Basolateral amygdala and stress-induced hyperexcitability affect motivated behaviors and addiction.

The amygdala integrates and processes incoming information pertinent to reward and to emotions such as fear and anxiety that promote survival by warning of potential danger. Basolateral amygdala (BLA) communicates bi-directionally with brain regions affecting cognition, motivation and stress responses including prefrontal cortex, hippocampus, nucleus accumbens and hindbrain regions that trigger norepinephrine-mediated stress responses. Disruption of intrinsic amygdala and BLA regulatory neurocircuits is often caused by dysfunctional neuroplasticity frequently due to molecular alterations in local GABAergic circuits and principal glutamatergic output neurons. Changes in local regulation of BLA excitability underlie behavioral disturbances characteristic of disorders including post-traumatic stress syndrome (PTSD), autism, attention-deficit hyperactivity disorder (ADHD) and stress-induced relapse to drug use. In this Review, we discuss molecular mechanisms and neural circuits that regulate physiological and stress-induced dysfunction of BLA/amygdala and its principal output neurons. We consider effects of stress on motivated behaviors that depend on BLA; these include drug taking and drug seeking, with emphasis on nicotine-dependent behaviors. Throughout, we take a translational approach by integrating decades of addiction research on animal models and human trials. We show that changes in BLA function identified in animal addiction models illuminate human brain imaging and behavioral studies by more precisely delineating BLA mechanisms. In summary, BLA is required to promote responding for natural reward and respond to second-order drug-conditioned cues; reinstate cue-dependent drug seeking; express stress-enhanced reacquisition of nicotine intake; and drive anxiety and fear. Converging evidence indicates that chronic stress causes BLA principal output neurons to become hyperexcitable.

Norepinephrine secretion in the hypothalamic paraventricular nucleus of rats during unlimited access to self-administered nicotine: An in vivo microdialysis study.

Norepinephrine (NE) secretion within the hypothalamic paraventricular nucleus (PVN) is pivotal to endocrine and behavioral responses. Activation of NE afferents to PVN also is necessary for the hypothalamo-pituitary-adrenal axis response to passively administered nicotine. The mode of drug delivery is a critical determinant of the dynamics of neurotransmitter secretion, yet the PVN NE response to nicotine self-administration (SA) is unknown. Herein, rats housed in operant chambers had unlimited 23 hr access to self-administered nicotine. In vivo microdialysis of PVN NE was performed, collecting consecutive 7 min samples over 9 hr sessions during three phases of nicotine SA: acquisition (day 1); early maintenance, once stable rates of SA were achieved (day 9.2 +/- 0.6); later maintenance (day 18.6 +/- 0.8). On d1, nicotine animals had an increased percentage of SA episodes (SAEs) in which NE levels were elevated (80 vs 30% with saline; p < 0.01). By early maintenance, a fourfold increase in such episodes was observed in nicotine animals (p < 0.01), and the overall NE level was greater (1.30 +/- 0.24 vs 0.63 +/- 0.07 pg/10 microl in saline; p < 0.05); NE increased during the first, but not the last, SAE. The pattern was similar during later maintenance, although NE responsiveness declined (overall NE level, 0.96 +/- 0.19 in nicotine vs 0.52 +/- 0.08 pg/10 microl in saline; p < 0.05). Therefore, nicotine SAEs were associated with sustained increases in NE secretion during all three phases of SA. However, the reduced NE responsiveness observed both within the dialysis session in each phase and by later versus early maintenance is consistent with progression of partial daily desensitization of PVN NE secretion to nicotine SA. Therefore, in rats chronically self-administering nicotine, the drug stimulates sustained PVN NE secretion that may alter neuroendocrine and behavioral responses mediated by the PVN. Compared with studies of chronic human smokers, our nicotine SA model may reflect the CNS noradrenergic responses that occur during human cigarette smoking.

Regulation of delta opioid receptor expression by anti-CD3-epsilon, PMA, and ionomycin in murine splenocytes and T cells.

