Luisa Ugedo

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.

Control of serotonergic neurons in rat brain by dopaminergic receptors outside the dorsal raphe nucleus.

We studied the control of dorsal raphe (DR) serotonergic neurons by dopaminergic transmission in rat brain using microdialysis and single unit extracellular recordings. Apomorphine (0.5–3.0 mg/kg s.c.) and quinpirole (0.5 mg/kg s.c.) increased serotonin (5‐HT) output in the DR and (only apomorphine) in striatum. These effects were antagonized by 0.3 mg/kg s.c. SCH 23390 (in DR and striatum) and 1 mg/kg s.c. raclopride (in DR). 5‐HT1A receptor blockade potentiated the 5‐HT increase produced by apomorphine in the DR. Apomorphine (50–400 µg/kg i.v.) increased the firing rate of most 5‐HT neurons, an effect prevented by SCH 23390 and raclopride. Quinpirole (40–160 µg/kg i.v.) also enhanced the firing rate of 5‐HT neurons. When applied in the DR, neither drug increased the 5‐HT output in the DR or striatum. Likewise, micropressure injection of quinpirole (0.2–8 pmol) failed to increase the firing rate of 5‐HT neurons. In situ hybridization showed that the dopamine (DA) D2 receptor transcript was almost absent in the DR and abundant in the substantia nigra (SN) and the periaqueductal grey matter (PAG). Using dual probe microdialysis, the application of tetrodotoxin or apomorphine in SN significantly increased the DR 5‐HT output. Thus, the discrepancy between local and systemic effects of dopaminergic agonists and the absence of DA D2 receptor transcript in 5‐HT neurons suggest that DA D2 receptors outside the DR control serotonergic activity.

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.

A Comprehensive Analysis of the Effect of DSP4 on the Locus Coeruleus Noradrenergic System in the Rat

Degeneration of the noradrenergic neurons in the locus coeruleus (LC) is a major component of Alzheimers (AD) and Parkinsons disease (PD), but the consequence of noradrenergic neuronal loss has different effects on the surviving neurons in the two disorders. Therefore, understanding the consequence of noradrenergic neuronal loss is important in determining the role of this neurotransmitter in these neurodegenerative disorders. The goal of the study was to determine if the neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4) could be used as a model for either (or both) AD or PD. Rats were administered DSP4 and sacrificed 3 days 2 weeks and 3 months later. DSP4-treatment resulted in a rapid, though transient reduction in norepinephrine (NE) and NE transporter (NET) in many brain regions receiving variable innervation from the LC. Alpha(1)-adrenoreceptors binding site concentrations were unchanged in all brain regions at all three time points. However, an increase in alpha(2)-AR was observed in many different brain regions 2 weeks and 3 months after DSP4. These changes observed in forebrain regions occurred without a loss in LC noradrenergic neurons. Expression of synthesizing enzymes or NET did not change in amount of expression/neuron despite the reduction in NE tissue content and NET binding site concentrations at early time points, suggesting no compensatory response. In addition, DSP4 did not affect basal activity of LC at any time point in anesthetized animals, but 2 weeks after DSP4 there is a significant increase in irregular firing of noradrenergic neurons. These data indicate that DSP4 is not a selective LC noradrenergic neurotoxin, but does affect noradrenergic neuron terminals locally, as evident by the changes in transmitter and markers at terminal regions. However, since DSP4 did not result in a loss of noradrenergic neurons, it is not considered an adequate model for noradrenergic neuronal loss observed in AD and PD.

Modulation of brain α2-adrenoceptor and μ-opioid receptor densities during morphine dependence and spontaneous withdrawal in rats

SummaryThe densities of brain α2-adrenoceptors and μ-opioid receptors, quantitated by means of the binding of the agonists [3H]clonidine and [3H]dihydromorphine, respectively, were studied during the development of morphine dependence and spontaneous withdrawal in the rat. The oral administration of morphine (12–130 mg/kg for 3–21 days) led to inconsistent changes in α2-adrenoceptor density while the density of μ-opioid receptors was down-regulated. In contrast, spontaneous opiate withdrawal (3–72 h) significantly increased the density of α2-adrenoceptors while the density of μ-opioid receptors was rapidly up-regulated to control values. In the hypothalamus, but not in other brain regions, the increase in α2-adrenoceptor density after withdrawal followed a time course (3–72 h) related to the severity of the abstinence syndrome. Thus, there was a positive and significant correlation between the severity of withdrawal and the density of α2-adrenoceptors in the hypothalamus. Short-term treatment with clonidine (2 × 0.5 mg/kg, i. p.) prevented the morphine withdrawal-induced increases in α2-adrenoceptor density in various brain regions, but not in the hypothalamus. The main results suggest that modulation of hypothalamic α2-adrenoceptor density during morphine withdrawal is a relevant physiological mechanism by which the opiate abstinence syndrome is counteracted.

