Joseba Pineda

Somatodendritic α2-Adrenoceptors in the locus coeruleus are involved in the in vivo modulation of cortical noradrenaline release by the antidepressant desipramine

Abstract: The effect of the antidepressant and selective noradrenaline reuptake blocker desipramine (DMI) on noradrenergic transmission was evaluated in vivo by dual‐probe microdialysis. DMI (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), DMI (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 α2‐adrenoceptor antagonist RX821002 (1 µM) was perfused in the LC, DMI (1 mg/kg, i.p.) no longer decreased but rather increased NA dialysate in the Cg. In electrophysiological experiments, DMI (1 mg/kg, i.p.) inhibited the firing activity of LC neurons by a mechanism reversed by RX821002. Local DMI (0.01–100 µM) into the LC increased concentration‐dependently NA levels in the LC and simultaneously decreased NA levels in the Cg. This decrease was abolished by local RX821002 administration into the LC. The results demonstrate in vivo that DMI inhibits NA reuptake at somatodendritic and nerve terminal levels of noradrenergic cells. The increased NA dialysate in the LC inhibits noradrenergic activity, which in part counteracts the effects of DMI on the Cg. The modulation of cortical NA release by activity of DMI at the somatodendritic level is mediated through α2‐adrenoceptors located in the LC.

Modulation of Anxiety-Like Behavior and Morphine Dependence in CREB-Deficient Mice

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 (Creb1NesCre). A genetically defined cohort of the previously described hypomorphic Creb1αΔ mice, in which the two major transcriptionally active isoforms (α and Δ) 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.

Neuroprotection by GDNF-secreting stem cells in a Huntington's disease model: optical neuroimage tracking of brain-grafted cells

The use of stem cells for reconstructive or neuroprotective strategies can benefit from new advances in neuroimaging techniques to track grafted cells. In the present work, we analyze the potential of a neural stem cell (NSC) line, which stably expresses the glial cell line-derived neurotrophic factor (GDNF) and the firefly luciferase gene (GDNF/Luc-NSC), for cell therapy in a Huntingtons disease mouse model. Our results show that detection of light photons is an effective method to quantify the proliferation rate and to characterize the migration pathways of transplanted NSCs. Intravenous administration of luciferine, the luciferase substract, into the grafted animals allowed the detection of implanted cells in real time by an optical neuroimaging methodology, overpassing the limits of serial histological analyses. We observed that transplanted GDNF/Luc-NSCs survive after grafting and expand more when transplanted in quinolinate-lesioned nude mouse striata than when transplanted in non-lesioned mice. We also demonstrate that GDNF/Luc-NSCs prevent the degeneration of striatal neurons in the excitotoxic mouse model of Huntingtons disease and reduce the amphetamine-induced rotational behavior in mice bearing unilateral lesions.

Stimulatory effects of clonidine, cirazoline and rilmenidine on locus coeruleus noradrenergic neurones: possible involvement of imidazoline-preferring receptors.

SummaryClonidine and related drugs not only interact with α2-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 α2-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 μg/kg, i.v.) (ED50 = 702 μg/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 μg/kg) and rilmenidine (0.3–10 mg/kg) also stimulatedneuronal activity(ED50 = 192 μg/kg, Emax = 102%, n = 5; ED50 = 1563 μg/kg, Emax = 70%, n = 1–5, respectively) by an α2-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.

Inhibition of 5-hydroxytryptamine reuptake by the antidepressant citalopram in the locus coeruleus modulates the rat brain noradrenergic transmission in vivo

