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Major depression is the largest contributor to global disability by years lived with disability. Selective serotonergic reuptake inhibitors, including fluoxetine (FLX), are the most commonly used antidepressant for the treatment of major depression. However, they are effective for remission in only 30% of patients. Recently, we observed that the N-terminal fragment of Galanin [GAL(1-15)] enhanced the antidepressant effects of FLX in naïve animals. In this work, we have analyzed in an animal model of depression, the olfactory bulbectomy (OBX) rats, the effect of GAL(1-15) on FLX-mediated responses in the forced swimming test (FST) and the sucrose preference test (SPT), tests related with despair and anhedonic behaviours. We have also studied the corticosterone levels in OBX rats after the coadministration of GAL(1-15)+FLX. Groups of rats received a subchronic pattern of FLX(10mg/Kg) alone or in combination with GAL(1-15)(1nmol)15min before the tests. Blood samples for corticosterone assay were collected 1h after the treatments. One-way ANOVA followed by Fisher ́s least significant difference test was used. Our results show that GAL(1-15) decreases the immobility time by 50% (p

Recently, we described that Galanin(1-15)[GAL(1-15)] enhanced Escitalopram(ESC) effectiveness in depression symptoms. Moreover GAL(1-15) induces a substantial reduction in alcohol consumption. To investigate the effect of GAL(1-15) on ESC-activity in depression-alcoholism comorbidity, we used the ethanol self-administration test and the forced swimming test(FST) in rats, after a chronic alcohol consumption. Also to study if GAL(1-15)+ESC modulate the reward system induced by different reinforcers we have analyzed this combination in the saccharine self-administration test. Groups of rats received three times intraperitoneal injections of ESC (2.5mg/Kg or 7.5mg/Kg) 23, 5 and 1h before the test and one icv injection of GAL(1-15) (0.3nmol or 1 nmol) 15 minutes before the test. One-way ANOVA followed by Fisher´s least significant difference test was used. In the saccharin self-administration test, the coadministration GAL(1-15)(1nmol) and ESC(2.5mg/Kg) induced a strong reduction in the number of reinforcements of saccharine (p

We previously described that Galanin (1-15) [GAL(1-15)] enhances the antidepressant-like effects induced by the SSRI Fluoxetine in the forced swimming test (FST) in naïve rats. In this work, we have analyzed in olfactory bulbectomy rats (OBX) the effect of GAL(1-15)-Escitalopram (ESC) combination in the FST and the involvement of GALR and 5-HT1A receptors in these effects. In the first set of experiments, OBX rats received three injections of ESC (10mg/Kg) (23, 5 and 1 hour) and a single injection of a threshold dose of GAL(1-15) (1nmol) and GALR2 antagonist M871 (3nmol) alone or in combination 15 minutes before the FST. Secondly, we have generated siRNA 5HT1A knockdown rats, and we have evaluated the effects of ESC and GAL(1-15) administration in the FST. One-way ANOVA followed by Fisher ́s least significant difference test was used. In the FST, GAL(1-15) (1nmol) enhanced the antidepressant-like effects of ESC, reducing immobility (p

The Galanin N-terminal fragment (1-15)[GAL(1-15)] induces depressant- and anxiogenic-like actions in behavioural tests in rats. Since anhedonia is a core feature of depression, we have analyzed GAL(1-15) actions in anhedonic-like behaviour tests: saccharin Self-administration, Sucrose Preference test (SPT), Novelty suppressed feeding (NSF) and Female urine sniffing test (FUST). Three sets of experiments were conducted in the saccharin Self-administration. First, a dose-response curve of GAL(1-15) 1nmol, 3nmol or vehicle was performed. We have also compared the effects in the number of saccharine reinforcements of GAL 3nmol and GAL(1-15) 3nmol. In the last experiments, rats received GAL(1-15) 3nmol and the GALR2 antagonist M871 3nmol. In SPT, NSF and FUST we have analyzed the effects of GAL(1-15) 3nmol in the sucrose intake and preference, the latency of the first feeding episode and the female urine sniffing duration respectively. GAL(1-15)3nmol significantly decreased the number of reinforcement of saccharin self-administer (p