Electrophysiological evidence for postsynaptic 5-HT1A receptor control of dorsal raphe 5-HT neurones

Postsynaptic 5-hydroxytryptamine(1A) (5-HT(1A)) receptors have been proposed to participate in the control of dorsal raphe 5-HT neurone activity. To further investigate this hypothesis we performed single-unit extracellular recordings in anaesthetized rats. Pertussis toxin (2 microg/4 microl/day; 2 days, 24-72 h before the experiment) was applied close to the dorsal raphe nucleus to uncouple somatodendritic 5-HT(1A) autoreceptors from their effector system. After this treatment the spontaneous firing rate was higher (approximately +60% P<0.005) than in the vehicle-pretreated group. In addition, intravenous administration of 8-hydroxy-2-(di-n-propylamino)tetralin HBr (8-OH-DPAT) inhibited 5 out of 11 cells of the pertussis toxin-pretreated group (ED(50)=1.65+/-0.94 microg/kg), whereas in the vehicle-pretreated group, all tested cells were inhibited (ED(50)=1.87+/-0.39 microg/kg). Local administration of 8-OH-DPAT did not affect cells (n=12) in pertussis toxin-pretreated rats, even at doses much higher than those needed to completely inhibit 5-HT cells in vehicle-pretreated rats (ED(50)=3.34+/-0.62 fmol). These results confirm the involvement of distal postsynaptic 5-HT(1A) receptors in the control of 5-HT neurone activity in the dorsal raphe nucleus. However, this control does not appear to be exerted on all 5-HT neurones, but rather on a subpopulation of them.

Locus coeruleus and dorsal raphe neuron activity and response to acute antidepressant administration in a rat model of Parkinson's disease

In addition to noradrenergic and serotonergic systems, dopaminergic neurotransmission seems to play an important role in the aetiopathogenesis of, and recovery from, depression. Moreover, the incidence of depression is higher in patients affected by diseases where the dopaminergic system is highly impaired, such us Parkinsons disease. Here, we investigated the effects of dopamine degeneration on the activity and response to antidepressants of locus coeruleus (LC) noradrenergic and dorsal raphe nucleus (DRN) serotonergic neurons. To this end, single-unit extracellular recordings were performed in control and 6-hydroxydopamine (6-OHDA)-lesioned animals. In this latter group, LC neurons showed a lower basal firing rate as well as less sensitivity to the administration of the serotonin reuptake inhibitor, fluoxetine. The rest of electrophysiological parameters and the response to the administration of the α2-adrenoceptor agonist, clonidine and the noradrenaline reuptake inhibitor, reboxetine remained unaltered. In the DRN, dopamine depletion did not modify the basal electrophysiological characteristics and the response to clonidine or fluoxetine administration. In contrast, the administration of reboxetine more efficiently induced an inhibitory effect in the lesioned group. In additional analyses it was observed that while in control animals, LC and DRN basal firing rate was significantly correlated, this relationship was lost after the 6-OHDA lesion. In conclusion, dopaminergic degeneration alters LC neuron basal activity, the relationship/synteny between both nuclei, and their response to antidepressants. These findings shed fresh light on our understanding of the role of dopamine in depression and the mechanism action of antidepressants.

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.

Increased antiparkinson efficacy of the combined administration of VegF- and gDNF-loaded nanospheres in a partial lesion model of Parkinson's disease

Current research efforts are focused on the application of growth factors, such as glial cell line-derived neurotrophic factor (GDNF) and vascular endothelial growth factor (VEGF), as neuroregenerative approaches that will prevent the neurodegenerative process in Parkinson’s disease. Continuing a previous work published by our research group, and with the aim to overcome different limitations related to growth factor administration, VEGF and GDNF were encapsulated in poly(lactic-co-glycolic acid) nanospheres (NS). This strategy facilitates the combined administration of the VEGF and GDNF into the brain of 6-hydroxydopamine (6-OHDA) partially lesioned rats, resulting in a continuous and simultaneous drug release. The NS particle size was about 200 nm and the simultaneous addition of VEGF NS and GDNF NS resulted in significant protection of the PC-12 cell line against 6-OHDA in vitro. Once the poly(lactic-co-glycolic acid) NS were implanted into the striatum of 6-OHDA partially lesioned rats, the amphetamine rotation behavior test was carried out over 10 weeks, in order to check for in vivo efficacy. The results showed that VEGF NS and GDNF NS significantly decreased the number of amphetamine-induced rotations at the end of the study. In addition, tyrosine hydroxylase immunohistochemical analysis in the striatum and the external substantia nigra confirmed a significant enhancement of neurons in the VEGF NS and GDNF NS treatment group. The synergistic effect of VEGF NS and GDNF NS allows for a reduction of the dose by half, and may be a valuable neurogenerative/neuroreparative approach for treating Parkinson’s disease.

Interaction between the 5-HT system and the basal ganglia: functional implication and therapeutic perspective in Parkinson's disease.

The neurotransmitter serotonin (5-HT) has a multifaceted function in the modulation of information processing through the activation of multiple receptor families, including G-protein-coupled receptor subtypes (5-HT1, 5-HT2, 5-HT4–7) and ligand-gated ion channels (5-HT3). The largest population of serotonergic neurons is located in the midbrain, specifically in the raphe nuclei. Although the medial and dorsal raphe nucleus (DRN) share common projecting areas, in the basal ganglia (BG) nuclei serotonergic innervations come mainly from the DRN. The BG are a highly organized network of subcortical nuclei composed of the striatum (caudate and putamen), subthalamic nucleus (STN), internal and external globus pallidus (or entopeduncular nucleus in rodents, GPi/EP and GPe) and substantia nigra (pars compacta, SNc, and pars reticulata, SNr). The BG are part of the cortico-BG-thalamic circuits, which play a role in many functions like motor control, emotion, and cognition and are critically involved in diseases such as Parkinsons disease (PD). This review provides an overview of serotonergic modulation of the BG at the functional level and a discussion of how this interaction may be relevant to treating PD and the motor complications induced by chronic treatment with L-DOPA.