrogen plus progesterone were coadministered at different ratios to determine whether the interaction of those hormones during the estrous cycle contributes to cocaine-induced alterations in behavior. Before cocaine (15 mg/kg) or saline administration, ovariectomized female rats received either vehicle or estrogen (10 microg or 50 microg) and progesterone (100 microg or 500 microg). Cocaine-induced locomotor activity was affected by estrogen plus progesterone ratio. While administration of 50 microg estrogen plus 500 microg progesterone increased total locomotor behavior, administration of 10 microg estrogen plus 500 microg progesterone inhibited total locomotor activity. This study suggests that alterations of estrogen and progesterone serum levels may underlie the observed changes in cocaine-induced behavior during the estrous cycle.

Effects of RU 486 and tamoxifen on cocaine-induced behavioral and endocrinologic activations in male and female Fischer rats.

Evidence suggests that sex differences in response to cocaine administration may be regulated by activation of progesterone and estrogen receptors. To test this hypothesis, rats were pretreated with either RU 486 (progesterone antagonist; 0, 3, or 25 mg/kg), tamoxifen (estrogen antagonist; 0, 1, or 3 mg/kg), or vehicle followed by saline or cocaine administration (15 mg/kg). Although RU 486 did not affect cocaine-induced locomotor activity in female rats, it dose-dependently decreased such activity in males (3 mg/kg significantly attenuated locomotor responses in cocaine-treated rats as compared with vehicle treatment or 25 mg/kg of RU 486). RU 486 also affected baseline serum levels of corticosterone. Males treated with 3 mg/kg of RU 486 plus cocaine had higher progesterone and corticosterone serum levels than vehicle-treated groups. In females, both doses (3 and 25 mg/kg) of RU 486 significantly attenuated corticosterone serum levels compared with vehicle treatment. For both sexes overall, tamoxifen neither significantly influenced cocaine-induced ambulatory and rearing responses nor altered cocaine-induced progesterone and corticosterone serum levels. Taken together, our results suggest that progesterone receptors have a sexually dimorphic role in cocaine-induced effects, but estrogen receptors have only a limited role. Moreover, both receptor antagonists modulate neurochemical responses differentially.

Testosterone plays a limited role in cocaine-induced conditioned place preference and locomotor activity in male rats.

Growing evidence suggests that sex differences in cocaine reward responses are regulated by endogenous gonadal hormones. However, few studies have addressed the role of testosterone on cocaine reward and psychomotor activation. This study aimed to determine whether testosterone influences the development of psychomotor and reward responses to cocaine. Castrated 8-week-old male Fisher rats received placebo or testosterone via Silastic capsules (1-3 capsules of 100% testosterone) or subcutaneous injections (400, 800, or 1200 microg/kg) concurrent with cocaine administration. Although chronic testosterone administration did not alter cocaine-induced conditioned place preference (CPP), concurrent administration of testosterone and cocaine affected the development of cocaine CPP dose-dependently; 400 microg/kg blocked the expression of cocaine-induced CPP. Testosterone did not affect cocaine-induced locomotor activity. Furthermore, testosterone-saline-treated controls did not develop CPP, suggesting that at these doses, testosterone does not produce rewarding or motor responses. These data suggest that testosterone may play a limited role in cocaine-induced reward associations and locomotor responses and thus has a limited effect in the previously reported sexually dimorphic responses to cocaine.

Effects of acute cocaine on ERK and DARPP-32 phosphorylation pathways in the caudate-putamen of Fischer rats.

Activation of extracellular signal-regulated kinase (ERK) and dopamine- and cAMP-regulated phosphoprotein (DARPP-32) pathways has been implicated in biochemical and behavioral effects induced by various drugs of abuse. In this study, we investigated the phosphorylation pathways of these two proteins in response to acute cocaine administration. A single cocaine administration (30 mg/kg) increased ERK-mediated signaling proteins, phosphoryation of cAMP response element-binding protein (CREB) kinase, pp90 ribosomal S6 kinase (RSK), and c-Fos protein levels in the caudate/putamen of Fischer rats. Acute cocaine administration also induced phosphorylation of the striatal-enriched protein tyrosine phosphatase (STEP) and decreased the phosphorylation of DARPP-32 protein at the Thr-75 site. The phosphorylation states of these inhibitors of ERK and DARPP-32 proteins may thus contribute to the effects of cocaine on ERK- and DARPP-32-mediated cascades, on gene expression and on behaviors.

Estradiol replacement in ovariectomized rats is antihyperalgesic in the formalin test.