The selective serotonergic reuptake inhibitors (SSRIs) are the most commonly used for the treatment of major depression. Recently, we observed that the Galanin N-terminal fragment (1-15) [GAL(1-15)] enhances the antidepressant-like effects induced by Fluoxetine (FLX) in the forced swimming test (FST) (Flores-Burgess et al, 2017). Therefore, we have analyzed the ability of GAL(1-15) to enhance the behavioural effects of Escitalopram (ESC), other SSRIs, in the FST and the tail suspension test (TST). In the first set of experiments, groups of rats received three injections (23, 5 and 1 hour) before FST of two different doses of ESC (5mg/Kg or 7,5mg/Kg) or vehicle to perform a dose-response curve in the FST. Secondly, different groups of rats received three injections of ESC (7,5mg/Kg) and a single injection of a threshold dose of GAL(1-15) (1nmol) or aCSF 15 minutes before the FST and TST. In the dose-response curve, ESC 5 mg/kg and 7,5 mg/kg significantly increase the swimming time (p

The Galanin N-terminal fragment (1-15) [GAL(1-15)] induces depressant- and anxiogenic-like actions in behavioral tests and these effects were significantly stronger than the ones induced by Galanin. Since anhedonia is a core feature of depression, we have analyzed GAL(1-15) actions in two anhedonic-like behavior tests: saccharin Self-administration and Sucrose Preference test (SPT). In order to investigate whether the effect of GAL(1–15) was associated with the reward circuit, we have studied the GAL(1-15) actions over the mesolimbic system by the expression of the C-Fos, Dat, Vmat2 and Dopamine and GAL receptors genes in VTA and NAc. GAL(1-15) 3nmol significantly decreased the number of reinforcement of saccharin self-administer (p

Supplemental material, Supplemental_Material for Role of the galanin N-terminal fragment (1-15) in anhedonia: Involvement of the dopaminergic mesolimbic system by Carmelo Millón, Antonio Flores-Burgess, Belén Gago, Francisco Alén, Laura Orio, Laura García-Durán, José A Narváez, Kjell Fuxe, Luis Santín and Zaida Díaz-Cabiale in Journal of Psychopharmacology

Major Depression is the most frequent mood disorder, with a lifetime prevalence that has been reported to range from 7% to 21%. It is associated with a substantial functional impairment, diminished quality of life, increased burden both for patients and caregivers, as well as with a higher risk of mortality. Although the underlying mechanisms have not yet been clearly defined in the last decade the importance of the role of neuropeptides, including Galanin (GAL) and the N-terminal fragment GAL(1-15) and/or their receptors in the treatment of stress-related mood disorders is becoming increasingly apparent. We have described that GAL(1-15) induces strong depression-related and anxiogenic-like effects in rats and these effects were significantly stronger than the ones induced by GAL. The GALR1-GALR2 heteroreceptor complexes in the dorsal hippocampus and dorsal raphe (DR) were involved in these effects and demonstrated also in cellular models. Although several neurotransmitter systems and brain areas have been implicated in depression, the pharmacological treatment of major depression is mainly based on drugs elevating serotonergic (5-HT) activity. Specifically, selective 5-HT reuptake inhibitors (SRRIs) are the most commonly used for treatment of major depression. In particular, Fluoxetine (FLX) is usually chosen for the treatment of symptoms of depression In view of these results the purpose of the current study was to assess the ability of GAL(1–15) to modulate the behavioral effects of the 5-HT1AR agonist 8-OH-DPAT and FLX. We have analyzed the effect of GAL (1–15) on the 5-HT1AR agonist 8-OH-DPAT and FLX-mediated responses in a behavioral test of depression. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. This work was supported by SAF2016-79008-P, PSI2017-82604-R (Grant BES-2014-068426).

The Galanin N-terminal fragment (1-15) [GAL(1-15)] induces depressant- and anxiogenic- like actions in several behavioral tests, and these effects were significantly stronger than the ones induced by Galanin (1-29). Since, anhedonia is a core feature of major depressive disor- der, we have analyzed GAL(1-15) actions in two anhedonic-like behaviour tests: saccharin Self-administration and Sucrose Preference test. Three sets of experimemts were conducted in the saccharin Self-administration test. In the first, a dose-response curve of GAL(1-15) 1nmol, 3nmol or vehicle was performed. We have also compared the effects in the number of saccharine reinforcements of Galanin (1-29) 3nmol and GAL(1-15) 3nmol. In the last experiments, rats received i.c.v. GAL(1-15) 3nmol and the GALR2 antagonist M871 3nmol. In Sucrose Preference test, we have analyzed the effects of GAL(1-15) 3nmol in the sucrose intake and preference after 2, 8 and 24 h. GAL(1-15) 3nmol significantly decreased the number of reinforcement of saccharin self- administer (p

