Chronic morphine administration increases levels of adenylyl cyclase and cAMP-dependent protein kinase (PKA) activity in the locus coeruleus (LC), which contributes to the severalfold activation of LC neurons that occurs during opiate withdrawal. A role for the transcription factor cAMP response elementbinding protein (CREB) in mediating the opiate-induced upregulation of the cAMP pathway has been suggested, but direct evidence is lacking. In the present study, we first demonstrated that the morphine-induced increases in adenylyl cyclase and PKA activity in the LC are associated with selective increases in levels of immunoreactivity of types I and VIII adenylyl cyclase and of the catalytic and type II regulatory subunits of PKA. We next used antisense oligonucleotides directed against CREB to study the role of this transcription factor in mediating these effects. Infusion (5 d) of CREB antisense oligonucleotide directly into the LC significantly reduced levels of CREB immunoreactivity. This effect was sequence-specific and not associated with detectable toxicity. CREB antisense oligonucleotide infusions completely blocked the morphine-induced upregulation of type VIII adenylyl cyclase but not of PKA. The infusions also blocked the morphine-induced upregulation of tyrosine hydroxylase but not of Gi␣, two other proteins induced in the LC by chronic morphine treatment. Electrophysiological studies revealed that intra-LC antisense oligonucleotide infusions completely prevented the morphine-induced increase in spontaneous firing rates of LC neurons in brain slices. This blockade was completely reversed by addition of 8-bromo-cAMP (which activates PKA) but not by addition of forskolin (which activates adenylyl cyclase). Intra-LC infusions of CREB antisense oligonucleotide also reduced the development of physical dependence to opiates, based on attenuation of opiate withdrawal. Together, these findings provide the first direct evidence that CREB mediates the morphine-induced upregulation of specific components of the cAMP pathway in the LC that contribute to physical opiate dependence.Key words: morphine; opiate withdrawal; gene expression; cAMP; adenylyl cyclase; protein kinase A; G-proteins; tyrosine hydroxylase; protein phosphorylationThe locus coeruleus (LC) has served as a usef ul model system in which to study the long-term actions of opiates on target neurons. The LC is the major noradrenergic nucleus in brain, located on the floor of the fourth ventricle in the anterior pons (Dahlstrom and Fuxe, 1965;Foote et al., 1983;Aston-Jones et al., 1996). Under normal conditions, the LC is implicated in controlling attention, vigilance, and activity of the autonomic nervous system. The LC also has been implicated in physical opiate dependence. Whereas acute opiate administration inhibits the activity of LC neurons, their firing rates recover toward control levels after chronic exposure and increase more than fourfold above control levels on administration of an opioid receptor antagonist in vivo (Aghajanian, 1978;Rasmuss...

No abstract

The transcription factor cAMP-responsive element binding protein (CREB) has been shown to regulate different physiological responses including drug addiction and emotional behavior. Molecular changes including adaptive modifications of the transcription factor CREB are produced during drug dependence in many regions of the brain, including the locus coeruleus (LC), but the molecular mechanisms involving CREB within these regions have remained controversial. To further investigate the involvement of CREB in emotional behavior, drug reward and opioid physical dependence, we used two independently generated CREB-deficient mice. We employed the Cre/loxP system to generate mice with a conditional CREB mutation restricted to the nervous system, where all CREB isoforms are lacking in the brain (Creb1 NesCre ). A genetically defined cohort of the previously described hypomorphic Creb1 aD mice, in which the two major transcriptionally active isoforms (a and D) are disrupted throughout the organism, were also used. First, we investigated the responses to stress of the CREB-deficient mice in several paradigms, and we found an increased anxiogenic-like response in the both Creb1 mutant mice in different behavioral models. We investigated the rewarding properties of drugs of abuse (cocaine and morphine) and natural reward (food) using the conditioned place-preference paradigm. No modification of motivational responses of morphine, cocaine, or food was observed in mutant mice. Finally, we evaluated opioid dependence by measuring the behavioral expression of morphine withdrawal and electrophysiological recordings of LC neurons. We showed an important attenuation of the behavioral expression of abstinence and a decrease in the hyperactivity of LC neurons in both Creb1 mutant mice. Our results emphasize the selective role played by neuronal CREB in emotional-like behavior and the somatic expression morphine withdrawal, without participating in the rewarding properties induced by morphine and cocaine.

The effect of the antidepressant and selective noradrenaline reuptake blocker desipramine (DM1) on noradrenergic transmission was evaluated in vivo by dual-probe microdialysis. DM1 (1, 3, and 10 mg/kg, i.p.) dose-dependently increased extracellular levels of noradrenaline (NA) in the locus coeruleus (LC) area. In the cingulate cortex (Cg), DM1 (3 and 10 mg/kg, i.p.) also increased NA dialysate, but at the lowest dose (1 mg/kg, i.p.) it decreased NA levels. When the s2-adrenoceptor antagonist RX821002 (1 1iM) was perfused in the LC, DM1 (1 mg/kg, i.p.) no longer decreased but rather increased NA dialysate in the 0g. In electrophysiological experiments, DM1 (1 mg/kg, i.p.) inhibited the firing activity of LC neurons by a mechanism reversed by RX821 002. Local DM1 (0.01-100 ,uM) into the LC increased concentration-dependently NA levels in the LC and simultaneously decreased NA levels in the 0g. This decrease was abolished by local RX821 002 administration into the LC. The results demonstrate in vivo that DM1 inhibits NA reuptake at somatodendritic and nerve terminal levels of noradrenergic cells. The increased NA dialysate in the LO inhibits noradrenergic activity, which in part counteracts the effects of DM1 on the 0g. The modulation of cortical NA release by activity of DM1 at the somatodendritic level is mediated through cn2-adrenoceptors located in the LO.

