Neurophormacology Vol. 31, No. Printed in Great Britain
2, pp.
169-175,
1992
0028-3908/92
$5.00
+ 0.00
Pergamon Press plc
PRIOR CHRONIC EXPOSURE TO COCAINE INHIBITS THE SEROTONERGIC STIMULATION OF ACTH AND SECRETION OF CORTICOSTERONE L. D. VAN DE KAR,’ ANNA M. BONADONNA,’P. A. RITTENHOUSE,’JANICEE. KERR,’ A. D. LEVY,’ LALITHA IYER,’ G. B. HERBERT,’MARIA C. ALVAREZ SANZ,’ STEPHANIE J. LENT*
and MOLLY CARNE~* ‘Department of Pharmacology, Stritch School of Medicine, Loyola University of Chicago, 2160 S. First Avenue, Maywood, IL 60153, U.S.A. and *University of Wisconsin, Department of Medicine and Middleton Memorial Veterans Hospital, Geriatric Section, Madison, WI 53705, U.S.A. (Accepted 25 July 1991)
Summary-The effect of long-term pretreatment with cocaine on serotonergic regulation of ACTH (adrenocorticotropic hormone; corticotropin) and secretion of corticosterone in rats was investigated. The following observations were made: (1) Pretreatment with cocaine had no significant effect on basal levels of ACTH and corticosterone in plasma. However, cocaine caused a reduction in the ability of the S-HT (S-hydroxytryptamine, serotonin) releaser p-chloroamphetamine (PCA) to increase corticosterone in plasma, 42 hr after the last injection of cocaine. (2) Exposure to cocaine for 7 days was sufficient to produce a maximal inhibition of the PCA-induced increase in ACTH in plasma. (3) The inhibitory effect of cocaine on PCA-induced release of ACTH was more marked than on corticosterone. (4) Conversely, the dose-dependent stimulatory effect of two 5-HT, agonists, RU 24969 (S-methoxy-3-(1,2,3,4-tetrahydro4-pyridinyl)-lH-indole) and m-CPP (m-chlorophenylpiperazine), on ACTH and corticosterone was not reduced by 7 days of exposure to cocaine. Taken together, these findings indicate that pretreatment with cocaine reduced the function of serotonergic nerve-terminals but not postsynaptic receptors, that stimulate ACTH and secretion of corticosterone. Key wordF--cocaine, ACTH, corticosterone, 5-HT, p-chloroamphetamine,
Cocaine is a psychostimulant, effect
by altering
thought to exert its neurotransmission in the brain.
In addition to uptake sites for dopamine and norepinephrine, cocaine binds with a high affinity to synaptic reuptake sites for serotonin (5-HT) (Reith, Sershen, Allen and Lajtha, 1983; Ritz, Cone and Kuhar, 1990). This prevents the reuptake of these neurotransmitters from the synaptic cleft and leads to an increased concentration of 5-HT (as well as dopamine and norepinephrine) in the synapse (Hanson, Matsuda and Gibb, 1987; Koe, 1976). Cocaine has a profound influence on the activity of serotonergic neurons. Acute intravenous injection of cocaine suppresses the firing rate of serotonergic neurons in the dorsal raphe nucleus (Pitts and Marwah, 1986; Cunningham and Lakoski, 1988; Lakoski and Cunningham, 1988). In vitro, addition of cocaine to slices of midbrain of the rat decreases the firing rate of dorsal raphe neurons by increasing the concentration of S-HT in the synapse (Pan and Williams, 1989). The cocaine-induced increase in the concentration of 5-HT in the synapse leads to a compensatory decrease in its synthesis (Galloway, 1990). Little agreement exists concerning the long-term effects of cocaine on serotonergic neurons. Two studies found that repeated injection of cocaine
S-HT receptors, 5-HT uptake.
reduced the content of 5-HT in the brain (Roy, Bhattacharyya, Pradhan and Pradhan, 1978; Taylor and Ho, 1977). However, another study (Kleven, Woolverton and Seiden, 1988) observed no change in the content of 5-HT in several regions of the brain after injection of a smaller dose of cocaine, for 10 days. Thus, additional studies are necessary to establish the long-term effects of cocaine on serotonergic function. The present study utilized the hormonal response to a release of 5-HT and 5-HT agonists, as indices of serotonergic function. Serotonergic neurons stimulate, either directly or indirectly, the release of many hormones, including adrenocorticotropic hormone (ACTH), /I-endorphin, corticosterone, prolactin, renin, oxytocin and vasopressin (for review see Van de Kar, 1991). The present study focused on the influence of cocaine on the serotonergic regulation of the hypothalamic-pituitaryadrenal axis. This axis is composed of corticotropin releasing factor (CRF)-containing neurons in the hypothalamic paraventricular nucleus, pituitary cells which are activated by CRF to release ACTH into the blood and adrenal cortical cells that respond to ACTH by secreting corticosterone. Serotonergic nerveterminals in the paraventricular nucleus stimulate the secretion of CRF (Liposits, Phelix and Paull, 1987; 169
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L.
D. VAN
Feldman, Conforti and Melamed, 1987). Release of ACTH and corticosterone can be stimulated by activation of either 5-HT,, or S-HT,c/5-HT, receptors (Gilbert, Brazell, Tricklebank and Stahl, 1988; Fuller and Snoddy, 1990; King, Brazell, Dourish and Middlemiss, 1989; Lorens and Van de Kar, 1987). Acute injections of cocaine stimulate the hypothalamic-pituitary-adrenal axis, through release of CRF (Calogero, Gallucci, Kling, Chrousos and Gold, 1989; Moldow and Fischman, 1987; Rivier and Vale, 1987). Both dopaminergic and serotonergic neurons mediate the acute effect of cocaine on the secretion of ACTH and corticosterone (Borowsky and Kuhn, 1991; Levy, Li, Kerr, Rittenhouse, Molonas, Cabrera, Battaglia, Alvarez Sanz and Van de Kar, 1991). Because acute administration of cocaine inhibits reuptake of 5-HT, it is likely that long-term administration of cocaine would modify normal serotonergic function. The dose and duration of exposure to cocaine that influenced the serotonergic regulation of secretion of ACTH was investigated, examining the endocrine response to the 5-HT-releasing drug p-chloroamphetamine (PCA). To examine whether the inhibitory effect of cocaine on serotonergic neurons was mediated post-synaptically, the effect of direct 5-HT, agonists RU 24969 and m-CPP (Glennon, 1987) on ACTH and corticosterone in plasma in cocaine-pretreated rats was tested. The rats were only pretreated with cocaine for 7 days, because the effect of PCA on ACTH was inhibited to the same extent after 7 days as after 30 days. METHODS
Animals
DE KAR et al.
for Drug Abuse (NIDA, Rockville, Maryland). These batches of cocaine were used for the first two experiments, i.e. the experiments involving injection of cocaine for 30 days, followed by a challenge with PCA. In all other experiments, the cocaine used was purchased from Sigma (St Louis, Missouri). The 5-HT releasing drug, PCA (Marsden, 198 1; Marsden, Conti, Strope, Curzon and Adams, 1979) was purchased from Sigma (St Louis, Missouri). The 5-HT, agonist, m -CPP (m -chlorophenylpiperazine) was purchased from Sigma (St Louis, Missouri). Another 5-HT, agonist, RU 24969 (5-methoxy-3-(1,2,3,4_tetrahydro-4-pyridinyl)-lH-indole) (Euvrard and Boissier, 1980) was a gift from Roussel Uclaf (Romainville, France). All doses of drugs are expressed as the salt. Experimental protocols Dose-response effect of cocaine. Cocaine was injected twice daily for 30 days, at doses of 0 (saline), 1, 5, 10 and 15 mg/kg, (i.p.). The rats received an injection of 8 mg/kg (i.p.) of PCA or saline, 42 hr after the last injection of cocaine. The rats remained in their home cage for 60min after the injection, at which time they were killed by decapitation. The effect of PCA on several hormones reaches a maximum 60 min after injection (Fuller and Snoddy, 1980; Van de Kar, Wilkinson and Ganong, 1981). Time course of the eflect of cocaine. Cocaine was administered twice daily for either 0, 1, 7 or 30 days, at a dose of 15 mg/kg, (i.p.). All the rats received injections for 30 days. The rats that received injections of cocaine for 2 days, first received injections of saline for 28 days, followed by 2 days of injections of cocaine. Similarly the rats that received injections of cocaine for 7 days, first received injections of saline for 23 days, followed by injections of cocaine. The rats subsequently received 8 mg/kg (i.p.) of PCA or saline, 42 hr after the last cocaine injection and were killed 60 min later by decapitation.
