María Torrecilla

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.

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.

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.

Morphine withdrawal is modified in pituitary adenylate cyclase-activating polypeptide type I-receptor-deficient mice

The pituitary adenylate cyclase-activating polypeptide type I-receptor (PAC1) is a G-protein-coupled receptor that is widely expressed in neurons of the central and peripheral nervous system. The strong expression of PAC1 in the second sensory neuron as well as in brainstem regions such as the locus coeruleus prompted us to elucidate the potential in vivo role of PAC1-mediated signalling in pain perception and opioid addiction using a PAC1-deficient mouse line. We observed a selective involvement of PAC1 in the mediation of visceral pain. While there was no impairment in acute somatic pain perception, PAC1-mutants exhibited a dramatically decreased response in the abdominal writhing test. These data in concert with data from the literature implicate PAC1 in the mediation of visceral and chronic pain. In addition, we observed that PAC1 did not influence the motivational aspects of opioid addictive properties, since morphine-induced rewarding effects and sensitization to locomotor responses were completely maintained in PAC1-deficient mice. However, there was a dramatic increase in physical withdrawal signs after naloxone-precipitated morphine withdrawal in PAC1 mutants. At the cellular level, electrophysiological examinations in locus coeruleus neurons from morphine-dependent wild-type and PAC1-deficient mice did not reveal any differences in firing rates. These data therefore suggested that most likely disruption of PAC1-mediated signalling in afferents towards the locus coeruleus but not within the intrinsic locus coeruleus system led to the enhancement of somatic withdrawal signs.

α2-Adrenoceptors mediate the acute inhibitory effect of fluoxetine on locus coeruleus noradrenergic neurons

So far, the mechanisms underlying the action of selective serotonin reuptake inhibitors, such as fluoxetine, are not completely understood. Thus, to clarify if fluoxetine has any effect on noradrenergic transmission, we measured the spontaneous firing rate of noradrenergic neurons in the locus coeruleus both in vivo and in vitro using single-unit extracellular recordings. In anesthetized rats, fluoxetine (2.5-20 mg/kg, i.v.) reduced the firing rate in a dose-dependent manner, reaching a maximal inhibition of 55 +/- 5% with respect to the basal value. This effect was not only completely reversed by the alpha(2)-adrenoceptor antagonist, RX 821002 (0.2 mg/kg, i.v.), but also prevented by previous application of both idazoxan (0.05 and 0.1 mg/kg, i.v.) and RX 821002 (6.25 microg/kg, i.v). Furthermore, when noradrenaline was depleted from axon terminals by means of the injection of alpha-methyl-DL-tyrosine (250 mg/kg, i.p.) 24 h prior to the experiment, fluoxetine failed to inhibit locus coeruleus activity. In rat brain slices, perfusion with fluoxetine (100 microM for 5 min) did not modify the firing rate of locus coeruleus neurons (n = 7). We conclude that fluoxetine inhibits locus coeruleus neurons in vivo through a mechanism involving noradrenaline interacting with alpha(2)-adrenoceptors. However, the lack of effect on brain slices would seem to indicate that afferents to the nucleus may be involved in the observed inhibitory effect.

Deletion of GIRK2 Subunit of GIRK Channels Alters the 5-HT1A Receptor-Mediated Signaling and Results in a Depression-Resistant Behavior.

Background: Targeting dorsal raphe 5-HT1A receptors, which are coupled to G-protein inwardly rectifying potassium (GIRK) channels, has revealed their contribution not only to behavioral and functional aspects of depression but also to the clinical response to its treatment. Although GIRK channels containing GIRK2 subunits play an important role controlling excitability of several brain areas, their impact on the dorsal raphe activity is still unknown. Thus, the goal of the present study was to investigate the involvement of GIRK2 subunit-containing GIRK channels in depression-related behaviors and physiology of serotonergic neurotransmission. Methods: Behavioral, functional, including in vivo extracellular recordings of dorsal raphe neurons, and neurogenesis studies were carried out in wild-type and GIRK2 mutant mice. Results: Deletion of the GIRK2 subunit promoted a depression-resistant phenotype and determined the behavioral response to the antidepressant citalopram without altering hippocampal neurogenesis. In dorsal raphe neurons of GIRK2 knockout mice, and also using GIRK channel blocker tertiapin-Q, the basal firing rate was higher than that obtained in wild-type animals, although no differences were observed in other firing parameters. 5-HT1A receptors were desensitized in GIRK2 knockout mice, as demonstrated by a lower sensitivity of dorsal raphe neurons to the inhibitory effect of the 5-HT1A receptor agonist, 8-OH-DPAT, and the antidepressant citalopram. Conclusions: Our results indicate that GIRK channels formed by GIRK2 subunits determine depression-related behaviors as well as basal and 5-HT1A receptor-mediated dorsal raphe neuronal activity, becoming alternative therapeutic targets for psychiatric diseases underlying dysfunctional serotonin transmission.