Previous studies have shown that low levels of delta opioid receptor (DOR) mRNA were detectable by reverse transcription polymerase chain reaction (RT-PCR) in unstimulated splenocytes from BALB/c female mice. This study demonstrates that DOR transcripts can be detected in freshly obtained splenocytes froin CD 1 female mice as well. The results of studies using quantitative competitive RT-PCR showed that DOR transcripts in splenic T cells increased from < 1 copy/cell to 22 and 42 copies/cell, respectively, after stimulation with anti-CD3-epsilon for 24 and 48 h compared to the level in freshly obtained T cells. In the presence of actinomycin D, anti-CD3-epsilon did not affect the rate of degradation of DOR mRNA, suggesting that its stability is not altered by anti-CD3-epsilon. After incubation with phorbol myristate acetate (PMA) and ionomycin, the expression of DOR mRNA in splenocytes and T cells was significantly reduced compared with unstimulated cells in culture. In addition, the inhibitory effect of PMA prevented anti-CD3-epsilon-stimulated DOR expression. These data suggest that signaling through the T cell receptor complex by anti-CD3-epsilon regulates DOR expression differently than PMA and ionomycin.

Morphine promotes the growth of HIV-1 in human peripheral blood mononuclear cell cocultures.

Because morphine has been shown to alter the function of human T lymphocytes and monocytes, we postulated that morphine would promote the growth of HIV-1 in these cells. To test this hypothesis, a coculture assay was used consisting of phytohemagglutinin (PHA)-activated peripheral blood mononuclear cells (PBMC) from normal donors and PBMC which had been infected with a viral isolate from an asymptomatic patient, HIV-1AT. The growth of HIV-1AT, as reflected by the concentration of p24 antigen in coculture supernatants, was markedly increased in cocultures that contained morphine. A bell-shaped dose-response curve was observed with three- to fourfold increased growth at a morphine concentration of 10(-12) M. Augmentation of HIV-1AT growth by morphine required an interaction with the PHA-activated donor PBMC. Furthermore, potentiation of HIV-1AT growth by morphine was stereospecific and was antagonized by naloxone and beta-funaltrexamine indicating involvement of an opiate receptor mechanism. These findings provide an additional explanation of how opiates could act as a cofactor in the pathogenesis of HIV-1 in intravenous drug users.

Prostaglandins mediate the adrenocorticotropin response to tumor necrosis factor in rats.

To determine whether prostaglandins (PGs) mediate the ACTH response to tumor necrosis factor-alpha (TNF alpha), indomethacin (Indo; 0.1-1.0 mg/kg, iv) was administered before TNF alpha (1 microgram, iv) in freely moving, alert rats. While Indo alone did not affect plasma ACTH levels, it dose-dependently blocked the ACTH response to TNF alpha. The highest dose of Indo abolished the ACTH response to TNF alpha [peak plasma ACTH values (mean +/- SEM): buffer/buffer, 137 +/- 34 pg/ml; Indo/buffer, 115 +/- 31; buffer/TNF alpha, 469 +/- 77; Indo/TNF alpha, 120 +/- 27] without modifying the ACTH response to CRF 1 microgram/kg, iv, demonstrating that pituitary responsiveness was unaffected. Since it has been reported that Indo elevates plasma corticosterone (B) levels, the effect of Indo could reflect rapid negative feedback by B, rather than the involvement of PGs. However, inhibition of ACTH secretion was shown to be dependent on the dose of Indo, whereas plasma B levels were elevated to the same degree, independent of the Indo dose. In addition, Indo failed to block the ACTH response to an unrelated ACTH stimulus, insulin-induced hypoglycemia (area under response curve: insulin alone, 31,131 +/- 2,794 pg/min.ml; Indo/insulin, 32,919 +/- 3,582 pg/min.ml). Finally, in adrenalectomized B-replaced rats, TNF alpha elevated ACTH to levels similar to those seen in sham animals, and Indo inhibited these ACTH responses to the same extent in both groups. Thus, Indo inhibited the ACTH response to TNF alpha by a mechanism independent of B feedback. These results indicate that acute systemic administration of TNF alpha stimulates ACTH secretion through a PG-dependent mechanism.

Detection by in vivo microdialysis of nicotine-induced norepinephrine secretion from the hypothalamic paraventricular nucleus of freely moving rats: dose-dependency and desensitization.