Male Sprague-Dawley rats (200-250 g) were purchased from Sasco-King Animal Laboratories (Oregon, Wisconsin). The rats were housed, 2 per cage, in a temperature-, humidity- and illumination(12: 12 hr light/dark cycle; lights on at 0700 hr) controlled room. Water and food (Wayne Lab Blox, Lab Mills Inc., Chicago, Illinois) were available ad libitum. The rats were killed by decapitation, in an area outside the rat room and trunk blood was collected into centrifuge tubes, containing 0.5 ml of 0.3 M ethylenediamine tetraacetic acid (EDTA; pH 7.4) for the hormone assays. All the experimental protocols were approved by Loyola University Animal Care and Use Committee.
as described above. Forty-two hours after the last injection of cocaine, the rats received an injection of one of several doses of the 5-HT agonists, RU 24969 (0.2, 1 or 5 mg/kg, i.p.) or m-CPP (1, 5 or 20 mg/kg, i.p.). Control rats received an injection of saline. All the rats were killed by decapitation 30 min after the last injection, which is the time when the 5-HT agonists produce their maximum neuroendocrine effects.
Drugs
Biochemical determinations
All drugs were dissolved in saline and injected in a volume of 1 ml/kg, intraperitoneally (i.p.). All injections of cocaine were performed twice a day (08:30 and 17:00) and all subsequent challenges with a 5-HT release or 5-HT agonists occurred 42 hr after the last injection of cocaine. This interval was chosen to eliminate cocaine from the circulation, prior to injection of the 5-HT releaser and S-HT agonists. Cocaine HCl was donated by the National Institute
Radioimmunassay of corticosterone in plasma. This was performed on un-extracted samples of plasma (2 and 5 ~1) in which the binding proteins had been denatured by boiling, using procedures and antiserum from Radioassay Systems Laboratories (Carson, California). The sensitivity limit was 0.02 ng/tube and the intra- and inter-assay variabilities were 4.5% and 11.9%, respectively (Van de Kar, Urban, Richardson and Bethea, 1985).
EfSect of direct S-HT agonists in cocaine-pretreated rats. Rats received cocaine (15 mg/kg, i.p.) for 7 days
Cocaine effect on ACTH and corticosterone Radioimmunassay of ACTH in plasma. This was performed on un-extracted samples of plasma (2050 PI), as previously described (Cames, Brownfield, Kalin, Lent and Barksdale, 1986). Briefly, the ACTH antiserum was obtained from IgG Corp (Nashville, Tennessee). The ACTH for standards (l-39) was obtained from Calbiochem and [‘2SI]-ACTH from INCSTAR (Stillwater, Minnesota). The sequence recognition of the antiserum was 5-18. In addition, this antiserum does not significantly recognize or-MSH, /I-MSH, /?-endorphin, /?-lipotropin, ACTH l-24 or ACTH 1-16-amide. The minimum detectable concentration was 0.25 pg/tube and the intraand inter-assay variations were 4.2% and 14.6%, respectively. Statistics
The data are represented as the group means and the standard errors of the mean (SEM). Statistical analysis of the data was performed by two-way analysis of variance (ANOVA) and individual group means were compared by Student-Newman-Keuls’ test (Steel and Torrie, 1960), using a computer program (NWA STATPAK, Portland, Oregon).
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rats (Fig. 1). The ability of PCA to increase corticosterone in plasma was reduced in rats pretreated with cocaine for 30 days. Although the statistical analysis (two-way ANOVA) did not reveal a significant interaction between treatment with PCA and cocaine, a post-hoc Newman-Keuls’ test indicated that the PCA-induced elevation of corticosterone in plasma was significantly lower in rats receiving chronic pretreatment with the 15 mg/kg dose of cocaine (P < 0.05). A second experiment examined the effects of different durations of pretreatment with cocaine, on PCA-induced elevation of the concentration of ACTH and corticosterone in plasma. The effect of PCA on the concentration of ACTH in plasma was abolished in rats pretreated for 7 and 30 days with cocaine but not in rats pretreated with cocaine for only 1 day (Fig. 2). Two-way ANOVA for ACTH revealed a significant interaction between treatment with cocaine and PCA (F[3,38] = 9.087, P < 0.0001). The post-hoc Newman-Keuis’ tests indicated that 7 and 30 days of exposure to cocaine significantly reduced the PCAinduced elevation of the concentration of ACTH in plasma (P < 0.01).
RESULTS
Pretreatment with cocaine had no significant effect on basal levels of ACTH and corticosterone in plasma. Injection of the 5-HT releaser, PCA, significantly increased the concentration of corticosterone in plasma in saline-pretreated and cocaine-pretreated
r
t z
a
,-
0
Cocaine
1
5
pretreatment
10
(mg/kg,
SALINE PCA
500 -
o 400 E : 300 Z .c 200 z
0 m
a
ioao-
7
15
i.p.)
Fig. 1. Pretreatment with cocaine inhibited the PCAinduced increase in corticosterone in plasma. Rats were pretreated with varying doses of cocaine (1-15 mg/kg, i.p., twice daily) for 30 days. Rats received 8 mg/kg (i.p.) of PCA, 42 hr after the final injection of cocaine, and were sacrificed 60 min after administration of PCA. Data represent mean f SEM of 8 rats per group. Levels of corticosterone in plasma were enhanced in all PCA-treated groups, compared with their corresponding saline-treated groups (P i 0.01 Newman-Keuls’ test). * Significant difference from the salinepretreated rats, that were challenged with PCA (P < 0.01 Newman-Keuls’ test).
a Cocaine
1
7
pretreatment
30
30
(days)
Fig. 2. Inhibition of PCA-induced elevation of ACTH (top) and corticosterone (bottom) in plasma by increasing durations of exposure to cocaine. Rats were pretreated with cocaine (15 mg/kg, i.p., twice daily) for 1, 7 or 30 days. Rats received 8 mg/kg PCA, 42 hr after the final injection of cocaine and were sacrificed 60min later. Data represent mean k SEM of 8 rats per group. *Significant difference between PCA- and the corresponding saline-treated rats, (P < 0.05, Newman-Keuls’ test). * Significant difference between cocaine- and saline-pretreated rats (P < 0.05, Newman-Keuls’ test).
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L. D.
VAN DE KAR et al.
Injections of cocaine for 1, 7 or 30 days, decreased the levels of ACTH (F[3,54] = 12.206, P < 0.0001) PCA-induced elevation of the concentration of and corticosterone (F[3,54] = 13.256, P < O.OOOl) in corticosterone in plasma. The two-way ANOVA for plasma both in saline- and cocaine- pretreated rats. corticosterone indicated that the interaction between Also, m-CPP elevated the levels of ACTH (F[3,55] = pretreatment with cocaine and injection of PCA 23.52, P < 0.0001) and corticosterone (F[3,53] = 30.8, approached but did not attain statistical significance P < 0.0001) in plasma (Fig. 4). Pretreatment with (F[3,38] = 2.353, P = 0.083). However, the Newmancocaine did not modify the effect of m-CPP on the Keuls’ test revealed that PCA-induced elevation of concentration of either ACTH or corticosterone in the concentration of corticosterone in plasma was plasma. reduced in rats pretreated with cocaine for 1, 7 or 30 days, compared to saline-pretreated rats (P < 0.05). DISCUSSION Additional studies were conducted to examine whether the inhibition of the effects of PCA were due Repeated administration of cocaine altered seroto down-regulation of postsynaptic 5-HT receptors. tonergic regulation of the hypothalamic-pituitaryRats pretreated with the same regimen of cocaine for adrenal axis. Pretreatment with cocaine, of at least 7 days, were challenged with increasing doses of the 7 days, reduced the elevation of levels of ACTH, direct SHT, agonists, RU 24969 and m-CPP. There produced by a 5-HT releaser (PCA) but not by the was no evidence of reduced responsiveness to the 5-HT, agonists, RU 24969 or m-CPP. These data, S-HT, agonists on the concentrations of ACTH therefore, suggest that repeated administration of an corticosterone in rats pretreated with cocaine. As cocaine or its withdrawal, impairs presynaptic but can be seen in Fig. 3, pretreatment with cocaine not postsynaptic serotonergic function. A reduction in the concentration of 5-HT could significantly potentiated the corticosterone response to 1 mg/kg of RU 24969 (P < 0.05, Newman-Keuls account for the reduced ability of PCA to stimulate the secretion of ACTH and corticosterone in cocainetest). The drug RU 24969 significantly increased pretreated rats. The 5-HT releasing agent, PCA depends upon intact stores of endogenous 5-HT to enhance the concentration of 5-HT in the synapse
_. i3 ._ -‘0
0
1s 0
41 0
“’
0.2
Dose of RU 24969
’
I.0
I,‘/
/
“‘-5.0
(mg/kg,
i.p.1
Fig. 3. Increase in ACTH (top) and corticosterone (bottom) in plasma, induced by RU 24969 in rats pretreated with cocaine (15 mg/kg, twice daily) for 7 days. The injection of RU 24969 was performed 42 hr after the last injection of cocaine, and samples of blood were collected 30 min later. Data represent mean + SEM of 8 rats per group. *Significant difference between RH 24969- and corresponding salinetreated rats (P i 0.05 Newman-Keuls’ test). #Significant difference between cocaine- and saline-pretreated rats, receiving RU 24969 (1 mg/kg), (P < 0.01, Newman-Keuls’ .\
test).