Galanin (1-15)[GAL(1-15)] induces depressant- and anxiogenic-like actions in several behavioural tests. Recently, we described that GAL(1-15) induces an anhedonic-like effect in saccharine self-administration and sucrose preference test in rats. In order to investigate whether the effect of GAL(1–15) in anhedonia was associated with the reward circuit, we have studied the GAL(1-15) actions over the mesolimbic system by PET for in vivo imaging and in the expression of the C-Fos, Dat, Vmat2 and Dopamine and Galanin receptors genes in VTA and NAc. In the PET experiments, the [18F]FDG at 30, 60 and 90min after GAL(1-15) administration was measure as indicative of brain glucose metabolism. In the qPCR experiments, groups of rats (n=5-6) were killed 1h after i.c.v. GAL(1-15) 3nmol or vehicle. The VTA and NAc were dissected and the mRNA expression of C-Fos, Dat, Vmat2 and D1, D2, D3, D5, GALR1, and GALR2 receptors were measured by RT-qPCR. The GAL(1-15) induced a decrease in [18F]FDG uptake in the hippocampus and thalamus at 30, 60 and 90min, and in the striatum at 30min, however, in the prefrontal cortex, an increase in [18F]FDG uptake was observed after 30 and 60min. GAL(1-15) at a dose of 3 nmol produced a significant decrease in the mRNA levels of Dat and Vmat2 (p

Los resultados de esta tesis indican que GAL(1-15) potencia los efectos antidepresivos de la FLX en el FST tanto con dosis subumbrales como efectivas del antidepresivo y que dicha potenciación es exclusiva de GAL(1-15). Los resultados también sugieren la participación del heterodímero GALR1-GALR2 en la acción de GAL(1-15), ya que tanto el antagonista específico para GALR2 M871 como los modelos de silenciamiento génico para los receptores GALR1(siGalR1) y GALR2(siGalR2) bloquearon el efecto potenciador de GAL(1-15) sobre la acción antidepresiva de la FLX. Describimos también un papel fundamental del 5-HT1AR en la interacción GAL(1-15)-FLX a nivel de comportamiento ya que el uso del antagonista WAY100635 bloqueó los efectos potenciadores de GAL(1-15) sobre la FLX en el FST. Y además, la coadministración de GAL(1-15) y FLX modificó la funcionalidad y expresión de los 5-HT1AR a nivel de hipocampo. Estos resultados confirman el papel de GAL(1-15) en la comunicación cerebral y en los trastornos del estado de ánimo y abren la posibilidad de utilizar el fragmento GAL(1-15), algún agonista o un fármaco bivalente como una estrategia farmacológica de potenciación junto con la FLX para potenciar sus efectos. Fecha de lectura de Tesis Doctoral: 31 de mayo 2019. La Galanina (GAL) es un péptido ampliamente distribuido en el SNC (Tatemoto et al., 1983) que media sus acciones a través de tres receptores. GALR1 y GALR3 activan principalmente las proteínas G inhibitorias Gi/Go y GALR2 activa a Gq/G11 (Branchek et al., 2000). El fragmento N-terminal de la GAL [GAL(1-15)] también presenta una actividad biológica específica y unos mecanismos de acción distintos a los de GAL. Recientemente hemos descrito como el fragmento GAL(1-15) actúa por medio de la formación del heterodímero GALR1-GALR2 (Borroto-Escuela et al., 2014; Millón et al., 2015), y que potencia los efectos antidepresivos del agonista del receptor serotoninérgico 5-HT1AR 8-0H-DPAT más intensamente que la GAL, modificando además la funcionalidad y expresión de dicho receptor en el rafe dorsal (RD) y el hipocampo de ratas, núcleos clave de la depresión (Millón et al., 2016). Por otro lado, el 5-HT1AR es fundamental en la mediación de los efectos antidepresivos de los ISRS como la fluoxetina y tiene un papel diferencial dependiendo de si actúa como autorreceptor en el RD o en el hipocampo (Estada-camarena et al., 2006; Descarries et al., 2012; Ferrés-Coy et al., 2016). En base a este papel diferencial, se han diseñado estrategias farmacológicas que permitan mejorar la acción de los ISRSs (Artigas et al., 1994). Uno de los objetivos de esta tesis doctoral es estudiar si GAL(1-15) puede potenciar los efectos antidepresivos de la FLX en ratas en base a la interacción entre GAL(1-15)-5-HT1AR previamente descrita y analizar el papel de los receptores GALR1, GALR2 y 5-HT1AR en dicha interacción a nivel conductual empleando el test de natación forzada (FST). Además analizamos si la coadministración de FLX y GAL(1-15) modifican las características funcionales y expresión del 5-HT1AR en las regiones del RD e hipocampo mediante experimentos de autorradiografía e hibridación in situ.