The ACTH response to many stimuli depends on the secretion of norepinephrine, which activates neurons in the hypothalamic paraventricular nucleus (PVN). Studies with adrenergic antagonists and inhibitors of catecholamine synthesis indicate that norepinephrine is a mediator of nicotine-induced ACTH secretion. To directly assess the effect of nicotine on PVN norepinephrine secretion, in vivo microdialysis was performed. Nicotine (0.5 mg/kg BW, ip) induced peak norepinephrine levels within 20 min (approximately 2 x basal). The central nicotinic receptor antagonist, mecamylamine, abolished this response, whereas hexamethonium, a peripheral antagonist, was ineffective. The norepinephrine response was dose dependent (ED50, approximately 0.25 mg/kg), and nicotine (0.5 mg/kg BW) induced maximal secretion. Rapid desensitization occurred, as evidenced by a significant reduction (approximately 50%) in the response to a second injection of nicotine (0.5 mg/kg BW). Animals also received four injections of nicotine to determine whether repetitive dosing leads to progressive reduction of the norepinephrine response. The responses to the third and fourth nicotine injections were similar. Thus, desensitization occurred after a single exposure to nicotine and was not progressive. In contrast, nicotine pretreatment did not affect the release of norepinephrine due to yohimbine. These results indicate that basal and nicotine-stimulated levels of norepinephrine can be detected in microdialysates from the PVN; rapid desensitization of the norepinephrine response to nicotine and inhibition by mecamylamine, but not hexamethonium, parallel the findings previously reported for ACTH.

Nerve growth factor enhances [3H]nicotine binding to a nicotinic cholinergic receptor on PC 12 cells.

Although nicotinic cholinergic agonists have functional effects on PC 12 cells, radioligand-binding sites have not been detected. We, therefore, studied PC 12 cells incubated in the presence of nerve growth factor (NGF) and determined that specific [3H]nicotine-binding sites were induced approximately 2.5-fold in the presence of NGF (50 or 100 ng/ml). Specific binding was maximal between the first (100 ng/ml NGF) and seventh (50 ng/ml NGF) days of treatment and was stable for 2 weeks with addition of NGF every 3 days. Using intact cells, average association and dissociation rates for [3H]nicotine were 0.00021 min-1 nM-1 (n = 2) and 0.048 min-1 (n = 2), respectively, at 4 C, yielding an average apparent Kd of 229 nM. At 22 C, stable equilibrium was not attained during association studies. A similar Kd value for broken cell preparations was obtained by kinetic analysis (i.e. an average association rate of 0.00042 min-1 nM-1 and dissociation rate of 0.087 min-1), yielding an average Kd value of 207 nM (n = 2) at 4 C. By saturation binding analysis of intact cells, an average Kd of 292 nM (n = 2) and a binding capacity (Bmax) of 15,118 molecules/cell were obtained. [3H]Nicotine binding was inhibited on an equimolar basis by L-(-)nicotine and N-methylcarbamylcholine. D-(+) Nicotine was 7-fold less potent, whereas alpha-bungarotoxin, mecamylamine, and atropine showed no significant inhibition. [3H]Nicotine binding was also inhibited quantitatively by mono-specific polyclonal antibodies raised against the predicted alpha 3-subunit sequence (amino acids 130-139) of the rat neuronal nicotinic cholinergic receptor. This study represents the first biochemical characterization of NGF-stimulated nicotine-binding sites on PC 12 cells and confirms previous evidence of the presence of functional nicotinic cholinergic receptors on these cells.

Catecholamines mediate nicotine-induced adrenocorticotropin secretion via alpha-adrenergic receptors.

Previous studies determined that iv nicotine stimulates ACTH secretion by acting on sites accessible from the fourth ventricle (IV), rather than directly on CRF-containing neurons in the hypothalamus. Brainstem catecholaminergic cell groups, which are accessible from the IV, project to the hypothalamus and stimulate the secretion of CRF and ACTH. Therefore, these studies investigated the role of catecholamines in nicotine-stimulated ACTH secretion. Experiments with the catecholaminergic neurotoxin, 6-hydroxydopamine, demonstrate that the ACTH response to nicotine delivered iv (0.03 or 0.05 mg/kg body wt) or instilled into the IV (1 or 2.5 micrograms) was significantly reduced in lesioned animals (P less than 0.01). Selective inhibitors of epinephrine synthesis, SKF 64139 and 2,3-dichloro-alpha-methylbenzylamine (DCMB), significantly reduced (P less than 0.01) the ACTH response to 0.05 mg/kg body wt nicotine iv, without affecting median eminence CRF content. Because controversy exists about the effect of DCMB as an alpha 2 adrenoreceptor antagonist in vivo, this was examined by administering norepinephrine into the third ventricle after DCMB ip; DCMB significantly reduced the ACTH response to norepinephrine 0.2 micrograms (P less than 0.05) but not to 0.5 micrograms. To determine whether alpha 2 receptors are indeed involved in the ACTH response to nicotine, yohimbine, an alpha 2 antagonist, was injected into the third ventricle before nicotine injection into the IV. Yohimbine significantly reduced the ACTH response. Thus, the secretion of both hypothalamic epinephrine and, to some extent, norepinephrine is involved in the ACTH response to the activation of catecholaminergic neurons in the IV. Further investigation of the adrenergic receptor(s) involved compared the ACTH response to nicotine (1 microgram) instilled into the IV after prazocin (alpha 1 antagonist), or propranolol (beta antagonist) was injected into the third ventricle. Prazocin significantly reduced (P less than 0.05) the ACTH response, whereas propranolol was ineffective. Thus, both alpha 1 and alpha 2 receptors are involved in mediating the ACTH response to nicotine.