c
*'
Dose of m-CPP(mg/kg,
i.p.)
Fig. 4. Increase in ACTH (top) and corticosterone (bottom) in plasma, induced by m-CPP, in rats pretreated with cocaine (15 mg/kg, twice daily) for 7 days. The injection of m-CPP was performed 42 hr after the last injection of cocaine and samples of blood were collected 30min later. Data represent mean + SEM of 8 rats per group. *Significant difference between m-CPP- and the corresponding saline-treated rats (P < 0.05, Newman-Keuls’ test).
Cocaine effect on ACTH and corticosterone
(Marsden et al., 1979; Marsden, 1981) which, in turn, elevates ACTH and corticosterone in plasma (Fuller and Snoddy, 1980). The literature regarding the chronic effects of cocaine on the concentration of S-HT in brain is inconsistent. One study found that injections of cocaine for 9 days reduced the concentrations of 5-HT in several regions of the brain (Roy et al., 1978), while in another study, chronic administration of cocaine did not alter concentrations of 5-HT (Kleven et al., 1988). It is therefore unclear whether the inhibition of the ability of PCA to stimulate secretion of ACTH and corticosterone was due to decreased concentrations of 5-HT in serotonergic nerve terminals. Pretreatment with cocaine could also reduce PCAstimulated secretion of ACTH and corticosterone by interfering with the uptake of PCA into serotonergic nerve terminals. p-~hloroamphetamine enters S-HT nerve terminals through the uptake mechanism for 5-HT (Fuller, 1980; Fuller and Snoddy, 1980). Consequently, the effect of PCA on ACTH would be diminished if the drug could not enter the serotonergic nerve terminals. Cocaine binds with a high affinity to the uptake sites for S-HT and it is a potent blocker of the uptake of 5-HT (Reith et uf., 1983; Ritz et al., 1990; Hanson et al., 1987; Koe, 1976). In the present experiments, rats were allowed 42 hr between the final injections of cocaine and the challenge with PCA. Nicolaysen, Pan and Justice (1988) determined that the levels of cocaine in the extracellular fluid in the brain of the rat reached a peak at about 30 min and disappeared 240 min after a single injection. By 42 hr after chronic pretreatment with cocaine, it is unlikely that the concentration of cocaine would be sufficiently large to inhibit the uptake of PCA into serotonergic nerve terminals. It is possible, however, that chronic pretreatment with cocaine could reduce the function of the uptake mechanism for S-HT. If the uptake mechanism is not functioning properly, the ability of PCA to enter the serotonergic nerve terminal would be decreased and the neuroendocrine response would be reduced. Reduced responses to PCA, following chronic administration of cocaine, is not likely to be due to reduced postsynaptic receptor function. After 7 days of administration of cocaine, the ~Ieuroendoc~ne responses to two 5-HT receptor agonists, RU 24969 and m-CPP were examined. The ability of these 5-HT agonists to enhance levels of ACTH and corticosterone were not reduced by pretreatment with cocaine, suggesting that down-regulation of postsynaptic receptors is not the mechanism by which chronic administration of cocaine alters .5-HT function. These animals actually exhibited an enhanced response of corticosterone to RU 24969. At large doses, RU 24969 may act to release 5-HT (Auerbach, Kamalakannan and Rutter, 1990). However, the neuroendocrine effects of RU 24969 are predominantly mediated through its 5-HT agonist properties; destruction of serotonergic neurons does not inhibit
I73
the effect of RU 24969 on ACTH or corticosterone in plasma (Van de Kar, Cames, Maslowski, Bonadonna, Rittenhouse, Kunimoto, Piechowski and Bethea, 1989). Therefore, the enhanced response of corticosterone to RU 24969 may be due to supersensitive postsynaptic 5-HT receptors. This conclusion would be premature, however, because of differences in endocrine responses to RU 24969 and m-CPP. The possibility that postsynaptic 5-HT receptors are supersensitive, following repeated exposure to cocaine requires more extensive examination. It is inte~sting to note that the changes in endocrine responses to PCA and RU 24969 after chronic exposure to cocaine were not entirely parallel between ACTH and corticosterone. The PCA-induced enhancement of levels of ACTH was greatly diminished by 7 and 30 days of exposure to cocaine while the response of corticosterone was only moderately reduced. Additionally, pretrea~ent with cocaine enhanced the effect of RU 24969 on corticosterone but not ACTH. Similar findings have been previously reported. Bagdy, Calogero, Murphy and Szemeredi (1989) observed that some 5-HT agonists produced maximum increases in levels of corticosterone at doses that produced only moderate increases in ACTH. Differential effects on ACTH and corticosterone in plasma could be explained by alterations in adrenal sensitivity to ACTH. Several studies have determined that adrenal sensitivity to ACTH is not constant (England, Bymes, Presnell and Gann, 1981; Kaneko, Kaneko, Shinsako and Dallman, 1981). The ability of these 5-HT agents to maintain high corticosterone responses suggests that the sensitivity of the adrenals to ACTH may have been enhanced. Serotonergic neurons innervating the hypothalamic paraventricular nucleus, make direct synaptic contact with CRF neurons (Liposits et al., 1987) and are responsible for serotonergic control of the hypothalamic-pituit~y-adrenal axis (Feldman et al., 1987). It is likely that the reduced ability of PCA to enhance secretion of ACTH and corticosterone, following repeated injections of cocaine is due to presynaptic changes in these serotonergic nerve terminals. However, the different duration necessary for pretreatment with cocaine to inhibit PCA-induced secretion of ACTH and corticosterone (see Fig. 2), suggests that an additional peripheral effect of pretreatment with cocaine on the adrenal gland could also occur. In conclusion, the data suggest that repeated injections of cocaine alter the function of 5-HT neurons that regulate the hypothalamic-pituitaryadrenal axis. The data also suggest that cocaine acts at presynaptic 5-HT nerve terminals, while it is unlikely that changes in postsynaptic receptors are responsible for the reduced neuroendocrine response to PCA, after chronic administration of cocaine. Acknowledgements-The authors thank MS Kayoko Kunimoto and Mr Joseph Yracheta for their excellent
L. D. VAN DE KAR et al.
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technical assistance. The authors thank the following for the generous gifts of drugs: NIDA (Rockville, Maryland.) for cocaine and Roussel Uclaf (Romainville, France) for RU 24969. Supported in part by USPHS grants DA04865 MH45812 and American Heart Association of Metropolitan Chicago (VDK) and the Veterans Administration (MC).
