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Psychoneuroendocrinology (2008) 33, 321–327

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Effects of acute systemic cocaine administrationon the cortisol, ACTH and prolactin levelsof black tufted-ear marmosets

Daniela Limaa, Daiana B. Spındolaa, Luana O. Diasa,Carlos Tomaza,b, Marilia Barrosa,b,�

aDepartment of Pharmaceutical Sciences, School of Health Sciences, University of Brasilia, CEP 70910-900 Brasilia, DF, BrazilbPrimate Center and Laboratory of Neuroscience and Behavior, Institute of Biology, University of Brasilia, Brazil

Received 31 July 2007; received in revised form 25 October 2007; accepted 28 November 2007

KEYWORDSMarmoset;Cocaine;Acute effect;Cortisol;ACTH;Prolactin

nt matter & 2007uen.2007.11.014

uthor at: DepaHealth Sciences,F, Brazil. Tel./fax

barros@unb.br (M

SummaryThe effect of acute systemic cocaine administration on total circulating cortisol,adrenocorticotropin hormone (ACTH) and prolactin levels of nine adult black tufted-earmarmoset monkeys (Callithrix penicillata) was investigated. For each subject, two bloodsamples were obtained after an i.p. injection of 10 and 20mg/kg of cocaine: one samplewas drawn 30-min post-injection, while the second was following a 60-min interval. Bloodsamples were also obtained 0, 30 and 60min following a saline injection. The 20mg/kgdose of cocaine induced a significant decrease in ACTH levels 60min after beingadministered. Circulating levels of cortisol, on the other hand, increased significantly60min after the 20mg/kg dose. Prolactin concentrations decreased significantly 30 and60min after both doses of cocaine had been administered (10 and 20mg/kg). Finally, thecontent of the three hormones analyzed remained constant 0, 30 and 60min post-salineadministrations. Therefore, an acute systemic cocaine challenge alters the basalcirculating levels of ACTH, cortisol and prolactin of marmoset monkeys. A distinctresponse pattern was observed for each hormone analyzed, possibly related to specificnegative feedback mechanisms and/or this species glucocorticoid-resistance. The use ofthis small neotropical primate may thus provide a unique opportunity to investigatecocaine-induced neuroendocrine effects.& 2007 Elsevier Ltd. All rights reserved.

Elsevier Ltd. All rights reserved.

rtment of PharmaceuticalUniversity of Brasilia, CEP

: +55 61 3347 4622.

. Barros).

1. Introduction

The acute administration of cocaine is not only known toinduce considerable behavioral and neurochemical effects(Muller et al., 2003), but significant physiological changes as

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well. This psychostimulant has, in fact, been shown toinhibit prolactin release in rodents (e.g. Mantsch et al.,2000), rhesus monkeys (e.g. Evans and Foltin, 2006) andhumans (e.g. Mendelson et al., 2003), resulting from thecocaine-induced increase in extra-cellular dopamine in thehypothalamus (e.g. Demaria et al., 2000). Acute cocaineexposure has also been found to increase plasma corticos-terone secretion in rodents (for review, see Mello andMendelson, 1997, 2002) and cortisol in rhesus monkeys andhumans (Baumann et al., 1995; Sarnyai et al., 1996). Thisresponse seems mainly due to the increased release ofhypothalamic corticotropin-releasing factor (CRF), thandirect effects upon the pituitary adrenocorticotropin hor-mone (ACTH) or corticosterone/cortisol from the adrenals(for review, see Sarnyai et al., 2001; Mello and Mendelson,2002).

Further studies, however, are essential to better eluci-date the hormonal effects of acute/chronic cocaine ex-posure as this response—particularly that of the HPAaxis—has been associated to the neural mechanisms ofcocaine reinforcement and relapse (e.g. Goeders, 1997;Deroche-Gamonet et al., 2003). For such studies, non-human primates may be an invaluable animal model,demonstrating a behavioral and physiological response tostress conditions similar to that of humans (Kalin, 1993), andreducing ethical and methodological issues regarding clin-ical trials (Levy et al., 1994; Mello and Mendelson, 1997).Neotropical primates may be of particular interest, as theycharacteristically exhibit a glucocorticoid-resistance devoidof symptoms related to excess circulating steroids (Chrousoset al., 1982). Hormonal studies using New World species arescarce however, focused mainly on the reinstatement ofcocaine-seeking behavior (Lee et al., 2003, 2004; Plattet al., 2007). In fact, to our knowledge, marmosets have notbeen previously tested. Therefore, in order to initiallycharacterize the cocaine-induced neuroendocrine responseof black tufted-ear marmosets (Callithrix penicillata), weinvestigated the dose and temporal-dependent effects of anacute systemic challenge on the circulating levels ofcortisol, ACTH and prolactin.