El Trabajo Fin de Grado (TFG), asignatura obligatoria que el estudiante debe realizar autónoma e individualmente, es un trabajo original que será presentado y defendido ante un Tribunal de Evaluación. Su implantación ha manifestado debilidades y carencias (comunicación oral y escrita) del alumnado. Se proponen actividades para mejorar estas capacidades. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Universidad de Málaga (PIE17-150).

In this work, we have studied the effects of GAL(1-15) on FLX- mediated effects on the NOR and the OLM, two tasks where FLX treatment impaired long-term memories 24h post-training. Since the mPFC is a core region for the interaction between emotional processing and cognition with a high density of 5-HT1AR and GALR1 and GALR2, we have also analyzed the binding characteristics and mRNA levels of 5-HT1AR in the mPFC after GAL(1-15)-FLX administration in rats. A discrimination index (DI) was calculated as: DI=(N-F)/(N+F), and represent the difference in exploration time expressed as a proportion of the total time spent exploring the two objects. To analyze the binding characteristics and mRNA levels of 5-HT1AR, group of animals (n=6) were injected with FLX(10mg/kg) and GAL(1-15)(1nmol) alone or in combination and coronal sections of the mPFC were obtained to perform a quantitative autoradiography and in situ hybridization experiments In the NOR task, GAL(1-15)+FLX reversed the impairment memory effect induced by FLX(10mg/Kg) (p

Galanin N-terminal fragment (1-15) [GAL(1-15)] modulates the antidepressant effects induced by the 5-HT1A receptor (5-HT1AR) agonist in the forced swimming test (FST) and the binding characteristics and mRNA levels of 5-HT1AR in the dorsal hippocampus and dorsal raphe (DR). Recently, we observed that GAL(1-15) enhanced the antidepressant-like effects induced by Fluoxetine (FLX) in the FST. In this work, we have studied whether the effects of GAL(1–15) on FLX action were mediated via 5-HT1AR, analyzing the effect of the 5-HT1AR antagonist WAY100635 in this effect and if the binding characteristics and mRNA levels of 5-HT1AR in the DR and dorsal hippocampus are modified by GAL(1-15)+FLX. Groups of rats (n=6-8) received three injections of sc FLX(10mg/kg) and 15 minutes before the FST a single icv injection of GAL(1-15) (1nmol) and 5HT1AR antagonist WAY100635(6nmol) icv alone or in combination. We also analyzed the effects of GAL(1-15)+FLX in the binding characteristics of the 5-HT1AR agonist [H3]-8-OH-DPAT and 5-HT1A mRNA levels in the DR, CA1 and Dentate Gyrus (DG). WAY100635 significantly blocked the reduction in immobility time (p

Galanin (GAL) is involved in drug abuse and addiction including alcohol intake. In this work, we have analysed the role of the N-terminal GAL fragment (1-15) [GAL(1-15)] in voluntary ethanol consumption in rats using the two-bottle choice paradigm as well as compare the effects of GAL(1-15) with GAL. The two-bottle choice test was used to determine the voluntary ethanol consumption of rats (Castilla-Ortega et al., 2016). Three sets of experiments were conducted. In the first set of experiments, a dose-response curve of GAL(1-15) was performed. Groups of rats (n=7-9) received i.c.v. GAL(1-15) 1 nmol, 3 nmol or vehicle 2, 14 and 24 hours before the measures. In the second set of experiments, the effects in two-bottle choice test of GAL 3 nmol, and GAL(1-15) 3 nmol were compared. In the last set of experiments rats received i.c.v. GAL(1-15) 3nmol combined with GALR2 antagonist M871 3 nmol 2 hours before the measures. GAL(1-15) 3nmol significantly decreased the alcohol intake 2 (p

