Mechanisms of cocaine-induced decreases in immune cell function.

Cocaine has previously been shown to decrease mitogen-induced T lymphocyte proliferation in rats following intravenous administration. However, in this report, it is demonstrated that central administration of cocaine (1-50 microg) had no effect on lymphocyte proliferation responses. Similarly, the quaternary derivative, cocaine methiodide, also suppressed lymphocyte proliferation only when administered peripherally (6.5 mg/kg), and not centrally (1-20 microg). These results suggest that the effects of cocaine were mediated through a peripheral mechanism. Since significant elevations in plasma corticosterone were observed with all routes of administration of cocaine, the effects of cocaine did not appear to be due entirely to activation of the HPA axis. Instead, the peripheral administration of the local anesthetic, lidocaine (5 mg/kg) or the monoamine reuptake inhibitor, RTI-55 (2-5 mg/kg), produced significant suppressive effects on proliferation. suggesting that both of these peripheral activities of cocaine may be involved in the alteration of lymphocyte responses.

Morphine-induced conditioned taste aversions in the LEW/N and F344/N rat strains.

Previous reports have shown that the LEW/N and F344/N inbred rat strains display a differential sensitivity to cocaine in a number of preparations, with the LEW/N rats displaying an increased sensitivity to both the reinforcing and aversive effects of cocaine (relative to the F344/N rats). Given that the LEW/N rats are also more sensitive to the reinforcing effects of morphine than the F344/N strain, the present experiment examined the ability of morphine to condition taste aversions in the LEW/N and F344/N strains to determine if the general sensitivity to cocaine generalizes to another drug of abuse. Specifically, on four conditioning trials, 35 LEW/N and 33 F344/N female rats were allowed access to a novel saccharin solution and then injected with varying doses of morphine (0, 10, 32 and 56 mg/kg). On intervening recovery days, subjects were allowed 20-min access to water. Following the fourth trial, a final aversion test was administered. The F344/N rats, but not the LEW/N rats, rapidly acquired morphine-induced taste aversions at all doses of morphine. Pharmacokinetic differences between the strains were also assessed. Specifically, 10 mg/kg morphine (or vehicle) was administered to subjects of both strains and plasma morphine levels were analyzed at 0.5, 2 and 4 h postinjection. No differences in plasma levels between the strains were observed. Unlike with cocaine, the LEW/N rats do not seem generally sensitive to morphine (relative to the F344/N rats). Rather, the differential sensitivity of the two strains to these compounds seems to be preparation dependent. Possible mechanisms underlying the differential sensitivity evident in the strains were discussed.

Reversal of acute effects of high dose morphine on lymphocyte activity by chlorisondamine.

To explore the mechanisms mediating the effects of acute morphine on the immune system, effects of ganglionic blockade with chlorisondamine on acute high dose morphine-induced alterations in blood lymphocyte proliferation, white blood cell counts, spleen lymphocyte proliferation and splenic natural killer (NK) cell cytolytic activity were examined in male Sprague--Dawley rats. Two hours after morphine (30 mg/kg, s.c.) administration, blood lymphocyte proliferation (ConA) was decreased 85%; this effect was antagonized by chlorisondamine (5 mg/kg, i.p.). Notably, however, such morphine exposure did not significantly decrease splenic lymphocyte proliferation, although depression of NK cell activity was also evident and appeared to be chlorisondamine-sensitive. Immune effects of morphine 1 h after treatment were somewhat different. In this case, blood lymphocyte proliferation decreased and plasma levels of corticosterone increased, with ED(50) values of 2.2 and 7.8 mg/kg, respectively. Splenic lymphocyte proliferation and NK activity were also significantly depressed in the 1-h exposure paradigm, but only after administration of 30 mg/kg morphine. These results indicate that chlorisondamine blocks the effects of relatively high doses of morphine on blood lymphocyte activity and indicate that blood lymphocyte proliferation is more sensitive to effects of acute morphine exposure than splenic lymphocyte proliferation, NK cell cytolytic activity and activation of the HPA axis.

Acute effects of morphine on blood lymphocyte proliferation and plasma IL-6 levels.