Beta-endorphin enhances Concanavalin-A-stimulated calcium mobilization by murine splenic T cells.

Intracellular calcium mobilization is an important early event involved in T cell activation. The endogenous opioid peptide beta-endorphin is known to modulate immune functions that depend on T cell activation, therefore its effect on intracellular calcium mobilization was investigated. The intracellular calcium concentration ([Ca2+]i) of T cell-enriched splenocytes was measured by flow cytofluorometric analysis using the calcium-sensitive dye, Fluo-3. By gating on the T cell marker, Thy-1, a 95%-pure population of T cells was identified for study. Cells preincubated with beta-endorphin showed significantly enhanced [Ca2+]i responses to the mitogen, Concanavalin A (Con A). This was detectable with concentrations of beta-endorphin as low as 10(-13) M; maximal enhancement required 10(-10) to 10(-9) M doses. The efficacy of beta-endorphin was dependent on the duration of pretreatment. beta-Endorphin amplified the Con A-induced increase in [Ca2+]i by reducing the lag time for the response to Con A and by increasing the mean [Ca2+]i of the cells. N-Ac-beta-endorphin, which shows minimal potency at neuronal opiate receptors, was unable to substitute for beta-endorphin. Naltrindole, a highly selective delta opiate receptor antagonist, inhibited the action of beta-endorphin, whereas a selective mu opiate receptor antagonist was ineffective. Although less potent than beta-endorphin, the delta opiate receptor agonist D-Ala2-D-Leu5-enkephalin also significantly enhanced [Ca2+]i responses. In summary, concentrations of beta-endorphin, within the physiological range found in the systemic circulation, modulate the increase in T cell [Ca2+]i induced by Con A. Both the efficacy of D-Ala2-D-Leu5-enkephalin alone and the antagonism of beta-endorphin by naltrindole suggest that a delta-type opiate receptor may mediate these effects.

Nicotine-induced cFos expression in the hypothalamic paraventricular nucleus is dependent on brainstem effects: correlations with cFos in catecholaminergic and noncatecholaminergic neurons in the nucleus tractus solitarius.

Systemically administered nicotine elicits ACTH release indirectly by acting on neurons in brainstem catecholaminergic regions known to send afferent projections to the paraventricular nucleus of the hypothalamus (PVN), the site of CRH neurons involved in initiating ACTH secretion. The present study in rats examined 1) the relationship between dose-dependent expression of cFos in the PVN and that in the nucleus of the solitary tract (NTS)-A2, NTS-C2 and locus coeruleus (LC), after iv nicotine (0.045-0.18 mg/kg, administered at 0.09 mg/kg per min); 2) the dependence of PVN cFos expression on the effects of nicotine in brainstem, using the nicotinic cholinergic antagonist, mecamylamine, administered into the fourth ventricle; and 3) the extent of catecholaminergic involvement in the effect of nicotine on the PVN, measured by immunocytochemical double-labeling for cFos and tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis. The results showed that the magnitude of cFos expression was dependent on the dose of nicotine in all regions studied (P < 0.0006); however, at the two lowest doses, only the NTS and CRH-containing region of the PVN expressed cFos, whereas the LC and the rest of the PVN were activated only by higher doses. Nicotine also elicited a dose-dependent increase in cFos expression in the TH+ neurons of the NTS, with C2 more sensitive than A2. Interestingly, the majority of NTS neurons expressing cFos were noncatecholaminergic, implicating other transmitter systems. Fourth ventricular mecamylamine completely blocked nicotine-induced cFos expression throughout the NTS, as well as the PVN. The results provide further support for the idea that catecholaminergic afferents from the NTS, but not the LC, play a significant, albeit not an exclusive, role in the activation of the PVN in response to nicotine.