REFERENCES
Auerbach S. B., Kamalakannan N. and Rutter J. J. (1990) TFMPP and RU24969 enhanced serotonin release from rat hippocampus. Eur. J. Pharmac. 190: 51-57. Bagdy G., Calogero A. E., Murphy D. L. and Szemeredi K. (1989) Serotonin agonists cause parallel activation of the sympathoadrenomedullary system and the hypothalamo pituitary-adrenocortical axis in conscious rats. Endocrinology 125: 2664-2669. Borowsky B. and Kuhn C. M. (1991) Monoamine mediation of cocaine-induced hypothalamopituitary-adrenal activation. J. Pharmac. exp. Ther. 256: 204-210. Calogero A. E., Gallucci W. T., Kling M. A., Chrousos G. P. and Gold P. W. (1989) Cocaine stimulates rat hypothalamic corticotropin-releasing hormone secretion in vitro. Brain Res. 505: 7-l I Carnes M., Brownfield M. S., Kahn N., Lent S. and Barksdale C. M. (1986) Episodic secretion of ACTH in rats. Peptides 7: 219-224. Cunningham K. A. and Lakoski J. M. (1988) Electrophysiology effects of cocaine and procaine on dorsal raphe serotonin neurons, Eur. J. Pharmac. 148: 4577 462. England W. C., Byrnes G. J., Presnell K. and Gann D. S. (1981) Adrenocortical sensitivity to adrenocorticotropin (ACTH) in awake dogs changes as a function of the time of observation and after hemorrhage independently of changes in ACTH. Endocrinology 108: 2149-2153. Euvrard C. and Boissier J. (1980) Biochemical assessment of the central 5-HT agonist activity RU 24969 (a piperidinyl indole). Eur. J. Pharmac. 63: 65-72. Feldman S., Conforti N. and Melamed E. (1987) Paraventricular nucleus serotonin mediates neurally stimulated adrenocortical secretion. Brain Res. Bull. 18: 165-168. Fuller R. W. (1980) Mechanism by which uptake inhibitors antagonize p-chloroamphetamine-induced depletion of brain serotonin. Neurochem. Res. 5: 241-245. Fuller R. W. and Snoddy H. D. (1980) Effect of serotoninreleasing drugs on serum corticosterone concentration in rats. Neuroendocrinology 31: 966100. Fuller R. W. and Snoddy H. D. (1990) Serotonin receptor subtypes involved in the elevation of serum corticosterone concentration in rats by directand indirect-acting serotonin agonists. Neuroendocrinology 52: 206-211. Galloway M. P. (1990) Regulation of dopamine and serotonin synthesis by acute administration of cocaine. Synapse 6: 63-72. Gilbert F., Braze11 C., Tricklebank M. D. and Stahl S. M. (1988) Activation of the 5-HT,, receptor subtype increases plasma ACTH concentration. Eur. J. Pharmac. 147: 431-439. Glennon R. A. (1987) Central serotonin receptors as targets for drug research. J. med. Chem. 30: I-12. Hanson G. R., Matsuda L. A. and Gibb J. W. (1987) Effect of cocaine on methamphetamine-induced neurochemical changes: characterization of cocaine as a monoamine uptake blocker. J. Pharmac. exp. Ther. 242: 5077513. Kaneko M., Kaneko K., Shinsako J. and Dalfman M. F. (1981) Adrenal sensitivity to adrenocorticotropin varies diurnally. Endocrinology 109: 70-75. King B. H., Braze11 C., Dourish C. T. and Middlemiss D. N. (1989) MK-212 increases rat plasma ACTH concentration by activation of the 5-HT,, receptor subtype. Neurosci. Lett. 105: 174-176.
Kleven M. S., Woolverton W. L. and Seiden L. S. (1988) Lack of long-term monoamine depletions following repeated or continuous exposure to cocaine. Brain Res. BUN. 21: 233-237. Koe B. K. (1976) Molecular geometry of inhibitors of the uptake of catecholamines and serotonin in synaptosomal preparations of rat brain. J. Pharmac. exp. Ther. 199: 6499661. Lakoski J. M. and Cunningham K. A. (1988) Cocaine interaction with central monoaminergic systems: Electrophysiological approaches. Trends Pharmac. Sci. 9: 177-180. Levy A. D., Li Q., Kerr J. E., Rittenhouse P. A., Milonas G., Cabrera T. M., Battaglia G., Alvarez Sanz M. C. and Van de Kar L. D. (1991) Cocaine-induced elevation of plasma ACTH and corticosterone is mediated by serotonergic neurons. J. Pharmac. exp. Ther. In press. Liposits Z., Phelix C. and Paul1 W. K. (1987) Synaptic interaction of serotonergic axons and corticotropin releasing factor (CRF) synthesizing neurons in the hypothalamic paraventricular nucleus of the rat. A light and electron microscopic immunocytochemical study. Histochemistry 86: 541-549. Lorens S. A. and Van de Kar L. D. (1987) Differential effects of serotonin (5HT,, and 5HT,) agonists and antagonists on renin and corticosterone secretion. Neuroendocrinology 45: 305-3 10. Marsden C. A. (1981) p-Chloroamphetamine and alphamethyltryptamine-correlation of behavioural response with 5HT release. In: Seroronin Current Aspects of Neurochemistry and Function (Haber B. and Gabai S., Eds), pp. 7777778. Plenum Press, New York. Marsden C. A., Conti J., Strope E., Curzon G. and Adams R. N. (1979) Monitoring 5-hydroxytryptamine release in the brain of the freely moving unanaesthetized rat using in vivo voltametry. Brain Res. 171: 85599. Moldow R. L. and Fischman A. J. (1987) Cocaine induced secretion of ACTH, p-endorphin, and corticosterone. Peptides 8: 819-822. Nicolaysen L. C., Pan H. T. and Justice J. B. J. (1988) Extracellular cocaine and dopamine concentrations are linearly related in rat striatum. Brain Res. 456: 317-323. Pan Z. Z. and Williams J. T. (1989) Differential actions of cocaine and amphetamine on dorsal raphe neurons in vitro. J. Pharmac. exp. Ther. 251: 56662. Pitts D. K. and Marwah J. (1986) Electrophysiological effects of cocaine on central monoaminergic neurons. Eur. J. Pharmar. 131: 95598. Reith M. E. A., Sershen H., Allen D. L. and Lajtha A. (1983) A portion of [‘HIcocaine binding in brain is associated with serotonergic neurons. Molec. Pharmac. 23: 600-606. Ritz M. C., Cone E. J. and Kuhar M. J. (1990) Cocaine inhibition of ligand binding at dopamine, norepinephrine and serotonin transporters: A structure-activity study. Life Sri. 46: 6355645. Rivier C. and Vale W. (1987) Cocaine stimulates adrenocorticotropin (ACTH) secretion through a corticotropinreleasing factor (CRF)-mediated mechanism. Brain Res. 422: 403-406. Roy S. N., Bhattacharyya A. K., Pradhan S. and Pradhan S. N. (1978) Behavioural and neurochemical effects of repeated administration of cocaine in rats. Neuropharmacology 17: 5599564. Steel R. G. D. and Torrie J. H. (1960) Principles and Procedures of Stabstics with Special Reference to the Biological Sciences. McGraw-Hill, New York. Taylor D. and Ho B. T. (1977) Neurochemical effects of cocaine following acute and repeated injection. J. Neurosci. 3: 95-101. Van de Kar L. D. (1991) Neuroendocrine pharmacology of serotonergic (5-HT) neurons. A. Rev. Pharmac. Toxic. 31: 2899320.
Cocaine effect on ACTH and corticosterone Van de Kar L. D., Carnes M., Maslowski R. J., Bonadonna A. M., Rittenhouse P. A., Kunimoto K., Piechowski R. A. and Bethea C. L. (1989) Neuroendocrine evidence for denervation supersensitivity of serotonin receptors: Effects of the S-HT agonist RU 24969 on corticotropin, corticosterone, prolactin and renin secretion. J. Pharmac. exp. Ther. 251: 428-434.
Van de Kar L. D., Urban J. H., Richardson K. D. and Bethea C. L. (1985) Fenfluramine causes elevation in
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plasma prolactin levels via a serotonergic mechanism but causes elevation in plasma corticosterone levels via a mechanism that is independent of serotonin. Neuroendocrinology 41: 283-288.
Van de Kar L. D., Wilkinson C. W. and Ganong W. F. (1981) Pharmacological evidence for a role of brain serotonin in the maintenance of plasma renin activity in unanesthetized rats. J. Pharmac. exp. Ther. 219: 85-94.