2. Methods

2.1. Subjects and maintenance

Nine experimentally naive adult (2–5 years old; three malesand six normal cycling females) black tufted-ear marmosets(C. penicillata) were used in this study. The stage of themenstrual cycle of the female marmosets was not known.Animals weighed 32078 g (mean7SEM; range: 285–360 g) atthe beginning of the experiment and were housed inseparate heterosexual groups in semi-indoor/outdoor cages(2� 1.3� 2m3) of the same colony room. Not all membersof the housing colony were used in this study. Maintenanceand testing of subjects were held at the Primate Center,University of Brasilia, under natural light, temperature andhumidity conditions. Food and water were available adlibitum. For further housing details, see Barros et al. (2004).All procedures were approved by the Animal EthicsCommittee of the Institute of Biology, University of Brasilia,Brazil and the Brazilian Health Vigilance Agency (ANVISA)/

Ministry of Health (no. 129/2005), and followed the‘Principles of Laboratory Animal Care’ (NIH Publication no.85-23, revised 1996).

2.2. Drugs

Cocaine hydrochloride (Sigma-Aldrich, USA) was dissolved in0.9% physiological saline and injected i.p. in a dose of 10 and20mg/kg. Saline was also used as vehicle and the injectionvolume 1ml/kg. An i.p. injection was employed given theease and speed in which it is given to these small monkeys,and the fast onset of action observed for compoundsadministered via this route. The dose range was based onprevious behavioral (de Sousa e Silva et al., 2006a, b) andhormonal studies (unpublished data) using cocaine inmarmosets.

2.3. Procedure

The marmosets’ basal circulating levels of ACTH, cortisoland prolactin were initially assessed by collecting threeblood samples: one at 0min, one at 30min and one at 60minfollowing a saline administration. Samples were taken in 7-day intervals. This procedure also provided an ade-quate assessment of the subjects’ hormonal response tothe capture, handling, i.p. injection and blood samplingprocedures.

A cross-over experimental design was then used toevaluate the hormonal effects of cocaine, for whichmarmosets were randomly divided into two groups. Initially,group 1 (n ¼ 5) was administered the dose of 10mg/kg ofcocaine and group 2 (n ¼ 4) received the dose of 20mg/kg.For each dose being tested, two blood samples were takenfrom each subject in a 7-day interval, one at 30min andanother at 60min following the 10 (group 1) or 20mg/kg(group 2) cocaine injection. The cross-over phase took place2 months after the first cocaine injection. Initially, a newblood sample was drawn from each subject immediately(0min) after a saline administration. This new salineevaluation aimed at assessing any possible residual effectsof the previous phase on the marmosets’ baseline hormoneprofile. Following a 7-day interval, each subject was onceagain submitted to a cocaine challenge. However, during thecross-over phase, group 1 was administered the dose of20mg/kg, while group 2 received the dose of 10mg/kg. Foreach dose being tested, two more blood samples were takenfrom each subject in a 7-day interval, one at 30min andanother at 60min following the 20mg/kg (group 1) or 10mg/kg (group 2) cocaine injection. A cross-over design was usedto reduce the number of marmosets employed in the study.

Thus, at the end of the experiment, eight blood sampleswere drawn from each subject; i.e. four following a salineinjection (two at 0min, one at 30min and one at 60min),two after the 10mg/kg cocaine challenge (one at 30min andone at 60min) and two after the 20mg/kg (one at 30minand one at 60min). Only animals belonging to the sameexperimental group (1 or 2) were tested on a same day,although the order in which subjects were tested variedrandomly on each test day. Blood was drawn between 10:00and 12:00 h to avoid the 08:00 h peak in cortisol levels.