UNLABELLED: A subcutaneous implant of 17beta-estradiol or progesterone provides steady-state serum hormone levels from 7 to 24 days after implantation and allows the evaluation of the effects of the replacement with these hormones on phase 1 and phase 2 formalin-induced behaviors in ovariectomized (OVX) rats. Graded doses of 17beta-estradiol (5% to 40%) reduce formalin-induced behavior by 35% to 49% during phase 2 but not during phase 1, as measured with an automated formalin apparatus. The maximal response is seen with 20% 17beta-estradiol. The antihyperalgesic effect of 20% 17beta-estradiol is significant at 8 days after implantation and persists at 21 days. In contrast, graded doses of progesterone have no effect on either phase of formalin. The estrogen receptor antagonist tamoxifen completely prevents the antihyperalgesic effect of the 20% 17beta-estradiol implant. Formalin-induced behaviors during phase 2 are significantly less in proestrus females and OVX rats given 20% 17beta-estradiol compared with OVX control rats. Also, the formalin-induced increase in serum corticosterone is attenuated in OVX control rats compared with proestrus females and OVX rats given 20% 17beta-estradiol. These results indicate that estrogen replacement in OVX rats restores the maximal corticosterone response to tonic pain and, by an estrogen receptor-mediated process, inhibits tonic pain. PERSPECTIVE: Hormone replacement (HR) therapy remains a widely used modality. We used a pharmacokinetically based rat HR model that results in continuous physiological levels of 17beta-estradiol to demonstrate the analgesic (antihyperalgesic) effects of estrogen replacement in an inflammatory pain model (formalin). These results suggest a potentially important consequence of HR therapy.

NMDAR dependent intracellular responses associated with cocaine conditioned place preference behavior.

The aim of this study was to investigate the intracellular responses associated with the acquisition and expression of cocaine-context associations. ERK (extracellular regulated kinase), CREB (cAMP responsive element binding protein), FosB and ΔFosB proteins were of particular interest due to their involvement in cocaine reward and in synaptic plasticity underlying learning and memory. We used the conditioned place preference (CPP) paradigm, which employs a Pavlovian conditioning procedure to establish an association between a drug-paired environment and the drug's rewarding effects, to study the role of these signaling pathways in cocaine-context associations. N-methyl-D-aspartate receptor (NMDAR) antagonism prior to cocaine administration during conditioning blocked the acquisition of cocaine CPP and reduced Nucleus Accumbens (NAc) phosphorylated-ERK (pERK) and phosphorylated CREB (pCREB) levels following the CPP test (drug-free). We also show that cocaine-induced increases in Caudate Putamen (CPu) FosB and ΔFosB levels are decreased after MK-801 pre-treatment during conditioning. In addition, our results provide evidence for the involvement of striatal SIRT (Silent Information Regulator of Transcription) proteins in cocaine-CPP. These results will aid in the advancement of general knowledge about the molecular formation and retrieval of cocaine-associated memories that can be used in the future when designing treatments for cocaine addiction that target both prevention and relapse.

Why are women from Venus and men from Mars when they abuse cocaine?

Both preclinical and clinical studies have shown sexually dimorphic patterns in behavioral responses to cocaine in all phases of the cocaine addiction process (induction, maintenance, and relapse). Thus, a clear picture is emerging which suggests that the biological basis of sex-specific differences in cocaine addiction resides in the disparate regulation of the CNS by male and female gonadal hormones. This review discusses the role that gonadal hormones play in these sexually dimorphic patterns of behavioral responses to cocaine.

Progesterone receptors activation after acute cocaine administration.

Cocaine modulates serum levels of progesterone in intact female and male rats, as well as in pregnant dams, and progesterone decreases or attenuates cocaine-induced behavioral and reward responses. It has been postulated that cocaine's modulation of serum progesterone levels may in turn alter progesterone receptor activity, thereby contributing to cocaine-induced alterations of neuronal functions and genomic regulations. To test this hypothesis, intact male rats received acute injections of saline or cocaine (15 or 30 mg/kg, dissolved in 0.9% saline, intraperitoneal). Progesterone serum levels, progesterone receptor (PR) protein levels, and PR-DNA binding complexes were measured in the striatum by radioimmunoassay, Western blot, and gel shift analyses, respectively. After injection of 15 mg/kg of cocaine, induction of progesterone serum levels was closely followed by an increase in receptor protein levels and DNA binding complexes. After injection of 30 mg/kg of cocaine, similar effects were observed along with an attenuation of receptor protein levels and DNA binding complexes at 60 min. Our results suggest that activation of progesterone receptors may be a mechanism by which cocaine mediates behavior through molecular alterations in the central nervous system.

Effects of short- and long-term estrogen and progesterone replacement on behavioral responses and c-fos mRNA levels in female rats after acute cocaine administration.

It is well established that there are estrous cycle differences in cocaine-induced behavioral activity, implicating fluctuations in levels of estrogen and progesterone throughout the cycle in these alterations in behavior. However, the mechanisms by which steroids alter cocaine-induced behavioral responses have yet to be determined. The aim of this study was to determine whether short- or long-term estrogen and progesterone administration differentially alters behavioral responses to cocaine. Estrogen (50 microg) was administered 30 min or 48 h before cocaine (15 mg/kg, i.p.) administration; progesterone (500 microg) was administered 30 min or 24 h before cocaine. Short-term estrogen replacement decreased cocaine-induced ambulations. Short-term progesterone decreased rearing, whereas long-term progesterone decreased ambulations. Although cocaine increased levels of c-fos mRNA, none of the estrogen or progesterone replacement paradigms affected this measure. Because long-term estrogen replacement has been shown to have no effect on locomotor activity after acute cocaine administration, our observations suggest that short-term estrogen may underlie behavioral alterations. These findings suggest that after acute cocaine administration, while estrogen may activate only membrane receptors to alter behavioral responses to cocaine, progesterone activates both nuclear and membrane receptors.