2, pp.
169-175,
1992
0028-3908/92
$5.00
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Pergamon Press plc
PRIOR CHRONIC EXPOSURE TO COCAINE INHIBITS THE SEROTONERGIC STIMULATION OF ACTH AND SECRETION OF CORTICOSTERONE L. D. VAN DE KAR,’ ANNA M. BONADONNA,’P. A. RITTENHOUSE,’JANICEE. KERR,’ A. D. LEVY,’ LALITHA IYER,’ G. B. HERBERT,’MARIA C. ALVAREZ SANZ,’ STEPHANIE J. LENT*
and MOLLY CARNE~* ‘Department of Pharmacology, Stritch School of Medicine, Loyola University of Chicago, 2160 S. First Avenue, Maywood, IL 60153, U.S.A. and *University of Wisconsin, Department of Medicine and Middleton Memorial Veterans Hospital, Geriatric Section, Madison, WI 53705, U.S.A. (Accepted 25 July 1991)
Summary-The effect of long-term pretreatment with cocaine on serotonergic regulation of ACTH (adrenocorticotropic hormone; corticotropin) and secretion of corticosterone in rats was investigated. The following observations were made: (1) Pretreatment with cocaine had no significant effect on basal levels of ACTH and corticosterone in plasma. However, cocaine caused a reduction in the ability of the S-HT (S-hydroxytryptamine, serotonin) releaser p-chloroamphetamine (PCA) to increase corticosterone in plasma, 42 hr after the last injection of cocaine. (2) Exposure to cocaine for 7 days was sufficient to produce a maximal inhibition of the PCA-induced increase in ACTH in plasma. (3) The inhibitory effect of cocaine on PCA-induced release of ACTH was more marked than on corticosterone. (4) Conversely, the dose-dependent stimulatory effect of two 5-HT, agonists, RU 24969 (S-methoxy-3-(1,2,3,4-tetrahydro4-pyridinyl)-lH-indole) and m-CPP (m-chlorophenylpiperazine), on ACTH and corticosterone was not reduced by 7 days of exposure to cocaine. Taken together, these findings indicate that pretreatment with cocaine reduced the function of serotonergic nerve-terminals but not postsynaptic receptors, that stimulate ACTH and secretion of corticosterone. Key wordF--cocaine, ACTH, corticosterone, 5-HT, p-chloroamphetamine,
Cocaine is a psychostimulant, effect
by altering
thought to exert its neurotransmission in the brain.
In addition to uptake sites for dopamine and norepinephrine, cocaine binds with a high affinity to synaptic reuptake sites for serotonin (5-HT) (Reith, Sershen, Allen and Lajtha, 1983; Ritz, Cone and Kuhar, 1990). This prevents the reuptake of these neurotransmitters from the synaptic cleft and leads to an increased concentration of 5-HT (as well as dopamine and norepinephrine) in the synapse (Hanson, Matsuda and Gibb, 1987; Koe, 1976). Cocaine has a profound influence on the activity of serotonergic neurons. Acute intravenous injection of cocaine suppresses the firing rate of serotonergic neurons in the dorsal raphe nucleus (Pitts and Marwah, 1986; Cunningham and Lakoski, 1988; Lakoski and Cunningham, 1988). In vitro, addition of cocaine to slices of midbrain of the rat decreases the firing rate of dorsal raphe neurons by increasing the concentration of S-HT in the synapse (Pan and Williams, 1989). The cocaine-induced increase in the concentration of 5-HT in the synapse leads to a compensatory decrease in its synthesis (Galloway, 1990). Little agreement exists concerning the long-term effects of cocaine on serotonergic neurons. Two studies found that repeated injection of cocaine
S-HT receptors, 5-HT uptake.
reduced the content of 5-HT in the brain (Roy, Bhattacharyya, Pradhan and Pradhan, 1978; Taylor and Ho, 1977). However, another study (Kleven, Woolverton and Seiden, 1988) observed no change in the content of 5-HT in several regions of the brain after injection of a smaller dose of cocaine, for 10 days. Thus, additional studies are necessary to establish the long-term effects of cocaine on serotonergic function. The present study utilized the hormonal response to a release of 5-HT and 5-HT agonists, as indices of serotonergic function. Serotonergic neurons stimulate, either directly or indirectly, the release of many hormones, including adrenocorticotropic hormone (ACTH), /I-endorphin, corticosterone, prolactin, renin, oxytocin and vasopressin (for review see Van de Kar, 1991). The present study focused on the influence of cocaine on the serotonergic regulation of the hypothalamic-pituitaryadrenal axis. This axis is composed of corticotropin releasing factor (CRF)-containing neurons in the hypothalamic paraventricular nucleus, pituitary cells which are activated by CRF to release ACTH into the blood and adrenal cortical cells that respond to ACTH by secreting corticosterone. Serotonergic nerveterminals in the paraventricular nucleus stimulate the secretion of CRF (Liposits, Phelix and Paull, 1987; 169
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Feldman, Conforti and Melamed, 1987). Release of ACTH and corticosterone can be stimulated by activation of either 5-HT,, or S-HT,c/5-HT, receptors (Gilbert, Brazell, Tricklebank and Stahl, 1988; Fuller and Snoddy, 1990; King, Brazell, Dourish and Middlemiss, 1989; Lorens and Van de Kar, 1987). Acute injections of cocaine stimulate the hypothalamic-pituitary-adrenal axis, through release of CRF (Calogero, Gallucci, Kling, Chrousos and Gold, 1989; Moldow and Fischman, 1987; Rivier and Vale, 1987). Both dopaminergic and serotonergic neurons mediate the acute effect of cocaine on the secretion of ACTH and corticosterone (Borowsky and Kuhn, 1991; Levy, Li, Kerr, Rittenhouse, Molonas, Cabrera, Battaglia, Alvarez Sanz and Van de Kar, 1991). Because acute administration of cocaine inhibits reuptake of 5-HT, it is likely that long-term administration of cocaine would modify normal serotonergic function. The dose and duration of exposure to cocaine that influenced the serotonergic regulation of secretion of ACTH was investigated, examining the endocrine response to the 5-HT-releasing drug p-chloroamphetamine (PCA). To examine whether the inhibitory effect of cocaine on serotonergic neurons was mediated post-synaptically, the effect of direct 5-HT, agonists RU 24969 and m-CPP (Glennon, 1987) on ACTH and corticosterone in plasma in cocaine-pretreated rats was tested. The rats were only pretreated with cocaine for 7 days, because the effect of PCA on ACTH was inhibited to the same extent after 7 days as after 30 days. METHODS
Animals
DE KAR et al.
for Drug Abuse (NIDA, Rockville, Maryland). These batches of cocaine were used for the first two experiments, i.e. the experiments involving injection of cocaine for 30 days, followed by a challenge with PCA. In all other experiments, the cocaine used was purchased from Sigma (St Louis, Missouri). The 5-HT releasing drug, PCA (Marsden, 198 1; Marsden, Conti, Strope, Curzon and Adams, 1979) was purchased from Sigma (St Louis, Missouri). The 5-HT, agonist, m -CPP (m -chlorophenylpiperazine) was purchased from Sigma (St Louis, Missouri). Another 5-HT, agonist, RU 24969 (5-methoxy-3-(1,2,3,4_tetrahydro-4-pyridinyl)-lH-indole) (Euvrard and Boissier, 1980) was a gift from Roussel Uclaf (Romainville, France). All doses of drugs are expressed as the salt. Experimental protocols Dose-response effect of cocaine. Cocaine was injected twice daily for 30 days, at doses of 0 (saline), 1, 5, 10 and 15 mg/kg, (i.p.). The rats received an injection of 8 mg/kg (i.p.) of PCA or saline, 42 hr after the last injection of cocaine. The rats remained in their home cage for 60min after the injection, at which time they were killed by decapitation. The effect of PCA on several hormones reaches a maximum 60 min after injection (Fuller and Snoddy, 1980; Van de Kar, Wilkinson and Ganong, 1981). Time course of the eflect of cocaine. Cocaine was administered twice daily for either 0, 1, 7 or 30 days, at a dose of 15 mg/kg, (i.p.). All the rats received injections for 30 days. The rats that received injections of cocaine for 2 days, first received injections of saline for 28 days, followed by 2 days of injections of cocaine. Similarly the rats that received injections of cocaine for 7 days, first received injections of saline for 23 days, followed by injections of cocaine. The rats subsequently received 8 mg/kg (i.p.) of PCA or saline, 42 hr after the last cocaine injection and were killed 60 min later by decapitation.