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2.4. Blood sampling and hormone assays

For each blood sample, the marmoset was captured in itshome-cage, conveyed to an adjacent procedure room whereit was administered a pre-established treatment (saline,cocaine 10mg/kg, cocaine 20mg/kg; i.p.) and then releasedback into its home-cage. Following a pre-established time-interval (30 or 60min), the subject was re-captured and takenonce again to the adjacent procedure room, where it wasanesthetized by placing a cotton-ball permeated withisoflurane (Fluranes) near its nose and mouth. For the twocontrol samples collected immediately (0min) after a salineadministration, the animal was taken directly to theprocedure room where it was injected with saline andimmediately anesthetized using the same procedure de-scribed above. The subject was considered anesthetizedwhen voluntary movements had ceased and respiratory ratevisibly decreased. A 1.0ml blood sample was then collectedvia femoral venipuncture and immediately divided into twovials: 0.4ml was placed in a chilled EDTA tube for total plasmaACTH analysis and 0.6ml was transferred to a chilled tubecontaining gel barrier and clot-promoting additives for totalserum cortisol and prolactin analyses. Following its recovery(o5min), the subject was released back into its home-cage,monitored for 15min and given an iron supplement. The totalduration of each blood sampling procedure (i.e. time elapsedbetween re-entering the home-cage to capture the animaland the end of the blood sampling procedure) was measuredin order to further assess any possible influence of thisprocedure on the hormone content observed. The latencyobserved was, on average, 4.0570.10min (mean range:3.18–4.42min). The longest latencies were measured duringthe 0min saline treatments, as the total duration included theinjection procedure. All samples, however, were obtained ino5min. The procedures were conducted in the presence ofthe Primate Center’s veterinarian.

Blood samples were kept on ice until centrifuged at3500 rpm, 4 1C, for 5min. Resulting plasma/serum wasimmediately transferred to vials and diluted with hormone-free diluent kits (Diagnostic Products Corp., Los Angeles, CA,USA) to 1:50 for cortisol assays (L2M1Z), 1:2 for ACTH (L2ACA2)and 1:8 for prolactin (L2PRZ). Dilution factor was based onprevious studies in this species using the same blood samplingand analysis procedure (unpublished data). Diluted plasma/serum was then analyzed by single chemiluminescenceenzyme-immunoassay (EIA) using commercial kits for theautomated IMMULITE 2000s (cortisol: L2KCO6, ACTH: L2KAC2,prolactin: L2KPR6; Diagnostic Products Corp., Los Angeles, CA,USA). Cortisol, ACTH and prolactin assay sensitivity was0.20ng/dl, 5.00pg/ml and 0.16ng/ml, respectively. Inter-assay coefficients of variation (CVs) from pooled plasma/serum for high (n ¼ 4) and low pools (n ¼ 3) were, respec-tively, 9.4% and 8.2% for cortisol, 6.1% and 10.0% for ACTH and7.9% and 6.9% for prolactin. Intra-assay CVs for the high andlow pools were, respectively, 7.4% and 6.1% for cortisol, 9.5%and 8.7% for ACTH and 2.3% and 2.2% for prolactin.

2.5. Statistical analysis

For each hormone assayed, data were analyzed to deter-mine possible differences: (1) within each post-injection

time-interval (0, 30 or 60min) by means of a one-wayrepeated measure analysis of variance (ANOVA); (2) in pre-planned comparisons within each cocaine treatment (10 or20mg/kg) using the paired t-test; and (3) between the twoexperimental groups via two-way ANOVA with repeatedfactors on treatment and experimental group. The latterwas used to determine whether the cross-over experimentaldesign influenced the hormonal pattern observed; i.e. if thefirst dose received confounded the results observed follow-ing the second dose. Significant ANOVA results were followedby pairwise comparisons using the appropriate errorvariance terms from the ANOVA summary tables with Tukey’stest. Also, for each treatment, the duration of the bloodsampling procedure was compared to the respective levelsof the HPA axis which were observed using the Pearson’scorrelation test. Significance level was set at pp0.05.