The in vivo effect of the serotonin (5-HT) reuptake inhibitor antidepressant citalopram, administered in the locus coeruleus (LC), on noradrenergic transmission was evaluated in the rat brain. In dual-probe microdialysis assays, citalopram (0.1-100 microM), in a concentration-dependent manner, increased extracellular noradrenaline (NA) in the LC and simultaneously decreased extracellular NA in the cingulate cortex (Cg). These effects of citalopram were abolished by pretreatment with the 5-HT synthesis inhibitor p-chlorophenylalanine (400 mg/kg, i.p.). When the alpha(2)-adrenoceptor antagonist RS79948 (1 microM) was perfused in the LC, local citalopram increased NA dialysate in the LC but no longer modified NA dialysate in the Cg. In electrophysiological experiments, the administration of citalopram (100 microM) in the LC by reversal dialysis, decreased the firing rate of LC neurones. The results demonstrate in vivo that local administration of citalopram in the LC leads to a decreased release of NA in the Cg. This modulation seems to be the result of an increase in NA concentration in the LC and the subsequent inhibition of LC neurones via alpha(2)-adrenoceptors. The effects of citalopram are dependent on the presence of endogenous 5-HT in the LC.

Attenuation of withdrawal-induced hyperactivity of locus coeruleus neurones by inhibitors of nitric oxide synthase in morphine-dependent rats

Electrophysiological, biochemical, and behavioural studies have suggested that opiate withdrawal is mediated, at least in part, by a hyperactivity of locus coeruleus (LC) neurones. The aim of this study was to evaluate, using single-unit extracellular recordings, the role of NO in the opiate withdrawal-induced hyperactivity of LC neurones in anaesthetized rats. In animals chronically treated with morphine (5 days), administration of naloxone caused an increase in the spontaneous firing rate of LC cells. Acute pretreatment with the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (30 mg kg(-1) i.p.) attenuated some signs of opiate withdrawal (total score reduced by 55%), and also the withdrawal-induced hyperactivity of LC neurones (hyperactivity reduced by approximately 50%). Acute pretreatment with 7-nitro indazole (50 mg kg(-1) i.p.), a selective inhibitor of neuronal NOS, caused a complete blockade of the withdrawal-induced hyperactivity of LC neurones. Application of 7-nitro indazole (30 microM) in the vicinity of the LC also caused a reduction (of approximately 60%) in the withdrawal-induced hyperactivity of LC cells. Intravenous administration of these NOS inhibitors (after naloxone challenge) did not produce comparable changes in the LC cell firing activity. 7-Nitro indazole failed to affect the development of tolerance of the LC to the morphine effect in opiate-dependent rats (i.e. morphine dose-effect curves were shifted to the right by morphine treatments to a similar degree in vehicle- and 7-nitro indazole-pretreated rats). The present data suggest that opiate withdrawal might be mediated by nitric oxide acting as an intermediate messenger in the LC.

Attenuation of acute and chronic effects of morphine by the imidazoline receptor ligand 2-(2-benzofuranyl)-2-imidazoline in rat locus coeruleus neurons

The aim of this study was to determine if 2‐(2‐benzofuranyl)‐2‐imidazoline (2‐BFI) interacts with the opioid system in the rat locus coeruleus, using single‐unit extracellular recordings. In morphine‐dependent rats, acute administration of the selective imidazoline receptor ligands 2‐BFI (10 and 40 mg kg−1, i.p. and 100 μg, i.c.v.) or valldemossine (10 mg kg−1, i.p.) did not modify the naloxone‐induced hyperactivity of locus coeruleus neurons compared with that observed in the morphine‐dependent control group. After chronic administration of 2‐BFI (10 mg kg−1, i.p., three times daily, for 5 days) and morphine, naloxone‐induced hyperactivity and tolerance to morphine were attenuated. This effect was not observed when a lower dose of 2‐BFI (1 mg kg−1, i.p.) or valldemossine (10 mg kg−1, i.p.) were used. Acute administration of 2‐BFI (10 and 40 mg kg−1, i.p. and 100 μg, i.c.v.) but not valldemossine (40 mg kg−1, i.p.) diminished the potency of morphine to inhibit locus coeruleus neuron activity in vivo (ED50 values increased by 2.3, 2.9; and 3.1 fold respectively). Similarly, the potency of Met5‐enkephalin to inhibit locus coeruleus neurons was decreased when 2‐BFI (100 μM) was applied to rat brain slices (EC50 increased by 5.6; P<0.05). The present data demonstrate that there is an interaction between 2‐BFI and the opioid system in the locus coeruleus. This interaction leads to an attenuation of both the hyperactivity of locus coeruleus neurons during opiate withdrawal and the development of tolerance to morphine when 2‐BFI is chronically administered. These results suggest that imidazoline drugs may prove to be useful agents for the management of opioid dependence and tolerance.