Protein kinase A regulates nicotinic cholinergic receptors and subunit messenger ribonucleic acids in PC 12 cells.

To delineate mechanisms regulating the expression of neuronal nicotinic cholinergic receptors (nAcChRs), we studied the cAMP-dependent second messenger system. PC 12 cells were grown in (Bu)2cAMP (0.001-1.0 mM) or vehicle for 7 days, and specific [3H] nicotine binding was measured. (Bu)2cAMP (0.1 mM) increased specific binding 2- and 4-fold at 3 and 7 days, respectively, whereas 1.0 mM enhanced binding 4-fold at both time intervals. Cells grown in 8-bromo-cAMP (1.0 mM) showed a 2-fold increase in [3H]nicotine binding at 3 days. Forskolin (10-100 microM), in combination with isobutyl-methylxanthine (1.0 mM), enhanced [3H]nicotine binding 2- to 3-fold at 7 days; forskolin or isobutyl-methylxanthine alone had no effect. Specific [3H] nicotine binding to PC 12 cell mutants (A126.1B2 and A123.7), deficient in cAMP-responsive protein kinase A types I and II, were unaffected by (Bu)2cAMP. Northern gel analysis of nAcChR subunit messenger RNAs showed that the alpha-3, alpha-5, and beta-4 subunits were significantly decreased by (Bu)2cAMP at 4 h. However, (Bu)2cAMP caused an increase in the beta-2 messenger RNA transcript at 4 h, which returned to baseline by 24 h. These studies indicate that the cAMP-protein kinase A system regulates expression of nAcChR by PC 12 cells. These studies also suggest that enhancement of [3H]nicotine binding by activated protein kinase A may not involve synthesis of new receptor subunit proteins.

Induction of the messenger ribonucleic acid for proenkephalin A in cultured murine CD4-positive thymocytes.

Although proenkephalin A (PEA) messenger RNA (mRNA) has been detected in many types of immune cells, little understanding exists about its role or the role of enkephalin peptides in immune responses. We have studied the expression of PEA mRNA during thymocyte maturation by identifying the subpopulation of thymocytes that expresses PEA mRNA. PEA mRNA was induced in unfractionated murine thymocytes after in vitro activation of these cells with the T cell mitogen, concanavalin A (Con-A). A slight induction of PEA mRNA was seen after 48 h of Con-A stimulation; however, the maximal response occurred after 72 h of culture with Con-A. Two PEA mRNA bands were present in unfractionated thymocytes which had been cultured with Con-A for 48 and 72 h. The predominant band was 1.4 kilobases (kb), and a second band was approximately 1.7 kb. Fractionation of thymocytes into CD4, CD8, and double negative subpopulations showed that only the 1.4 kb PEA mRNA was inducible in the mature CD4 subpopulation. Induction required the presence of antigen-presenting cells in addition to CD4 thymocytes. Neither the 1.4 kb nor the 1.7 kb PEA mRNA was induced in the CD8 or double negative subpopulations. In contrast to the action of Con-A on murine thymocytes, PEA mRNA was not induced by this mitogen in murine splenic mononuclear cells at 24, 48, or 72 h. The regulated expression of PEA mRNA in murine thymocytes, but not in peripheral T lymphocytes, suggests a role for PEA mRNA and its peptides in thymocyte maturation.

Adrenocorticotropin response and nicotine-induced norepinephrine secretion in the rat paraventricular nucleus are mediated through brainstem receptors.