3. Results

The marmosets’ plasma ACTH levels (Figure 1) remainedunaltered 30min after both 10 and 20mg/kg of cocaine hadbeen injected, compared to the 30-min post-saline control(F(8,2) ¼ 1.20, p ¼ 0.33). After 60min, however, ACTHplasma concentration decreased significantly in responseto both doses of cocaine, compared to the 60min post-salinecontrol (F(8,2) ¼ 7.14, po0.05). Furthermore, within-dosecomparisons revealed that ACTH levels were significantlylower 60min after 10mg/kg of cocaine had been adminis-tered, compared to its respective 30-min interval (t ¼ 2.96,df ¼ 7, po0.05). Following the 20mg/kg dose, a significanteffect was not observed (t ¼ 83.00, df ¼ 8, p ¼ 0.13). Inaddition, compared to the 30-min interval, circulating ACTHcontent was significantly lower 60-min post-saline injection(F(8,2) ¼ 4.47, po0.05). In fact, the ACTH content ob-served 60min after the saline treatment was significantlycorrelated with the duration of the blood samplingprocedure; i.e. the longer it took to obtain the bloodsample, the higher the ACTH concentration was (r ¼ 0.80,po0.05). However, no significant group (F(7,1) ¼ 3.27,p ¼ 0.08), treatment (F(7,1) ¼ 1.74, p ¼ 0.12) or interac-tion effects (F(7,1) ¼ 0.93, p ¼ 0.50) were found betweenthe two experimental groups (Table 1).

For cortisol (Figure 1), a significant change was onlyobserved 60min after 20mg/kg of cocaine had beenadministered, when circulating levels increased relative tothe 60-min post-saline control (10mg/kg: F(8,2) ¼ 0.59,p ¼ 0.56; 20mg/kg: F(8,2) ¼ 10.21, po0.05). When eachdose was compared between its 30 and 60-min intervals,however, significant differences were not observed (10mg/kg: t ¼ �0.37, df ¼ 7, p ¼ 0.72; 20mg/kg: t ¼ 1.56, df ¼7, p ¼ 0.16). Importantly, cortisol levels remained unaltered0, 30 and 60-min post-saline injection (F(8,2) ¼ 1.27,p ¼ 0.31), and the duration of the blood sampling procedurewas not significantly correlated to the levels of cortisolobserved for any of the treatments. Between-group analysis(group 1 vs. group 2; Table 1) revealed only a significanttreatment effect (treatment: F(7,1) ¼ 2.41, po0.05; group:F(7,1) ¼ 0.76, p ¼ 0.39; interaction: F(7,1) ¼ 0.70, p ¼0.68), which followed the same pattern for both groups asthat of the general analysis described above (statistical datanot shown).

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Figure 1 Effect of acute systemic cocaine (10 and 20mg/kg) or saline administration on the mean+S.E.M. plasma/serum ACTH,cortisol and prolactin levels in marmoset monkeys (n ¼ 9) measured immediately (0min) after the injection, and/or 30 and 60-minpost-injection.*po0.05 vs. 30-min post-saline; **po0.05 vs. 60-min post-saline; ***po0.05 vs. 60-min post-saline and 60-min post-cocaine 10mg/kg; +po0.05 vs. 60-min post-cocaine 10mg/kg.

Table 1 Effect of acute systemic cocaine administration on the circulating ACTH, cortisol and prolactin levels of marmosetsof experimental groups 1 and 2.�

Hormoney Treatment

Saline Cocaine (10mg/kg) Cocaine (20mg/kg)

0min 30min 60min 30min 60min 30min 60min

ACTHGroup 1 na 52.00710.52 24.6678.26 45.74715.27 12.5674.44 58.16724.09 9.6772.38Group 2 62.95715.98 83.65726.09 53.63713.11 94.43736.73 27.70711.27 267.137168.00 33.53712.35

CortisolGroup 1 145.64741.30 153.40730.63 139.36718.87 163.60723.70 143.20718.19 189.56760.61 269.50719.88z

Group 2 132.6379.73 178.00728.61 147.00713.98 207.00711.10 256.67750.93 183.7575.38 273.75763.66