Administration of morphine (10 mg/kg) to rats was found to decrease the proliferative potential of blood lymphocytes by 60-80% and concurrently elevate circulating levels of the cytokine, interleukin-6 (IL-6), 2- to 4-fold. Both parameters were similarly altered upon the central administration of morphine and were blocked upon pretreatment of animals with the opioid receptor antagonist, naltrexone. These results suggest that the activation of central opioid receptors is involved in morphine-induced inhibition of lymphocyte proliferation as well as increases in circulating levels of IL-6. Studies addressing the potential peripheral mechanisms demonstrated that intact ganglionic transmission was required for both effects of morphine. Although the suppression by morphine of lymphocyte proliferation appeared to be largely independent of stimulation of the hypothalamic-pituitary-adrenal axis, the elevation of IL-6 was completely abolished in adrenalectomized animals. Collectively, these results suggest that central opioid receptor activation results in changes in different immune parameters that can be mediated through distinct peripheral mechanisms.

Specific serotonin reuptake inhibitor-induced decreases in lymphocyte activity require endogenous serotonin release.

OBJECTIVES: We have previously reported that acute administration of the specific serotonin reuptake inhibitor (SSRI), fluoxetine, resulted in a decrease in mitogen-induced blood lymphocyte proliferation. The present studies further examine the specificity of this response to serotonin reuptake systems and the potential role of endogenous serotonin in mediating these effects. METHODS: Male Sprague-Dawley rats were treated intraperitoneally with the SSRIs, fluoxetine (6-10 mg/kg) or sertraline (20 mg/kg), dopamine and norepinephrine reuptake inhibitors, GBR 12909 and desipramine, respectively (6 mg/kg) or the serotonin precursor, 5-hydoxytryptophan (5-HTP, 50 mg/kg) 2 h prior to sacrifice. The serotonin-depleting agents, p-chlorophenylalanine (PCPA, 2 x 200 mg/kg) or the serotonin-lesioning agent, p-chloroamphetamine (PCA, 2 x 10 mg/kg) were administered intraperitoneally 5-7 days prior to fluoxetine (10 mg/kg) administration. RESULTS: Unlike the SSRIs, which significantly suppressed lymphocyte proliferation responses, selective norepinephrine or dopamine reuptake inhibition had no effect on lymphocyte proliferation. Elevation of extracellular serotonin levels with the serotonin precursor, 5-HTP, resulted in a similar decrease in lymphocyte proliferation as that seen with SSRI administration. Conversely, decreases in endogenous serotonin following PCA or PCPA treatment prevented the fluoxetine-induced decreases in lymphocyte proliferation. CONCLUSIONS: These results suggest that decreases in mitogen-induced lymphocyte proliferation following acute fluoxetine administration were due to elevations in extracellular serotonin following reuptake inhibition.

The effects of morphine, nicotine and epibatidine on lymphocyte activity and hypothalamic-pituitary-adrenal axis responses.

Acute administration of morphine alters various neuroendocrine and immune parameters via opioid receptors located within the central nervous system. Similar effects have been reported after systemic nicotine treatment. To examine the possible relationship between opioid and nicotinic receptor activation on the immune system, we compared the effects of morphine with both nicotine and the highly selective nicotinic agonist, epibatidine. Male Sprague-Dawley rats were treated with either morphine (10 mg/kg, s.c.), nicotine (2.85 mg/kg, s.c. = 1 mg/kg freebase), or epibatidine (5 microg/kg, s.c.) and sacrificed 2 hours later. Each drug increased plasma corticosterone levels and decreased the magnitude of the peripheral blood lymphocyte proliferation response to the T cell mitogen concanavalin A. None of the treatments had a significant effect on splenic or thymic lymphocyte responses. The effects of nicotine treatment were dose-dependent. Pretreatment with the quaternary ganglionic antagonist chlorisondamine (0.5 mg/kg, i.p.), completely blocked the effect of epibatidine on blood lymphocytes without altering the elevation of corticosterone levels. Although naltrexone (10 mg/kg, s.c.) blocked all effects of morphine, the effects of epibatidine were not blocked by the opioid receptor antagonist. Furthermore, in contrast to morphine (), central injection of neither nicotine (30 or 240 nmol) nor epibatidine (5, 50, or 500 ng) altered blood lymphocyte responses. These results suggest that, like morphine, nicotinic agonists decrease blood lymphocyte proliferation responses, apparently independent of elevated corticosterone. However, unlike morphine, nicotinic agonists appear to act predominantly at peripheral receptors, suggesting that nicotinic receptors are downstream of opioid receptors in a centrally mediated opioid-induced immunomodulatory pathway.

Evidence for central opioid receptors in the immunomodulatory effects of morphine: review of potential mechanism(s) of action.