Cannabinoids enhance N-methyl-d-aspartate-induced excitation of locus coeruleus neurons by CB1 receptors in rat brain slices

We studied the effect of cannabinoids on the activity of N-methyl-d-aspartate (NMDA) receptors in the locus coeruleus from rat brain slices by single-unit extracellular recordings. As expected, NMDA (100 microM) strongly excited (by nine fold) the cell firing activity of the locus coeruleus. Perfusion with the endocannabinoid anandamide (1 and 10 microM) or the anandamide transport inhibitor AM 404 (30 microM) enhanced the NMDA-induced excitation of locus coeruleus neurons. Similarly, the synthetic agonists R(+)-WIN 55212-2 (10 microM) and CP 55940 (30 microM) enhanced the effect of NMDA. In the presence of the CB(1) receptor antagonists SR 141716A (1 microM) or AM 251 (1 microM), the enhancement induced by anandamide (10 microM) was blocked. Our results suggest that cannabinoids modulate the activity of NMDA receptors in the locus coeruleus through CB(1) receptors.

Effect of the CB1 receptor antagonists rimonabant and AM251 on the firing rate of dorsal raphe nucleus neurons in rat brain slices

Background and purpose:  Previous studies have suggested a regulation of 5‐hydroxytryptamine (5‐HT) neurons by the endocannabinoid system. The aim of our work was to examine the effect of two CB1 receptor antagonists, SR141716A (rimonabant, Sanofi‐Synthélabo Recherche, Montpellier, France) and N‐(piperidin‐1‐yl)‐5‐(4‐iodophenyl)‐1‐(2,4‐dichlorophenyl)‐4‐methyl‐1H‐pyrazole‐3‐carboxamide (AM251, Tocris Cookson, Bristol, UK), on the firing rate of dorsal raphe nucleus (DRN) neurons.

Comparative study of the effects of desipramine and reboxetine on locus coeruleus neurons in rat brain slices

Several studies have suggested that the locus coeruleus may play an important role in the pathophysiology of depression. The aim of this study was to characterize, using single-unit extracellular recordings, the in vitro effects of the noradrenaline reuptake inhibitors desipramine and reboxetine, on locus coeruleus neurons from control rats and from those chronically treated with desipramine. Bath application of desipramine (1-100 microM) and reboxetine (0.1-10 microM) decreased the firing rate of locus coeruleus neurons in a concentration-dependent manner and the alpha(2)-adrenoceptor antagonist RX 821002 (10 microM) reversed these effects. In addition, reserpine (5 mg/kg, 3 h before the experiment) almost completely blocked the inhibitory effect of desipramine. Both drugs (1 microM desipramine and 0.1 microM reboxetine) potentiated the inhibitory effect of noradrenaline (10 microM). A 7-day treatment with desipramine (3 mg/kg/12 h, i.p.) caused a decrease in sensitivity to the alpha(2)-adrenoceptor agonist bromoxidine (EC(50) increased by 3.3-fold), but not to noradrenaline or reboxetine. In contrast, this treatment potentiated the inhibitory effect of desipramine with respect to control. Moreover, 14-day treatment with desipramine (3 mg/kg/12 h, i.p.) or reboxetine (10 mg/kg/12 h, i.p.) also potentiated the in vitro effect of desipramine without modifying the in vitro effect of reboxetine. These results show that desipramine and reboxetine modulate the activity of locus coeruleus neurons by noradrenaline acting on alpha(2)-adrenoceptors, and reveal that alpha(2)-adrenoceptor-independent mechanisms may also underlie the action of noradrenaline uptake inhibitors.