ProlactinGroup 1 6.3870.62 6.6370.56y 5.8970.52: 3.4670.65 1.6670.66 4.9270.32 1.4270.79yy

Group 2 7.4370.58 7.3570.90z 6.1970.48�� 4.6570.10yy 4.4671.64 0.6270.41 1.1370.29

na: not assayed.�Group 1 was initially administered cocaine 10mg/kg and 2 months later the dose of 20mg/kg, while group 2 was first given cocaine

20mg/kg and then 10mg/kg.yPlasma levels of ACTH (pg/ml), and serum concentrations of cortisol (mg/dl) and prolactin (ng/ml).zpo0.05 vs. 60-min post-saline and 60-min post-cocaine 10mg/kg.ypo0.05 vs. 30-min post-cocaine 10mg/kg (group 1).zpo0.05 vs. 30-min post-cocaine 10 and 20mg/kg (group 2).:po0.05 vs. 60-min post-cocaine 10 and 20mg/kg (group 1).��po0.05 vs. 60-min post-cocaine 20mg/kg (group 2).yypo0.05 vs. 30-min post-cocaine 20mg/kg (group 1 or 2).

D. Lima et al.324

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Cocaine, at both doses tested, significantly reduced themarmosets’ prolactin concentrations 30-min post-adminis-tration, compared to the 30-min post-saline control(F(8,2) ¼ 10.14, po0.01; Figure 1). This effect persisted60min after cocaine had been injected, relative to the 60-min post-saline interval (F(8,2) ¼ 17.77, po0.01). However,when each dose was compared between its 30 and 60-minintervals, similar levels were observed (dose 10mg/kg:t ¼ 1.51, df ¼ 7, p ¼ 0.18; dose 20mg/kg: t ¼ 1.75, df ¼ 7,p ¼ 0.12), as well 0, 30 and 60-min post-saline injection(F(8,2) ¼ 3.40, p ¼ 0.06). Between-group analysis (group 1vs. group 2; Table 1) also showed no significant group effect(F(7,1) ¼ 0.015, p ¼ 0.91), while treatment (F(7,1) ¼12.72, po0.01) and interaction effects were significant(F(7,1) ¼ 3.11, po0.05), which followed a similar pattern asthat in the general analysis (statistical data not shown).

4. Discussion

An acute systemic cocaine challenge induced significantchanges in the marmosets’ baseline hormone profile. On theHPA axis, specifically, a significant increase in serum cortisolwas observed 60min following the injection of the highestdose (20mg/kg) of cocaine. A similar response, due to anacute cocaine exposure, has also been reported in rodents,other monkey species and humans (for review, see Mello andMendelson, 1997, 2002). On the other hand, 10 and 20mg/kg of cocaine induced a significant decrease in themarmosets’ plasma ACTH levels 60min after having beeninjected. The decline in the circulating levels of thishormone—particularly for the 20mg/kg dose—may bepartly related to the negative corticoid feedback uponACTH-release (Torres and Rivier, 1992), even if the marmo-sets’ HPA hormone levels were not priorly elevated (i.e. 30-min post-cocaine). In fact, an enhanced glucocorticoidrelease, in response to (small) undetectable prior changesin ACTH content, was also reported in rodents (Levy et al.,1994). Similarly, in cocaine-naıve men, Heesch et al. (1995)failed to detect prior changes in ACTH levels, althoughcortisol increased 60min after cocaine had been adminis-tered. Then again, the order in which doses were givencould have contributed to the present rise in cortisol 60minfrom the initial 20mg/kg dose injection. The higherresponse seen in group 2 vs. group 1 suggests that theinitial high dose exposure in the former animals may haveinfluenced their response to the subsequent new challengeheld 2 months later with the lower dose. Chronic use hasindeed been shown to persistently alter the neuroendocrineresponse to cocaine (for review, see Mello and Mendelson,1997). However, the present regime—i.e. two injectionsover the course of 2 months—does not correspond to achronic regime and even five i.p. injections of 10mg/kg at72-h intervals did not induce long-lasting behavioral effectsin this primate (de Sousa e Silva et al., 2006a, b).