This review will discuss studies demonstrating that activation of opioid receptors within the central nervous system alters various immune system parameters. Specifically, natural killer cell cytolytic activity and lymphocyte proliferative responses to mitogen appear to be modulated predominantly, if not exclusively, through central opioid receptors. The potential mechanisms by which central opioid receptors appear to modulate these peripheral immune functions will be examined by evaluating the role of both the hypothalamic-pituitary-adrenal (HPA) axis and the autonomic nervous system. The studies discussed below indicate that acute administration of morphine or related compounds appears to primarily alter peripheral immune function through the sympathetic nervous system, while more prolonged exposure to opioids alter the immune system predominantly by activation of the HPA axis. Finally, the potential clinical relevance of these observations are discussed in relationship to both the therapeutic use, as well as the abuse of opioid compounds.

In vivo effects of cocaine on immune cell function.

Cocaine use has been shown to increase the risk of HIV infection in humans, and this increased risk cannot be explained by i.v. drug use alone. It is thought that this increased susceptibility may be a result of decreased immune responsiveness in cocaine addicts. Scientists are now using animal models to study the effects of cocaine on immune function in vivo under controlled conditions. Many facets of the immune system are being examined, which include immune cell number and distribution, cellular- and humoral-mediated immunity, cytokine production, and immunocompetence to challenges such as infection and tumor growth. The effects of cocaine on many of these functions are not yet clear. Often there are variations in the response of the immune system to cocaine. Potential confounding factors include variations in dose, duration of treatment, and route of administration of cocaine, as well as variations in assay protocols. In addition, there appear to be species differences in immune responses to cocaine. Although it is clear that more research is necessary to resolve the discrepancies, a sufficient number of trends are starting to emerge. This review will systematically evaluate the reported effects of cocaine on immune cell function in vivo. In addition, the possible mechanisms that may be contributing to the immune modulation observed with cocaine in vivo will be addressed. Further, data will be presented describing the effects of cocaine on the autonomic nervous system and the neuroendocrine system suggesting that inhibition of serotonin uptake may be an important component of the overall effects of cocaine on the immune system.

Role of central opioid receptor subtypes in morphine-induced alterations in peripheral lymphocyte activity.

Morphine has been shown to decrease proliferative responses of rat T-lymphocytes via central opioid receptors, however, the specific receptor subtype(s) mediating this effect have not been established. To determine the potential role of central mu opioid receptors in morphine-mediated suppression of T-lymphocyte proliferation, 20 nmol/2 microliters of either morphine sulfate or DAMGO (mu-selective agonist) were administered into the lateral ventricle of freely moving Sprague-Dawley rats. Lymphocyte proliferative response to the polyclonal T cell mitogen concanavalin A (ConA), changes in splenic natural killer cell (NK) cytolytic activity, activation of the hypothalamic-pituitary-adrenal (HPA) axis and antinociception (tail-flick latency) were examined. Results indicated that like morphine, DAMGO decreased blood lymphocyte proliferative responses by 80% and elevated both tail-flick latency and plasma corticosterone when compared to saline-treated animals. The proliferation response of lymphocytes from the spleen or thymus and splenic NK cell activity were not significantly altered by either morphine or DAMGO treatment. The effects of DAMGO were determined to be dose-dependent and completely antagonized by naltrexone pretreatment. Central administration of DPDPE (delta-selective agonist) and U-50488 (kappa-selective agonist) produced between 40-50% suppression of blood lymphocyte proliferation responses only at a dose five times greater (100 nmol) than DAMGO treatment, without altering antinociception or activation of the HPA axis. To determine the central opioid receptor subtype(s) involved in the effects of morphine, selective opioid antagonists were microinjected into the lateral ventricle prior to morphine treatment (6 mg/kg, s.c.). CTOP (mu-selective antagonist, 5 micrograms/2 microliters) completely blocked the effects of morphine on all parameters measured, however, naltrindole (delta-selective antagonist, 2 micrograms/2 microliters) or nor-binaltorphimine (kappa-selective antagonist, 73.5 micrograms/2 microliters) failed to block the effects of morphine. Collectively, these results provide evidence that morphine acts primarily through central mu receptors to modulate peripheral blood lymphocyte proliferation responses. Further, the antinociception and blood lymphocyte effects show greater sensitivity to opioids than either natural killer cell cytolytic activity or activation of the HPA axis.

Modulation of immune cell function following fluoxetine administration in rats.