Nicotine is a potent stimulus for the secretion of ACTH, and norepinephrinergic neurons originating in the brainstem are involved. Prior reports using in vivo microdialysis in alert rats have shown that nicotine, administered i.p. or into the fourth ventricle, stimulated the release of norepinephrine (NE) into the hypothalamic paraventricular nucleus (PVN), the site of neurons containing CRH. In the present studies, rats received an i.v. infusion of nicotine into the jugular vein on alternate days during their active (dark) phase; therefore, direct correlations between the levels of NE microdialyzed from the PVN and plasma ACTH could be made in each animal. Nicotine administered i.v. (0.045-0.135 mg/kg) elicited dose-dependent increases in both NE and ACTH (P < 0.01). A significant correlation was found between nicotine-stimulated NE release in the PVN and ACTH secretion (r = 0.91, P < 0.01). To address whether the site(s) of action of nicotine was on presynaptic receptors on NE terminals in the PVN or on receptors on neurons in brainstem regions accessible from the fourth ventricle, the nicotinic cholinergic antagonist, mecamylamine (0.1-4.8 microg), was microinjected directly into the PVN or into the fourth ventricle before nicotine infusion. Fourth-ventricular administration of mecamylamine (1.6 microg) or higher, before i.v. nicotine (0.09 mg/kg), completely blocked both NE release in the PVN (IC50 = 0.64 microg) and ACTH secretion (IC50 = 0.40 microg) (P < 0.01, compared with vehicle before nicotine), whereas it was ineffective when injected directly into the PVN. The results demonstrate that the nicotinic cholinergic receptors in the brainstem, rather than presynaptic receptors within the PVN itself, mediate nicotine-stimulated PVN NE release and ACTH secretion.

The adrenocorticotropin response to interleukin-1 beta instilled into the rat median eminence depends on the local release of catecholamines.

Interleukin-1 beta (IL-1 beta) is a potent ACTH secretagogue which activates the release of hypothalamic CRF but is unable to cross the blood brain barrier. Recently, it was reported that IL-1 beta, instilled directly into the hypothalamic median eminence (ME), rapidly induced ACTH secretion. Thus, due to the lack of a blood brain barrier the ME appears to be a site whereby iv IL-1 beta can access the brain to stimulate CRF and, consequently, ACTH secretion. To evaluate the role of central catecholamines in this process, 250 g male rats were lesioned with 6-hydroxy-dopamine instilled into the lateral ventricle. Ten days later, rats received recombinant h-IL-1 beta (10 or 30 ng) into the ME and blood was sampled via indwelling jugular cannulae. The ACTH response to IL-1 beta, 30 ng, was reduced by approximately 50% in the lesioned rats (P = 0.05), and it was abolished in those receiving IL-1 beta, 10 ng (P less than 0.05). Moreover, in rats given iv IL-1 beta (1 microgram), 6-hydroxy-dopamine significantly reduced the ACTH response by more than 50% (P less than 0.001). To determine the effect of acute epinephrine depletion, 2,3 dichloro-alpha-methylbenzylamine (DCMB, 60 mg/kg BW) and SKF64139 (SKF) (100 mg/kg BW), inhibitors of the enzyme which converts norepinephrine to epinephrine, were administered ip 4 h before intra-ME IL-1 beta (30 ng). DCMB reduced the ACTH response by 80% and SKF reduced it to its own baseline. LY 10853, which acutely depletes both norepinephrine and epinephrine, also reduced the ACTH response by 80%. Because of the reported capacities of DCMB and SKF to block alpha 2 adrenoreceptors in vitro, yohimbine, an alpha 2 receptor antagonist, was studied. Intra-ME yohimbine failed to inhibit the ACTH response to intra-ME IL-1 beta. In contrast, a significant (P less than 0.01) dose-dependent reduction in the ACTH response to intra-ME IL-1 beta (30 ng) was observed in rats pretreated with either a nonselective alpha or beta adrenoreceptor antagonist (phentolamine, 2-40 micrograms or propranolol, 2-20 micrograms, respectively, into the ME). In contrast, intra-ME phentolamine, 20 micrograms, failed to reduce the ACTH response to iv CRF, 1 microgram/kg BW. Thus, the secretion of ACTH stimulated by the action of IL-1 beta at the ME depends, in part, on the local secretion of norepinephrine and epinephrine interacting with both alpha and beta adrenergic receptors.

Murine splenocytes express a naloxone-insensitive binding site for beta-endorphin.