We have described that Galanin N-terminal fragment (1-15) [GAL(1-15)] is associated with depressive effects and also modulates the antidepressant effects induced by the 5-HT1A receptor (5-HT1AR) agonist 8-OH-DPAT in the forced swimming test (FST). Importantly, the mechanism underlying this action involved interactions at the receptor level in the plasma membrane with changes also at the transcriptional level. Thus, GAL(1–15) affected the binding characteristics as well as the mRNA levels of 5-HT1AR in the dorsal hippocampus and dorsal raphe (DR). Recently, we observed that GAL(1-15) enhanced the antidepressant-like effects induced by Fluoxetine (FLX) in the FST. In view of this, we have studied whether the effects of GAL(1–15) on FLX action were mediated via 5-HT1AR, analyzing the effect of the 5-HT1AR antagonist WAY100635 in the GAL(1-15)-mediated effect in the FST. We have also determined the binding characteristics and mRNA levels of 5-HT1AR in the DR and dorsal hippocampus after GAL(1-15)-FLX administration. To study if the effects of GAL(1-15) on FLX action were mediated via the 5HT1AR, groups of rats (n=6-8 rats per groups) received three subcutaneous injections (sc) of FLX(10mg/kg) and 15 minutes before the FST a single intracerebroventricular injection (icv) of GAL(1-15) (1nmol) and 5HT1AR antagonist WAY100635(6nmol) icv alone or in combination. In the second set of experiments, we analyzed the effects of the three injections of sc FLX or Vehicle and a single icv injection of GAL(1-15) or aCSF and sacrified 30 min later to analyze the binding characteristics of the 5-HT1AR agonist [H3]-8-OH-DPAT and 5-HT1A mRNA levels in sections of the DR and Dorsal Hippocampus, specifically in the Ammon’s horn 1 (CA1) and Dentate Gyrus (DG). The results confirmed that the 5HT1AR participates in this interaction as the 5HT1AR antagonist WAY100635 (6nmol) significantly blocked the reduction in immobility time (p

The selective serotonergic (5- HT) reuptake inhibitors, including Fluoxetine (FLX), are the most commonly used for treatment of major depression. However, the understanding of the mechanism of action of FLX beyond its effect of elevating 5-HT is limited. The interaction between 5-HT system and neuropeptides signaling could be a key aspect. The neuropeptide Galanin(1-15) [GAL(1-15)], induced a strong depression-like and anxiogenic-like effects in the forced swimming test (FST), the tail suspension test, the open field and the light/dark test. The GALR1-GALR2 heteroreceptor complexes in the dorsal hippocampus and in the dorsal raphe were involved in these effects. We have analyzed the effect of GAL(1–15) on FLX-mediated responses in the FST. We tested the involvement of GALR in the GAL(1–15) effect with the selective GALR2 antagonist M871 and using siRNA GALR2 or GALR1 knockdown rats. Groups of rats received three injections of sc FLX(2.5mg/Kg) or FLX(10mg/Kg) and a single icv injection of a threshold dose of GAL(1-15)(1nmol) 15 minutes before the FST. In a second set of experiments, we determined the involvement of GALR1 and GALR2 in the effect of GAL(1-15) on FLX-mediated action. Groups of rats received three injections of sc FLX(10mg/kg), a single icv injection of GAL(1-15) (1nmol) and the GALR2 antagonist M871 (3nmol) icv alone or in combination. Also, in siRNA GALR1 or GALR2 knockdown rats we coadministered FLX(10mg/Kg) and GAL(1-15)(1nmol). The coadministration of sc FLX(2.5mg/Kg) and icv injection of GAL(1-15)(1nmol) induced antidepressant-like effects with a significant decrease in the immobility (p

Previously, we have described the Galanin (GAL) and Neuropeptide Y Y1 (NPYY1) interactions through GAL receptor 2 and NPYY1 receptor 1 (GALR2/NPYY1R) heterodimers in the Dentate Gyrus (DG) of the Hippocampus, using autoradiographic, in situ hybridization and in situ proximity ligation assay(PLA) (1,2). The current work is to evaluate GALR2 and NPYY1R interactions in relation to depression-like behaviour and c-Fos expression in the Hippocampal DG. Rats (n=6-8) were forced to swim for a 15-min period (pre-test) and 24 h later were subjected to a 5-min swimming session (test) 15 min after the administration alone or in combination of GAL, the NPYY1R agonist [Leu31,Pro34]NPY and the GALR2 antagonist M871. The total duration of immobility, swimming, and climbing periods were scored during the test. For c-Fos immunohistochemistry, experimental groups of rats were anesthetized with sodium pentobarbital (100 mg/kg, i.p.) and perfused with 4 % Paraformaldehyde 90 min after icv injections. Then, brains were coronally sliced and immunostained. As primary antibodies, an antibody against c-Fos protein (1:5000, sc- 52, Santa Cruz Biotechnology, CA, USA), revealed with 3,3´-Diaminobenzidine (DAB) plus nickel, was used as an indirect marker of neural activity. The antibody to Calbindin-D28 k (1:1000, Santa Cruz Biotecnology, CA, USA), revealed with DAB, was used to outline the granular region since it marks mainly hippocampal granule cells. Sections were analyzed using the optical fractionator stereological method. We observed that icv injection of GAL and NPYY1R agonist significantly enhanced the decrease in the immobility (p