Fluoxetine (FLX) and other specific serotonin uptake inhibitors (SSRIs) have become the drugs of choice for treating depression. However, only a limited number of studies have addressed the effects of FLX on immune cell function. Our lab has measured the effects of both acute and chronic FLX administration on two functions of cell-mediated immunity, mitogen-induced lymphocyte proliferation (MILP) and natural killer cell cytolytic activity (NKCA). In this article we report that acute FLX administration (10 mg/kg) resulted in a dose- and time-dependent decrease in MILP and NKCA. MILP was more sensitive than NKCA to FLX, requiring lower doses for inhibition; however, the effects were more transient. Following chronic FLX administration, these effects were no longer observed, suggesting that an apparent tolerance to these particular measures of cell-mediated immunity had developed. Finally, a single microinjection of FLX directly into the lateral ventricle produced similar suppressive effects on MILP and NKCA, suggesting that the immunomodulatory mechanism may have a central component.

Potential role of the autonomic nervous system in the immunosuppressive effects of acute morphine administration.

These studies investigated the role of the autonomic nervous system in mediating the immunosuppressive effect of morphine on blood lymphocyte proliferation in rats. To determine the contribution of the autonomic nervous system, rats were pretreated with the ganglionic blocker chlorisondamine (5 mg/kg) prior to morphine (7 mg/kg) administration. Ganglionic blockade with chlorisondamine completely antagonized the inhibitory actions of morphine, suggesting that intact ganglionic transmission was required for the inhibition to occur. Blockade of postganglionic parasympathetic neurotransmission with atropine methylbromide (1 mg/kg) or blockade of sympathetic neurotransmission with the alpha-adrenoceptor antagonist phentolamine (1 mg/kg) did not attenuate the suppressive effect of morphine. Blockade of beta-adrenoceptors with propranolol (2.5 mg/kg) resulted in partial antagonism, but this action was not shared by the peripherally acting beta-adrenoceptor antagonist nadolol (6 mg/kg). These results suggest that the inhibitory effect of morphine on blood lymphocyte proliferation may be mediated through activation of the autonomic nervous system; however, individual blockade of either the parasympathetic or sympathetic division of the autonomic nervous system was not sufficient to antagonize this immunosuppressive effect.

Tolerance and crosstolerance to the suppressive effects of cocaine and morphine on lymphocyte proliferation.

The effects of acute or daily exposure to either cocaine or morphine on lymphocyte proliferative responses and NK cytolytic activity were determined. Two hours following the IV infusion of cocaine (5 mg/kg), blood lymphocyte proliferative responses were found to be suppressed by 75%. Cocaine had no effect on proliferative responses of thymic or splenic lymphocytes or cytolytic activity of splenic NK cells following acute or 5-day repetitive dosing. Similar to the effects of cocaine, morphine (10 mg/kg) administration was also accompanied by a suppressed blood lymphocyte response, which was no longer apparent 8 days following repeated morphine injections. Animals that had received daily injections of either morphine of cocaine were also found to be resistant to the inhibitory effects of a single dose of morphine or cocaine, respectively. These data suggest repeated exposure to either morphine or cocaine results in the development of an apparent crosstolerant state to further suppression of blood lymphocyte proliferative responses by either drug.

Mechanisms of morphine-induced immunosuppression: effect of acute morphine administration on lymphocyte trafficking.

These studies investigated the potential mechanisms by which acute morphine administration inhibits peripheral blood lymphocyte activity in the rat. As reported previously, blood lymphocyte proliferative responses to concanavalin A were found to be suppressed by 70% 2 hr after administration of morphine (10 mg/kg). In the present study, a more selective mu receptor agonist, fentanyl (0.05 mg/kg), was found to similarly inhibit blood lymphocyte proliferation. Pretreatment with the opioid receptor antagonist naltrexone (5 mg/kg) completely blocked the inhibitory actions of morphine (7 mg/kg). Several different approaches were undertaken to determine whether the depressed blood lymphocyte proliferative response after opioid receptor stimulation was due to an effect on circulating lymphocyte number. First, it was found that morphine administration was accompanied by a 30% decrease in the absolute number of circulating lymphocytes. Fluorescence-activated cell sorter analysis revealed that the decrease was not selective for any specific subpopulation of T lymphocyte. Furthermore, the relative distribution of circulating monocytes, neutrophils or eosinophils was not altered by morphine treatment. The morphine-induced lymphopenia was abolished in adrenalectomized rats, suggesting that this redistribution effect was mediated by the release of adrenal hormones. However, on correcting for the decrease in lymphocyte number by using Ficoll-separated lymphocyte cultures, the proliferative responses of blood lymphocytes remained significantly depressed compared with control values. Collectively, these data suggest that both an adrenal-dependent lymphopenia and an opioid-induced decrease in responsiveness to mitogenic stimulation contribute to the overall antiproliferative effect of morphine on blood lymphocytes.