Altered neuronal activity and differential sensitivity to acute antidepressants of locus coeruleus and dorsal raphe nucleus in Wistar Kyoto rats: A comparative study with Sprague Dawley and Wistar rats

The Wistar Kyoto rat (WKY) has been proposed as an animal model of depression. The noradrenergic nucleus, locus coeruleus (LC) and the serotonergic nucleus, dorsal raphe (DRN) have been widely implicated in the ethiopathology of this disease. Thus, the goal of the present study was to investigate in vivo the electrophysiological properties of LC and DRN neurons from WKY rats, using single-unit extracellular techniques. Wistar (Wis) and Sprague Dawley (SD) rats were used as control strains. In the LC from WKY rats the basal firing rate was higher than that obtained in the Wis and SD strain, and burst firing activity also was greater compared to that in Wis strain but not in SD. The sensitivity of LC neurons to the inhibitory effect of the α2-adrenoceptor agonist, clonidine and the antidepressant reboxetine was lower in WKY rats compared to Wis, but not SD. Regarding DRN neurons, in WKY rats burst activity was lower than that obtained in Wis and SD rats, although no differences were observed in other firing parameters. Interestingly, while the sensitivity of DRN neurons to the inhibitory effect of the 5-HT1A receptor agonist, 8-OH-DPAT was lower in the WKY strain, the antidepressant fluoxetine had a greater inhibitory potency in this rat strain compared to that recorded in the Wis group. Overall, these results point out important electrophysiological differences regarding noradrenergic and serotonergic systems between Wis and WKY rats, supporting the utility of the WKY rat as an important tool in the research of cellular basis of depression.

Role of GIRK channels on the noradrenergic transmission in vivo: an electrophysiological and neurochemical study on GIRK2 mutant mice

Dysfunctional noradrenergic transmission is related to several neuropsychiatric conditions, such as depression. Nowadays, the role of G protein-coupled inwardly rectifying potassium (GIRK)2 subunit containing GIRK channels controlling neuronal intrinsic excitability in vitro is well known. The aim of this study was to investigate the impact of GIRK2 subunit mutation on the central noradrenergic transmission in vivo. For that purpose, single-unit extracellular activity of locus coeruleus (LC) noradrenergic neurons and brain monoamine levels using the HPLC technique were measured in wild-type and GIRK2 mutant mice. Girk2 gene mutation induced significant differences among genotypes regarding burst activity of LC neurons. In fact, the proportion of neurons displaying burst firing was increased in GIRK2 heterozygous mice as compared to that recorded from wild-type mice. Furthermore, this augmentation was even greater in the homozygous genotype. However, neither the basal firing rate nor the coefficient of variation of LC neurons was different among genotypes. Noradrenaline and serotonin basal levels were altered in the dorsal raphe nucleus from GIRK2 heterozygous and homozygous mice, respectively. Furthermore, noradrenaline levels were increased in LC projecting areas such as the hippocampus and amygdale from homozygous mice, although not in the prefrontal cortex. Finally, potency of clonidine and morphine inhibiting LC activity was reduced in GIRK2 mutant mice, although the efficacy remained unchanged. Altogether, the present study supports the role of GIRK2 subunit-containing GIRK channels on the maintenance of tonic noradrenergic activity in vivo. Electric and neurochemical consequences derived from an altered GIRK2-dependent signalling could facilitate the understanding of the neurobiological basis of pathologies related to a dysfunctional monoaminergic transmission.

NO synthase inhibitors reduce opioid desensitization in rat locus coeruleus neurons in vitro.

The aim of this study was to examine by electrophysiological techniques whether nitric oxide (NO) is involved in the development of desensitization to the opioid agonist Met5-enkephalin (ME) in locus coeruleus neurons from rat brain slices. Bath perfusion with ME (0.05–1.6 μM) caused a concentration-dependent reduction in the firing rate of locus coeruleus cells, whereas perfusion with a high concentration of ME (10 μM) desensitized the inhibitory effect of subsequent ME (0.8 μM) applications. However, in slices perfused with the NO synthase inhibitors 7-NI (100 μM), L-NAME (100 μM) or L-NA (100 μM) the ME (10 μM)-induced opioid desensitization was strongly attenuated. The effect of L-NAME was prevented by administration of l-arginine (100 μM). These results suggest that nitric oxide may contribute to opioid desensitization in locus coeruleus neurons.