Naloxone-resistant binding sites for beta-endorphin have previously been observed on transformed peripheral blood mononuclear cells and on the EL4-thymoma cell line. These sites may be related to the naloxone-insensitive immunomodulatory effects of beta-endorphin. The present study was performed 1) to determine whether these sites are present on normal splenocytes and 2) to characterize them. Ficoll-hypaque-purified murine splenocytes were used in a RRA with [125I]beta-endorphin. Neither fresh intact cells obtained from viral antibody-free mice nor membrane preparations showed evidence of binding. However, splenocytes cultured in 5% fetal bovine serum for 24-96 h showed sites on intact cells or membranes (after 3 h in culture no sites were present). Intact cultured splenocytes demonstrated a saturable binding isotherm, and Scatchard analysis showed a single site (Kd = 4.1 X 10(-9) M). Competition studies showed that N-acetyl-beta-endorphin (N-Ac-beta-endorphin)-(1-31) was equipotent to beta-endorphin-(1-31). beta-Endorphin-(6-31) and beta-endorphin-(28-31) were approximately 10- and 1000-fold less potent, respectively, whereas beta-endorphin-(1-27) and naloxone were completely ineffective. Covalent cross-linking of [125I]beta-endorphin to splenocytes and resolution by gel electrophoresis showed bands at 66K and 57K which were displaced equipotently by increasing amounts of beta-endorphin and N-Ac-beta-endorphin. beta-Endorphin-(18-31) or (28-31) were less potent, and naloxone or other opioid ligands selective for receptor subtypes were ineffective. Thus, high affinity, naloxone-insensitive binding sites for beta-endorphin, which show competition characteristics distinctively different from brain opiate receptors, are inducible on normal mouse splenocytes. N-Ac-beta-endorphin, presumed to be an inactivation product of beta-endorphin because it fails to bind brain opiate receptors, may be functional at this naloxone-insensitive binding site.

Regulation of the messenger ribonucleic acid for corticotropin-releasing factor in the paraventricular nucleus and other brain sites of the rat.

CRF, from the paraventricular nucleus of the hypothalamus (PVN), is the major hypothalamic releasing factor that controls pituitary ACTH. Recently, the mRNA for CRF and the CRF peptide have been detected in other brain sites. However, there is little information on the function and regulation of CRF in brain sites outside the paraventricular nucleus. We investigated the content of CRF mRNA in the PVN, the central nucleus of the amygdala (CN), the bed nucleus of the stria terminalis (BN), and the supraoptic nucleus (SON). Northern gel analysis showed that the mRNA for CRF is present in the BN, CN, and SON as well as the PVN, and that all are the same size. In response to adrenalectomy, the level of hybridizable mRNA increased 2.75-fold over 7 days in the PVN; there was no change in the CN, BN, or SON. High dose dexamethasone decreased, but did not eliminate, the PVN CRF mRNA; it was without effect in the other sites. Glucocorticoid replacement with constant low blood levels of corticosterone (5.6 +/- 0.3 micrograms/100 ml) suppressed plasma ACTH and decreased thymus weight while reducing, but not eliminating, PVN CRF mRNA. We conclude that the same sized mRNA for CRF is synthesized in the PVN, BN, CN, and SON, but only the PVN mRNA responds to alterations of peripheral glucocorticoid status. This may imply that only CRF from the PVN is involved in control of the hypothalamic-pituitary-adrenal axis.

Nicotine stimulates the expression of cFos protein in the parvocellular paraventricular nucleus and brainstem catecholaminergic regions.

The rapid secretion of ACTH in response to nicotine is mediated by a central mechanism involving brainstem catecholaminergic regions. To identify specific brainstem regions involved in activating the hypothalamo-pituitary-adrenal axis and other areas of the brain by iv nicotine, immunocytochemical detection of cFos protein was used as a marker for neuronal activation. Nicotine (0.05 mg/kg) stimulated cFos expression in the parvocellular paraventricular nucleus (pcPVN; containing CRH-positive neurons mediating ACTH secretion); this correlated with the expression of cFos in the A2 (norepinephrinergic) and C2 (epinephrinergic) regions of the brainstem nucleus tractus solitarius, which project directly to the pcPVN. The selectivity of this brainstem activation was shown by the absence of responses in the locus coeruleus (LC), A1, and C1 catecholaminergic regions to this low dose of nicotine. In contrast, a high dose of nicotine (0.1 mg/kg), which produced a brief episode of tremor, was required for expression of cFos in the LC. This was associated with a further increase in the number of cFos-positive cells in the PVN, primarily through recruitment in the magnocellular region, a known projection field of LC. The higher dose of nicotine also induced cFos in the vasopressinergic region of the supraoptic nucleus (SON), whereas the lower dose of nicotine exclusively induced cFos in the oxytocinergic region of the SON. Limbic regions that receive catecholaminergic inputs, such as the the central nucleus of the amygdala (involved in PVN regulation) and the cingulate gyrus of the cortex, showed a dose-dependent increase in the number of cFos-positive cells after nicotine, whereas the dentate gyrus of the hippocampus only responded to the high dose. Thus, nicotine is a potent and selective stimulus for neuronal activation in brainstem catecholaminergic regions and their projection fields in the pcPVN and SON, which regulate the hypothalamo-pituitary-adrenal axis and vasopressin/oxytocin secretion, respectively.