Male Sprague-Dawley rats (200-250 g) were purchased from Sasco-King Animal Laboratories (Oregon, Wisconsin). The rats were housed, 2 per cage, in a temperature-, humidity- and illumination(12: 12 hr light/dark cycle; lights on at 0700 hr) controlled room. Water and food (Wayne Lab Blox, Lab Mills Inc., Chicago, Illinois) were available ad libitum. The rats were killed by decapitation, in an area outside the rat room and trunk blood was collected into centrifuge tubes, containing 0.5 ml of 0.3 M ethylenediamine tetraacetic acid (EDTA; pH 7.4) for the hormone assays. All the experimental protocols were approved by Loyola University Animal Care and Use Committee.
as described above. Forty-two hours after the last injection of cocaine, the rats received an injection of one of several doses of the 5-HT agonists, RU 24969 (0.2, 1 or 5 mg/kg, i.p.) or m-CPP (1, 5 or 20 mg/kg, i.p.). Control rats received an injection of saline. All the rats were killed by decapitation 30 min after the last injection, which is the time when the 5-HT agonists produce their maximum neuroendocrine effects.
Drugs
Biochemical determinations
All drugs were dissolved in saline and injected in a volume of 1 ml/kg, intraperitoneally (i.p.). All injections of cocaine were performed twice a day (08:30 and 17:00) and all subsequent challenges with a 5-HT release or 5-HT agonists occurred 42 hr after the last injection of cocaine. This interval was chosen to eliminate cocaine from the circulation, prior to injection of the 5-HT releaser and S-HT agonists. Cocaine HCl was donated by the National Institute
Radioimmunassay of corticosterone in plasma. This was performed on un-extracted samples of plasma (2 and 5 ~1) in which the binding proteins had been denatured by boiling, using procedures and antiserum from Radioassay Systems Laboratories (Carson, California). The sensitivity limit was 0.02 ng/tube and the intra- and inter-assay variabilities were 4.5% and 11.9%, respectively (Van de Kar, Urban, Richardson and Bethea, 1985).
EfSect of direct S-HT agonists in cocaine-pretreated rats. Rats received cocaine (15 mg/kg, i.p.) for 7 days
Cocaine effect on ACTH and corticosterone Radioimmunassay of ACTH in plasma. This was performed on un-extracted samples of plasma (2050 PI), as previously described (Cames, Brownfield, Kalin, Lent and Barksdale, 1986). Briefly, the ACTH antiserum was obtained from IgG Corp (Nashville, Tennessee). The ACTH for standards (l-39) was obtained from Calbiochem and [‘2SI]-ACTH from INCSTAR (Stillwater, Minnesota). The sequence recognition of the antiserum was 5-18. In addition, this antiserum does not significantly recognize or-MSH, /I-MSH, /?-endorphin, /?-lipotropin, ACTH l-24 or ACTH 1-16-amide. The minimum detectable concentration was 0.25 pg/tube and the intraand inter-assay variations were 4.2% and 14.6%, respectively. Statistics
The data are represented as the group means and the standard errors of the mean (SEM). Statistical analysis of the data was performed by two-way analysis of variance (ANOVA) and individual group means were compared by Student-Newman-Keuls’ test (Steel and Torrie, 1960), using a computer program (NWA STATPAK, Portland, Oregon).
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rats (Fig. 1). The ability of PCA to increase corticosterone in plasma was reduced in rats pretreated with cocaine for 30 days. Although the statistical analysis (two-way ANOVA) did not reveal a significant interaction between treatment with PCA and cocaine, a post-hoc Newman-Keuls’ test indicated that the PCA-induced elevation of corticosterone in plasma was significantly lower in rats receiving chronic pretreatment with the 15 mg/kg dose of cocaine (P < 0.05). A second experiment examined the effects of different durations of pretreatment with cocaine, on PCA-induced elevation of the concentration of ACTH and corticosterone in plasma. The effect of PCA on the concentration of ACTH in plasma was abolished in rats pretreated for 7 and 30 days with cocaine but not in rats pretreated with cocaine for only 1 day (Fig. 2). Two-way ANOVA for ACTH revealed a significant interaction between treatment with cocaine and PCA (F[3,38] = 9.087, P < 0.0001). The post-hoc Newman-Keuis’ tests indicated that 7 and 30 days of exposure to cocaine significantly reduced the PCAinduced elevation of the concentration of ACTH in plasma (P < 0.01).
RESULTS
Pretreatment with cocaine had no significant effect on basal levels of ACTH and corticosterone in plasma. Injection of the 5-HT releaser, PCA, significantly increased the concentration of corticosterone in plasma in saline-pretreated and cocaine-pretreated
r
t z
a
,-
0
Cocaine
1
5
pretreatment
10
(mg/kg,
SALINE PCA
500 -
o 400 E : 300 Z .c 200 z
0 m
a
ioao-
7
15
i.p.)
Fig. 1. Pretreatment with cocaine inhibited the PCAinduced increase in corticosterone in plasma. Rats were pretreated with varying doses of cocaine (1-15 mg/kg, i.p., twice daily) for 30 days. Rats received 8 mg/kg (i.p.) of PCA, 42 hr after the final injection of cocaine, and were sacrificed 60 min after administration of PCA. Data represent mean f SEM of 8 rats per group. Levels of corticosterone in plasma were enhanced in all PCA-treated groups, compared with their corresponding saline-treated groups (P i 0.01 Newman-Keuls’ test). * Significant difference from the salinepretreated rats, that were challenged with PCA (P < 0.01 Newman-Keuls’ test).
a Cocaine
1
7
pretreatment
30
30
(days)
Fig. 2. Inhibition of PCA-induced elevation of ACTH (top) and corticosterone (bottom) in plasma by increasing durations of exposure to cocaine. Rats were pretreated with cocaine (15 mg/kg, i.p., twice daily) for 1, 7 or 30 days. Rats received 8 mg/kg PCA, 42 hr after the final injection of cocaine and were sacrificed 60min later. Data represent mean k SEM of 8 rats per group. *Significant difference between PCA- and the corresponding saline-treated rats, (P < 0.05, Newman-Keuls’ test). * Significant difference between cocaine- and saline-pretreated rats (P < 0.05, Newman-Keuls’ test).
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VAN DE KAR et al.
Injections of cocaine for 1, 7 or 30 days, decreased the levels of ACTH (F[3,54] = 12.206, P < 0.0001) PCA-induced elevation of the concentration of and corticosterone (F[3,54] = 13.256, P < O.OOOl) in corticosterone in plasma. The two-way ANOVA for plasma both in saline- and cocaine- pretreated rats. corticosterone indicated that the interaction between Also, m-CPP elevated the levels of ACTH (F[3,55] = pretreatment with cocaine and injection of PCA 23.52, P < 0.0001) and corticosterone (F[3,53] = 30.8, approached but did not attain statistical significance P < 0.0001) in plasma (Fig. 4). Pretreatment with (F[3,38] = 2.353, P = 0.083). However, the Newmancocaine did not modify the effect of m-CPP on the Keuls’ test revealed that PCA-induced elevation of concentration of either ACTH or corticosterone in the concentration of corticosterone in plasma was plasma. reduced in rats pretreated with cocaine for 1, 7 or 30 days, compared to saline-pretreated rats (P < 0.05). DISCUSSION Additional studies were conducted to examine whether the inhibition of the effects of PCA were due Repeated administration of cocaine altered seroto down-regulation of postsynaptic 5-HT receptors. tonergic regulation of the hypothalamic-pituitaryRats pretreated with the same regimen of cocaine for adrenal axis. Pretreatment with cocaine, of at least 7 days, were challenged with increasing doses of the 7 days, reduced the elevation of levels of ACTH, direct SHT, agonists, RU 24969 and m-CPP. There produced by a 5-HT releaser (PCA) but not by the was no evidence of reduced responsiveness to the 5-HT, agonists, RU 24969 or m-CPP. These data, S-HT, agonists on the concentrations of ACTH therefore, suggest that repeated administration of an corticosterone in rats pretreated with cocaine. As cocaine or its withdrawal, impairs presynaptic but can be seen in Fig. 3, pretreatment with cocaine not postsynaptic serotonergic function. A reduction in the concentration of 5-HT could significantly potentiated the corticosterone response to 1 mg/kg of RU 24969 (P < 0.05, Newman-Keuls account for the reduced ability of PCA to stimulate the secretion of ACTH and corticosterone in cocainetest). The drug RU 24969 significantly increased pretreated rats. The 5-HT releasing agent, PCA depends upon intact stores of endogenous 5-HT to enhance the concentration of 5-HT in the synapse
_. i3 ._ -‘0
0
1s 0
41 0
“’
0.2
Dose of RU 24969
’
I.0
I,‘/
/
“‘-5.0
(mg/kg,
i.p.1
Fig. 3. Increase in ACTH (top) and corticosterone (bottom) in plasma, induced by RU 24969 in rats pretreated with cocaine (15 mg/kg, twice daily) for 7 days. The injection of RU 24969 was performed 42 hr after the last injection of cocaine, and samples of blood were collected 30 min later. Data represent mean + SEM of 8 rats per group. *Significant difference between RH 24969- and corresponding salinetreated rats (P i 0.05 Newman-Keuls’ test). #Significant difference between cocaine- and saline-pretreated rats, receiving RU 24969 (1 mg/kg), (P < 0.01, Newman-Keuls’ .\
test).