In terms of prolactin release, the acute injection of bothdoses of cocaine was equally effective at significantlydecreasing its circulating levels 30 and 60min after havingbeen administered. In marmosets, prolactin content wasalready reduced after 20min (unpublished data), remaininginhibited for up to 90–120min in rhesus monkeys (Mello etal., 1993; Evans and Foltin, 2006). Similar results have also

been observed in rodents and humans (for review, seeSarnyai et al., 2001; Mello and Mendelson, 2002, 1997).

However, given that both doses of cocaine tested signifi-cantly altered the baseline levels of prolactin, while the HPAaxis hormones responded consistently only to the high dose, thispsychostimulant seems to exert a distinct hormone-dependenteffect. For ACTH/cortisol, more specifically, the generalabsence of a low dose effect may have been partly due tothe marmosets’ notably high baseline level of circulatingglucocorticoids, compared to Old World primates (includinghumans) and possibly even other neotropical species (Yamamo-to et al., 1977; Chrousos et al., 1982; Klosterman et al., 1986).Actually, a reduced binding affinity to its receptor is suggestedto underlie the marmosets’ glucocorticoid-resistance (e.g.Chrousos et al., 1982; however, see Cassorla et al., 1982;Klosterman et al., 1986; Scammell et al., 2001). In this primate,a 10mg/kg i.p. dose was also unable to significantly stimulatethe HPA axis after a 20-min interval (unpublished data),although prolactin suppression (unpublished data) and beha-vioral effects were seen (de Sousa e Silva et al., 2006a,b). Insquirrel monkeys, another glucocorticoid-resistant neotropicalprimate, cocaine priming injections dose-dependently rein-stated drug-seeking behavior, even if salivary cortisol remainedunaltered (Lee et al., 2003; Platt et al., 2007), while higherdoses of CRF than ACTH were required to induce significantincreases in salivary cortisol (Tiefenbacher et al., 2003).

Importantly, the hormonal changes observed do not seem tobe associated to possible stress-related methodological issues(i.e. capture, restrain, injection, blood sampling), as theprocedure was held in o5min (Saltzman et al., 1994), theACTH/cortisol levels observed were overall not significantlycorrelated to the duration of the procedures, and the hormonecontent assayed 0, 30 and 60min post-saline injectionremained generally constant and at mean concentrationssimilar to those previously reported for marmosets (0-minpost-saline; cortisol: 139.86723.45mg/dl; ACTH: 64.94712.91pg/ml; prolactin: 6.8570.46ng/ml) (Pryce et al., 2002;Boere et al., 2005; Almond et al., 2006; Saltzman et al., 2006).Caution should be taken when generalizing the results,however, as ACTH levels 60-min post-saline were significantlylower than at 30min, as well as the fact that the response ofgroups 1 and 2 differed for some hormones; i.e. first exposuremay have influenced the cortisol response following a secondinjection 2 months later. Also, due to the small male samplesize, a gender analysis was not performed, which has beenfound to be an influencing factor in primates (e.g. Broadbear etal., 1999). Finally, a possible influence of the ovarian hormonesand estrous cycle on the cocaine-induced rise of the activity ofthe HPA axis (Walker et al., 2001) could not be determined asthe stage of the females’ menstrual stage was not known.Cocaine, nonetheless, induced a distinct hormone-dependentresponse pattern in the marmosets, possibly related to specificnegative feedback mechanisms and/or this species glucocorti-coid-resistance. The use of this small neotropical primate maythus provide a unique opportunity to investigate cocaine-induced neuroendocrine effects.

Role of the funding sources

Funding for this study was provided by FINATEC/Brazil andCAPES/DAAD/PROBAL; the funding sources had no further

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role in study design; in the collection, analysis andinterpretation of data; in the writing of the report; and inthe decision to submit the paper for publication.

Conflict of interest

None declared.

Acknowledgments

This study was supported by FINATEC (to C.T. and M.B.). C.T.was a recipient of a CNPq research fellowship (no. 300364/1986-5). The authors would like to thank Drs. R. de Oliveiraand D. Teixeira for their assistance during blood samplings,N. Bessa for assistance during testing, G.V. da Silva foranimal care and maintenance and the Laboratorio Sabin deAnalises Clınicas de Brasilia for technical support of thehormone assays.

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