Expression and function of proenkephalin A messenger ribonucleic acid in murine fetal thymocytes.

In recent years we examined the function of the endogenous enkephalins encoded by PEA messenger RNA (mRNA) expressed in murine thymocytes, the precursor cells to T lymphocytes, which are the primary effector cells in cell- mediated immune responses. In the present study, we examined the expression and function of PEA mRNA and enkephalins in fetal thymocytes. By Northern gel and in situ hybridization techniques, we show that PEA mRNA is constitutively expressed in fetal thymocytes early in gestation, with maximal expression occurring on day 15. By birth, PEA mRNA is no longer constitutively expressed, but can be induced by culturing newborn thymocytes with the T cell-specific mitogen, Concanavalin-A. Both a delta-opioid receptor antagonist, naltrindole, and a PEA mRNA-specific antisense complementary DNA enhance the spontaneous proliferation of day 15, but not day 14, fetal thymocytes, consistent with the observation that PEA mRNA is expressed in thymocytes on day 15, but not day 14, of gestation. The enhanced proliferation of day 15 fetal thymocytes is reversed by a delta-opioid receptor agonist, deltorphin. The data suggest that endogenous enkephalins encoded by PEA mRNA expressed in day 15 fetal thymocytes act to inhibit the spontaneous proliferation of these cells, perhaps so that their differentiation into mature T lymphocytes can occur.

Selective administration of nicotine into catecholaminergic regions of rat brainstem stimulates adrenocorticotropin secretion.

Nicotine (Nic) is a potent stimulus for ACTH secretion, and this response appears to be mediated by central catecholamine secretion. We have previously shown that fourth ventricular administration of Nic rapidly elevated plasma ACTH levels, that a nicotinic cholinergic antagonist, mecamylamine, instilled into the fourth ventricle inhibited the ACTH response to iv Nic, and that Nic stimulated norepinephrine secretion in the hypothalamic paraventricular nucleus. Thus, the present investigations sought to identify Nic-responsive regions in the brainstem that give rise to ascending catecholaminergic afferents resulting in ACTH secretion. Chronic brain and jugular cannulae were implanted, and Nic (50 nl over 30 sec) was infused into the locus coeruleus (LC), nucleus of the solitary tract (NTS -C2 or -A2 regions), C1, or A1 cell regions of freely moving, adult male rats. Injection of Nic (free base, 0.25-10 micrograms) into either the C2 or A2 region of NTS resulted in a dose-dependent increase in plasma ACTH. In contrast, C1 was unresponsive and A1 only showed responses to the highest doses of Nic (5 or 10 micrograms). In LC, Nic in doses of 2.5 micrograms or higher was required to elevate plasma ACTH. This dose is approximately 10-fold greater than that required in NTS-A2. Finally, mecamylamine (0.25 mg/kg body wt, iv), administered 2 min before Nic, abolished the ACTH responses in both C2 and A2 and significantly reduced the 7-min peak ACTH response in LC (P < 0.05). In summary, microinjection of Nic selectively activated the brainstem regions under investigation, with a rank order of sensitivity to Nic that was NTS-A2 > NTS-C2 > LC > A1 > C1 = cerebrospinal fluid. Therefore, systemically administered Nic appears to activate multiple catecholaminergic brainstem regions that are involved in mediating ACTH secretion.

Opioid peptides rapidly stimulate superoxide production by human polymorphonuclear leukocytes and macrophages.

Opioid peptides found in the general circulation can modulate several functions of phagocytic cells that are related to their microbicidal and cytotoxic activity. Since reactive oxygen species are crucial to these activities, the affect of opioid peptides on superoxide (O-2) generation was evaluated with the use of lucigenin-enhanced chemiluminesence (CL). beta-Endorphin and dynorphin stimulate the production of O-2 in human polymorphonuclear leukocytes (PMN) and peritoneal macrophages (PMO) at peptide concentrations that prevail systemically (10(-14)-10(-12)M). There is an inverse dose-response relation for PMN but not PMO. The effect is rapid and sustained in PMN (peak CL at 2-4 min, duration greater than 15 min), whereas it is rapid but brief in PMO (peak 1 min, duration less than 3 min). Naloxone inhibits CL responses by greater than 75% in both cell types.