c
*'
Dose of m-CPP(mg/kg,
i.p.)
Fig. 4. Increase in ACTH (top) and corticosterone (bottom) in plasma, induced by m-CPP, in rats pretreated with cocaine (15 mg/kg, twice daily) for 7 days. The injection of m-CPP was performed 42 hr after the last injection of cocaine and samples of blood were collected 30min later. Data represent mean + SEM of 8 rats per group. *Significant difference between m-CPP- and the corresponding saline-treated rats (P < 0.05, Newman-Keuls’ test).
Cocaine effect on ACTH and corticosterone
(Marsden et al., 1979; Marsden, 1981) which, in turn, elevates ACTH and corticosterone in plasma (Fuller and Snoddy, 1980). The literature regarding the chronic effects of cocaine on the concentration of S-HT in brain is inconsistent. One study found that injections of cocaine for 9 days reduced the concentrations of 5-HT in several regions of the brain (Roy et al., 1978), while in another study, chronic administration of cocaine did not alter concentrations of 5-HT (Kleven et al., 1988). It is therefore unclear whether the inhibition of the ability of PCA to stimulate secretion of ACTH and corticosterone was due to decreased concentrations of 5-HT in serotonergic nerve terminals. Pretreatment with cocaine could also reduce PCAstimulated secretion of ACTH and corticosterone by interfering with the uptake of PCA into serotonergic nerve terminals. p-~hloroamphetamine enters S-HT nerve terminals through the uptake mechanism for 5-HT (Fuller, 1980; Fuller and Snoddy, 1980). Consequently, the effect of PCA on ACTH would be diminished if the drug could not enter the serotonergic nerve terminals. Cocaine binds with a high affinity to the uptake sites for S-HT and it is a potent blocker of the uptake of 5-HT (Reith et uf., 1983; Ritz et al., 1990; Hanson et al., 1987; Koe, 1976). In the present experiments, rats were allowed 42 hr between the final injections of cocaine and the challenge with PCA. Nicolaysen, Pan and Justice (1988) determined that the levels of cocaine in the extracellular fluid in the brain of the rat reached a peak at about 30 min and disappeared 240 min after a single injection. By 42 hr after chronic pretreatment with cocaine, it is unlikely that the concentration of cocaine would be sufficiently large to inhibit the uptake of PCA into serotonergic nerve terminals. It is possible, however, that chronic pretreatment with cocaine could reduce the function of the uptake mechanism for S-HT. If the uptake mechanism is not functioning properly, the ability of PCA to enter the serotonergic nerve terminal would be decreased and the neuroendocrine response would be reduced. Reduced responses to PCA, following chronic administration of cocaine, is not likely to be due to reduced postsynaptic receptor function. After 7 days of administration of cocaine, the ~Ieuroendoc~ne responses to two 5-HT receptor agonists, RU 24969 and m-CPP were examined. The ability of these 5-HT agonists to enhance levels of ACTH and corticosterone were not reduced by pretreatment with cocaine, suggesting that down-regulation of postsynaptic receptors is not the mechanism by which chronic administration of cocaine alters .5-HT function. These animals actually exhibited an enhanced response of corticosterone to RU 24969. At large doses, RU 24969 may act to release 5-HT (Auerbach, Kamalakannan and Rutter, 1990). However, the neuroendocrine effects of RU 24969 are predominantly mediated through its 5-HT agonist properties; destruction of serotonergic neurons does not inhibit
I73
the effect of RU 24969 on ACTH or corticosterone in plasma (Van de Kar, Cames, Maslowski, Bonadonna, Rittenhouse, Kunimoto, Piechowski and Bethea, 1989). Therefore, the enhanced response of corticosterone to RU 24969 may be due to supersensitive postsynaptic 5-HT receptors. This conclusion would be premature, however, because of differences in endocrine responses to RU 24969 and m-CPP. The possibility that postsynaptic 5-HT receptors are supersensitive, following repeated exposure to cocaine requires more extensive examination. It is inte~sting to note that the changes in endocrine responses to PCA and RU 24969 after chronic exposure to cocaine were not entirely parallel between ACTH and corticosterone. The PCA-induced enhancement of levels of ACTH was greatly diminished by 7 and 30 days of exposure to cocaine while the response of corticosterone was only moderately reduced. Additionally, pretrea~ent with cocaine enhanced the effect of RU 24969 on corticosterone but not ACTH. Similar findings have been previously reported. Bagdy, Calogero, Murphy and Szemeredi (1989) observed that some 5-HT agonists produced maximum increases in levels of corticosterone at doses that produced only moderate increases in ACTH. Differential effects on ACTH and corticosterone in plasma could be explained by alterations in adrenal sensitivity to ACTH. Several studies have determined that adrenal sensitivity to ACTH is not constant (England, Bymes, Presnell and Gann, 1981; Kaneko, Kaneko, Shinsako and Dallman, 1981). The ability of these 5-HT agents to maintain high corticosterone responses suggests that the sensitivity of the adrenals to ACTH may have been enhanced. Serotonergic neurons innervating the hypothalamic paraventricular nucleus, make direct synaptic contact with CRF neurons (Liposits et al., 1987) and are responsible for serotonergic control of the hypothalamic-pituit~y-adrenal axis (Feldman et al., 1987). It is likely that the reduced ability of PCA to enhance secretion of ACTH and corticosterone, following repeated injections of cocaine is due to presynaptic changes in these serotonergic nerve terminals. However, the different duration necessary for pretreatment with cocaine to inhibit PCA-induced secretion of ACTH and corticosterone (see Fig. 2), suggests that an additional peripheral effect of pretreatment with cocaine on the adrenal gland could also occur. In conclusion, the data suggest that repeated injections of cocaine alter the function of 5-HT neurons that regulate the hypothalamic-pituitaryadrenal axis. The data also suggest that cocaine acts at presynaptic 5-HT nerve terminals, while it is unlikely that changes in postsynaptic receptors are responsible for the reduced neuroendocrine response to PCA, after chronic administration of cocaine. Acknowledgements-The authors thank MS Kayoko Kunimoto and Mr Joseph Yracheta for their excellent
L. D. VAN DE KAR et al.
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technical assistance. The authors thank the following for the generous gifts of drugs: NIDA (Rockville, Maryland.) for cocaine and Roussel Uclaf (Romainville, France) for RU 24969. Supported in part by USPHS grants DA04865 MH45812 and American Heart Association of Metropolitan Chicago (VDK) and the Veterans Administration (MC).