Nitric oxide (NO) and carbon monoxide (CO) have been identified as two diffusible signaling messengers in the brain, capable of stimulating soluble guanylate cyclase. Locus coeruleus (LC) is rich in the alpha 1 and beta 1 subunits of soluble guanylate cyclase. Therefore, the possible role of the cGMP pathway in the regulation of LC neurons was investigated with electrophysiological techniques in rat brain slices. Bath application of various NO donors or CO-containing solutions increased the firing rate of most LC neurons. This activation was reversed by the NO scavenger hemoglobin, but not by methemoglobin. Bath or intracellular application of selective activators of cGMP-dependent protein kinase also caused increases in LC cell firing rate. The actions of NO donors and kinase activators were mutually occlusive and reversed by H8, an inhibitor of the cGMP-dependent protein kinase. Hemoglobin and H8 reduced the firing rate of LC neurons, but no change was found with inhibitors or activators of the NO synthase. In intracellular and whole-cell recordings, NO effect was associated with an inward current and an increase in the input conductance (mean reversal potential = -27 mV); these effects were abolished using a low-sodium buffer. Spontaneous EPSCs of LC cells were not modified with the NO donor administration. Taken together, these data suggest that NO and CO activate noradrenergic neurons of LC via a cGMP-dependent protein kinase and a nonselective cationic channel. It also is proposed that these effects occur at the postsynaptic level and that there may be a tonic regulation of LC neuronal firing by the cGMP pathway.

Seventeen patients with papillary thyroid cancer whose serum thyroglobulin (Tg) levels were elevated when hypothyroid, but whose diagnostic whole body scans were negative, were treated with 150-300 mCi 131I. All patients had total thyroidectomy and 131I ablation for thyroid remnants. Before the study, 9 patients had 131I therapy for tumor recurrence and/or metastases, and 5 patients had excisions of nonfunctioning metastasis. Radiological studies did not reveal evidence of metastases. In the initial evaluation, Tg levels ranged from 8-480 ng/mL (24 pmol/L to 1.5 nmol/L), and posttherapy whole body scans (RxWBS) revealed undiagnosed local recurrence and/or metastases in 16 of 17 patients. Follow-up from 6 months to 5 yr is available in 16 patients. RxWBS after a second treatment was positive in 8 of 13 patients, and after a third treatment in 5 of 5 patients, although in 3 cases, uptake in distant metastasis had disappeared. In 8 patients, Tg fell to 5 ng/mL or less. In 1 patient, RxWBS became negative, but Tg remained elevated; subsequent treatment revealed local and mediastinal uptake, but previous lung uptake had disappeared. In 8 patients, RxWBS remains positive, and elevated Tg persists. A total of 35 RxWBS were performed; 29 were positive. Follow-up Tg concentrations decreased in 81% of patients after the first treatment, in 90% after the second treatment, and in 100% of the patients after the third treatment. Tg (mean +/- SE) decreased from 74 +/- 33 ng/mL in the first evaluation to 62 +/- 32 ng/mL in the second study and 32 +/- 20 ng/mL in the third study. The therapeutic effectiveness of 131I treatment in patients with elevated Tg and negative diagnostic whole body scans is indicated by the conversion to negative RxWBS, the statistically significant decrease in the mean Tg level, and the reduction of serum Tg to 5 ng/mL or less in 50% of patients. Further experience with this therapeutic approach is required to evaluate its effectiveness in improving prognosis and survival.

Clonidine and related drugs not only interact with alpha 2-adrenoceptors but also recognise non-adrenoceptor sites in the brain. The involvement of these imidazoline-preferring receptors in the regulation of the activity of locus coeruleus noradrenergic neurones (NA-LC) was investigated after inactivation of alpha 2-adrenoceptors with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). In EEDQ-pretreated rats (6 mg/kg, i.p., 6 h), the characteristic inhibitory effect of low doses of clonidine on these neurones was abolished and a paradoxical, dose-dependent increase in firing rate was observed at higher doses (640-5120 micrograms/kg, i.v.) (ED50 = 702 micrograms/kg, Emax = 83%, n = 14). Guanfacine (0.3-20 mg/kg) did not modify neuronal activity but antagonised the stimulatory effect of clonidine. Cirazoline (80-640 micrograms/kg) and rilmenidine (0.3-10 mg/kg) also stimulated neuronal activity (ED50 = 192 micrograms/kg, Emax = 102%, n = 5; ED50 = 1563 micrograms/kg, Emax = 70%, n = 1-5, respectively) by an alpha 2-adrenoceptor-independent mechanism. The results suggest that these drugs can modulate the activity of locus coeruleus noradrenergic neurones through the activation of I1-imidazoline-preferring receptors.

No abstract