Acute infusions of cocaine result in time- and dose-dependent effects on lymphocyte responses and corticosterone secretion in rats.

In the present study, we investigated the effect of intravenously (i.v.) administered cocaine on mitogen-induced lymphocyte proliferation and NK cytolytic activity in rats implanted with indwelling jugular cannula. To assess whether the effects of cocaine were accompanied by adrenal gland activation, plasma corticosterone concentrations were also determined. It was found that the i.v. infusion of cocaine resulted in both a time- and dose-dependent decrease in both blood and splenic Con-A-stimulated lymphocyte proliferative responses. Within 60 minutes, blood responses were maximally inhibited by more than 60% with 5 mg/kg cocaine. By 4 h, the suppression of blood lymphocyte responses was no longer significant. In contrast to these findings, there were no significant effects observed with splenic lymphocyte responses until 4 h after drug administration. At this time, cocaine at doses of 5 and 10 mg/kg inhibited splenic proliferative responses by 50 and 75%, respectively. These effects appeared to be selective, since no concurrent decreases in NK cell activity were observed with 5 mg/kg at either 2 or 4 h. Within 30 min, plasma corticosterone concentrations were maximally increased by 10-fold with 5 and 10 mg/kg doses of cocaine. At lower doses of cocaine (1 mg/kg), neither changes in lymphocyte proliferative responses, NK cytolytic activity nor plasma corticosterone levels were apparent. This study demonstrates that a single i.v. infusion of cocaine results in a selective dose- and time-dependent immunosuppression which is preceded by transient increases in circulating levels of corticosterone.

Enhanced susceptibility of the immune system to stress in morphine-tolerant rats.

The purpose of the present study was to determine the potential consequences to the immune system of the combined exposure of rats to stressor and morphine. Within 30 min following either morphine (5 mg/kg) injection or restraint stress (30 min) maximal analgesic responses as measured by tail-flick assay were observed. However, only morphine treatment was accompanied by a significant suppression (50%) in mitogen-stimulated lymphocyte proliferative responses. Restraint stress for either a 30-min or 2-h duration had no effect on lymphocyte responses. Exposure to a combination of restraint stress and acute morphine (5 mg/kg) resulted in a 50% suppression of lymphocyte responses which was similar in magnitude to that observed with morphine administration alone. When rats were injected twice daily for 4 days with increasing doses of morphine ranging from 10 mg/kg to 40 mg/kg, morphine (10 mg/kg) administration on Day 5 was not accompanied by either analgesia or depressed blood lymphocyte proliferative responses. These results indicated that tolerance had developed to both the analgesic and the immunosuppressive effects of morphine. However, upon exposure of morphine-tolerant animals to restraint stress, significant analgesic responses were retained. Furthermore, in contrast to the lack of suppression following restraint stress on lymphocyte responses in saline-injected animals, restraint for 30 min produced greater than a 70% suppression in morphine-tolerant animals. These data suggest that morphine tolerance may be accompanied by an enhanced susceptibility to the immunosuppressive effects of stress.

In vitro effects of cocaine, lidocaine and monoamine uptake inhibitors on lymphocyte proliferative responses.

Cocaine was found to inhibit in vitro mitogen-stimulated rat B and T lymphocyte proliferation in a dose-dependent manner. The IC50 for B lymphocytes (70 microM) was 2 to 4 fold lower than that obtained with T lymphocytes. To determine whether ion channel blockade or inhibition of monoamine uptake produced a similar suppression of lymphocyte proliferation, the effects of pharmacological agents sharing each of these properties with cocaine were examined. Lidocaine (0.5 mM), a sodium channel blocker, had no significant effect on B and T cell proliferation. By comparison, cocaine inhibited lymphocyte responses by greater than 80 percent at this concentration. Monoamine uptake inhibitors were also found to suppress lymphocyte proliferation in a dose-dependent manner similar to that obtained with cocaine. Of those tested, desipramine and fluoxetine were considerably more potent than cocaine, nomifensine and nisoxetine. These data demonstrated the addition of cocaine directly to lymphocyte cultures resulted in a dose-dependent inhibition of proliferation which was not due to Na+ channel blockade. Instead, the resemblance of monoamine uptake inhibitors to the action of cocaine suggests that lymphocytes may be intrinsically sensitive to these agents.