REFERENCES
Auerbach S. B., Kamalakannan N. and Rutter J. J. (1990) TFMPP and RU24969 enhanced serotonin release from rat hippocampus. Eur. J. Pharmac. 190: 51-57. Bagdy G., Calogero A. E., Murphy D. L. and Szemeredi K. (1989) Serotonin agonists cause parallel activation of the sympathoadrenomedullary system and the hypothalamo pituitary-adrenocortical axis in conscious rats. Endocrinology 125: 2664-2669. Borowsky B. and Kuhn C. M. (1991) Monoamine mediation of cocaine-induced hypothalamopituitary-adrenal activation. J. Pharmac. exp. Ther. 256: 204-210. Calogero A. E., Gallucci W. T., Kling M. A., Chrousos G. P. and Gold P. W. (1989) Cocaine stimulates rat hypothalamic corticotropin-releasing hormone secretion in vitro. Brain Res. 505: 7-l I Carnes M., Brownfield M. S., Kahn N., Lent S. and Barksdale C. M. (1986) Episodic secretion of ACTH in rats. Peptides 7: 219-224. Cunningham K. A. and Lakoski J. M. (1988) Electrophysiology effects of cocaine and procaine on dorsal raphe serotonin neurons, Eur. J. Pharmac. 148: 4577 462. England W. C., Byrnes G. J., Presnell K. and Gann D. S. (1981) Adrenocortical sensitivity to adrenocorticotropin (ACTH) in awake dogs changes as a function of the time of observation and after hemorrhage independently of changes in ACTH. Endocrinology 108: 2149-2153. Euvrard C. and Boissier J. (1980) Biochemical assessment of the central 5-HT agonist activity RU 24969 (a piperidinyl indole). Eur. J. Pharmac. 63: 65-72. Feldman S., Conforti N. and Melamed E. (1987) Paraventricular nucleus serotonin mediates neurally stimulated adrenocortical secretion. Brain Res. Bull. 18: 165-168. Fuller R. W. (1980) Mechanism by which uptake inhibitors antagonize p-chloroamphetamine-induced depletion of brain serotonin. Neurochem. Res. 5: 241-245. Fuller R. W. and Snoddy H. D. (1980) Effect of serotoninreleasing drugs on serum corticosterone concentration in rats. Neuroendocrinology 31: 966100. Fuller R. W. and Snoddy H. D. (1990) Serotonin receptor subtypes involved in the elevation of serum corticosterone concentration in rats by directand indirect-acting serotonin agonists. Neuroendocrinology 52: 206-211. Galloway M. P. (1990) Regulation of dopamine and serotonin synthesis by acute administration of cocaine. Synapse 6: 63-72. Gilbert F., Braze11 C., Tricklebank M. D. and Stahl S. M. (1988) Activation of the 5-HT,, receptor subtype increases plasma ACTH concentration. Eur. J. Pharmac. 147: 431-439. Glennon R. A. (1987) Central serotonin receptors as targets for drug research. J. med. Chem. 30: I-12. Hanson G. R., Matsuda L. A. and Gibb J. W. (1987) Effect of cocaine on methamphetamine-induced neurochemical changes: characterization of cocaine as a monoamine uptake blocker. J. Pharmac. exp. Ther. 242: 5077513. Kaneko M., Kaneko K., Shinsako J. and Dalfman M. F. (1981) Adrenal sensitivity to adrenocorticotropin varies diurnally. Endocrinology 109: 70-75. King B. H., Braze11 C., Dourish C. T. and Middlemiss D. N. (1989) MK-212 increases rat plasma ACTH concentration by activation of the 5-HT,, receptor subtype. Neurosci. Lett. 105: 174-176.
Kleven M. S., Woolverton W. L. and Seiden L. S. (1988) Lack of long-term monoamine depletions following repeated or continuous exposure to cocaine. Brain Res. BUN. 21: 233-237. Koe B. K. (1976) Molecular geometry of inhibitors of the uptake of catecholamines and serotonin in synaptosomal preparations of rat brain. J. Pharmac. exp. Ther. 199: 6499661. Lakoski J. M. and Cunningham K. A. (1988) Cocaine interaction with central monoaminergic systems: Electrophysiological approaches. Trends Pharmac. Sci. 9: 177-180. Levy A. D., Li Q., Kerr J. E., Rittenhouse P. A., Milonas G., Cabrera T. M., Battaglia G., Alvarez Sanz M. C. and Van de Kar L. D. (1991) Cocaine-induced elevation of plasma ACTH and corticosterone is mediated by serotonergic neurons. J. Pharmac. exp. Ther. In press. Liposits Z., Phelix C. and Paul1 W. K. (1987) Synaptic interaction of serotonergic axons and corticotropin releasing factor (CRF) synthesizing neurons in the hypothalamic paraventricular nucleus of the rat. A light and electron microscopic immunocytochemical study. Histochemistry 86: 541-549. Lorens S. A. and Van de Kar L. D. (1987) Differential effects of serotonin (5HT,, and 5HT,) agonists and antagonists on renin and corticosterone secretion. Neuroendocrinology 45: 305-3 10. Marsden C. A. (1981) p-Chloroamphetamine and alphamethyltryptamine-correlation of behavioural response with 5HT release. In: Seroronin Current Aspects of Neurochemistry and Function (Haber B. and Gabai S., Eds), pp. 7777778. Plenum Press, New York. Marsden C. A., Conti J., Strope E., Curzon G. and Adams R. N. (1979) Monitoring 5-hydroxytryptamine release in the brain of the freely moving unanaesthetized rat using in vivo voltametry. Brain Res. 171: 85599. Moldow R. L. and Fischman A. J. (1987) Cocaine induced secretion of ACTH, p-endorphin, and corticosterone. Peptides 8: 819-822. Nicolaysen L. C., Pan H. T. and Justice J. B. J. (1988) Extracellular cocaine and dopamine concentrations are linearly related in rat striatum. Brain Res. 456: 317-323. Pan Z. Z. and Williams J. T. (1989) Differential actions of cocaine and amphetamine on dorsal raphe neurons in vitro. J. Pharmac. exp. Ther. 251: 56662. Pitts D. K. and Marwah J. (1986) Electrophysiological effects of cocaine on central monoaminergic neurons. Eur. J. Pharmar. 131: 95598. Reith M. E. A., Sershen H., Allen D. L. and Lajtha A. (1983) A portion of [‘HIcocaine binding in brain is associated with serotonergic neurons. Molec. Pharmac. 23: 600-606. Ritz M. C., Cone E. J. and Kuhar M. J. (1990) Cocaine inhibition of ligand binding at dopamine, norepinephrine and serotonin transporters: A structure-activity study. Life Sri. 46: 6355645. Rivier C. and Vale W. (1987) Cocaine stimulates adrenocorticotropin (ACTH) secretion through a corticotropinreleasing factor (CRF)-mediated mechanism. Brain Res. 422: 403-406. Roy S. N., Bhattacharyya A. K., Pradhan S. and Pradhan S. N. (1978) Behavioural and neurochemical effects of repeated administration of cocaine in rats. Neuropharmacology 17: 5599564. Steel R. G. D. and Torrie J. H. (1960) Principles and Procedures of Stabstics with Special Reference to the Biological Sciences. McGraw-Hill, New York. Taylor D. and Ho B. T. (1977) Neurochemical effects of cocaine following acute and repeated injection. J. Neurosci. 3: 95-101. Van de Kar L. D. (1991) Neuroendocrine pharmacology of serotonergic (5-HT) neurons. A. Rev. Pharmac. Toxic. 31: 2899320.
Cocaine effect on ACTH and corticosterone Van de Kar L. D., Carnes M., Maslowski R. J., Bonadonna A. M., Rittenhouse P. A., Kunimoto K., Piechowski R. A. and Bethea C. L. (1989) Neuroendocrine evidence for denervation supersensitivity of serotonin receptors: Effects of the S-HT agonist RU 24969 on corticotropin, corticosterone, prolactin and renin secretion. J. Pharmac. exp. Ther. 251: 428-434.
Van de Kar L. D., Urban J. H., Richardson K. D. and Bethea C. L. (1985) Fenfluramine causes elevation in
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plasma prolactin levels via a serotonergic mechanism but causes elevation in plasma corticosterone levels via a mechanism that is independent of serotonin. Neuroendocrinology 41: 283-288.
Van de Kar L. D., Wilkinson C. W. and Ganong W. F. (1981) Pharmacological evidence for a role of brain serotonin in the maintenance of plasma renin activity in unanesthetized rats. J. Pharmac. exp. Ther. 219: 85-94.