Acute immunosuppressive effects of morphine: lack of involvement of pituitary and adrenal factors.

We have previously reported that morphine inhibits Concanavalin A-stimulated blood lymphocyte proliferation in a dose-dependent manner. Maximal (80%) inhibition by morphine occurred with a dose of 10 mg/kg 2 hours after drug administration. Concurrent with this suppressive effect was a 2- to 4-fold increase in plasma corticosterone concentrations. In the present study, we examined the potential contribution of the hypothalamic-pituitary-adrenal axis to the suppressive effects of acute morphine exposure. To assess the role of glucocorticoids, rats were pretreated with the steroid receptor antagonist RU486 (20 mg/kg) 30 min before morphine (10 mg/kg) administration. A significant inhibition of lymphocyte activity occurred with morphine in the absence or presence of RU486 pretreatment. Consistent with a mechanism independent of glucocorticoids, adrenalectomy also failed to attenuate the inhibitory actions of morphine. To examine the potential role of pituitary hormones in the suppressive effect, similar experiments were carried out in hypophysectomized animals. In sham-operated or hypophysectomized animals, morphine was found to be equally effective in suppressing lymphocyte proliferation. These results suggest that factors elaborated from intact pituitary or adrenal glands are not required for the acute inhibitory effects of morphine on peripheral blood lymphocyte activity.

Immunosuppression by morphine is mediated by central pathways.

We reported previously that a single systemic injection of morphine (10 mg/kg) to rats profoundly suppressed mitogen-induced proliferation of blood lymphocytes by a receptor-mediated mechanism. The present study examined whether this immunosuppressive effect of morphine is mediated by opioid receptors located at either peripheral or central sites. First, the effects of systemic morphine administration on analgesia, mitogen-stimulated lymphocyte proliferation and corticosterone secretion were compared to those observed after the systemic administration of N-methylmorphine, a quaternary derivative which does not readily penetrate the blood-brain barrier. In contrast to systemically administered morphine, the i.p. injection of N-methyl-morphine (20 mg/kg) was without any effect on lymphocyte proliferation, plasma corticosterone concentrations or analgesic responses. Secondly, the effects of morphine and N-methylmorphine after central administration were compared. Within 2 hr after the microinjection of either morphine (10 micrograms/2 microliters) or N-methylmorphine (15 micrograms/2 microliters) into the third ventricle, blood lymphocyte responses were inhibited by 70%, plasma corticosterone concentrations were significantly elevated and maximal analgesic responses were present. Finally, microinjection of morphine (1 microgram/0.2 microliter) into the anterior hypothalamus inhibited blood lymphocyte proliferation by 50% without producing analgesia or a significant increase in plasma corticosterone. These findings suggest that central opioid pathways are involved in the immunosuppressive effects of morphine and these pathways may be distinct from those participating in opioid-induced analgesia and adrenal activation.

Increases of CCK mRNA and peptide in different brain areas following acute and chronic administration of morphine.

The present study examined whether either acute or chronic administration of morphine resulted in changes in the content of CCK mRNA and CCK immunoactive peptide in selective areas of the rat brain and spinal cord. Two hours after a single injection of morphine (10 mg/kg, s.c.), CCK mRNA significantly increased in the hypothalamus (0.8-fold) and spinal cord (2-fold) relative to the CCK mRNA content in saline-injected controls. No significant differences in CCK mRNA were observed in the frontal cortex, hippocampus, midbrain or brainstem. There were no significant alterations in CCK immunoreactivity in any brain regions and spinal cord after the acute treatment with morphine. Upon repeated morphine administration, the content of CCK mRNA in both the hypothalamus and the spinal cord was further elevated by at least 3-fold. A significant increase of CCK mRNA content in brain stem (2.8-fold) was also observed following chronic morphine administration. In contrast to the acute exposure to morphine, chronic administration resulted in significant increases in CCK immunoactive peptide in hypothalamus (2.6-fold), spinal cord (2.1-fold) and brainstem (1.6-fold), but not in the other brain areas. These results demonstrate that morphine, especially following repeated administrations, stimulates endogenous CCK biosynthesis in selective brain regions.