Galanin and Galanin (1-15) [GAL(1-15)] are implicated in anxiety- and depression related behaviors. Moreover, Galanin modulates 5-HT1A receptor (5-HT1AR) function at autorreceptor and postsynaptic level in the brain. In this study, we have analysed the ability of GAL(1-15) to modulate the effects of the 8-OH-DPAT agonist in the Forced Swimming Test (FST). Groups of rats were assessed in the FST. In the first set of experiments, to evaluate the interactions of 8-OH-DPAT and GAL(1-15), rats received subcutaneously (s.c) the effective doses of 8-OH-DPAT (0.25mg/Kg) 60min before the test and intracerebroventricularly (icv) GAL(1-15)1nmol 15min before the tests alone or in combination. In the second set of experiments, groups of rats received s.c. 8-OH-DPAT (0.125mg/Kg), icv GAL(1-15) 1nmol and icv the GALR2 antagonist M871 3 nmol alone or in combination. The locomotor activity was analysed in the open field test. GAL(1-15) 1nmol enhanced the antidepressant-like effects mediated by the effective dose of the 8-OH-DPAT. GAL(1-15) significantly decreased the immobility (p

Previously, we have described Galanin(GAL) and Neuropeptide Y Y1(NPYY1) interactions at behavioural, cellular and receptor levels through GALR2/NPYY1R heterodimers in the amygdala. The aim of this work was to study GAL/NPYY1R interactions in the Dentate Gyrus (DG) of the Hippocampus, using autoradiographic, in situ hybridization and in situ proximity ligation assay (PLA). Rats (n=6) were sacrificed 15 minutes or 5 hours after icv injections of GAL (3nmol) and DG sections were incubated with NPYY1R agonist [I125]-[Leu31,Pro34]PYY (25 pM) or NPYY1R-33PdATP specific probe, for autoradiography and in situ hybridization respectively. Autoradiograms were analyzed using NIH image analysis system and Student’s unpaired t-test was used. For PLA, DG sections were incubated with anti-GALR2 Rabbit (1:100) and anti-NPYY1R Goat (1:200). PLA signals were detected with PLA PLUS or MINUS probes for rabbit or goat/mouse antibodies. PLA signals were visualized by using a confocal microscope Leica TCS-SL confocal microscope (Leica). We observed that GAL significant increased the NPYY1R agonist [I125]-[Leu31,Pro34]PYY binding in the DG by 20% (p

Mood disorders, including depression and anxiety, are among the most prevalent mental illnesses with high socioeconomic impact. Although the underlying mechanisms have not yet been clearly defined in the last decade the importance of the role of neuropeptides, including Galanin (GAL), and/or their receptors in the treatment of stress-related mood disorders is becoming increasingly apparent. GAL is involved in mood regulation, including depression-related and anxiety-like behaviors. Activation of GALR1 and GALR3 receptors results in a depression like behavior while stimulation of GALR2 receptor leads to anti-depressant-like effects. Moreover, GAL modulates 5-HT1A receptors (5-HT1AR), a key receptor in depression at autoreceptor and postsynaptic level in the brain. This interaction can in part be due to the existence of GALR1-5-HT1AR heteroreceptor complexes in discrete brain regions [1]. Not only GAL but also the N-terminal fragments like GAL(1-15) are active in the Central Nervous System [2, 3]. Recently, we described that GAL(1-15) induces strong depression-related and anxiogenic-like effects in rats, and these effects were significantly stronger than the ones induced by GAL [4]. The GALR1-GALR2 heteroreceptor complexes in the dorsal hippocampus and especially in the dorsal raphe (DR), areas rich in GAL(1-15) binding sites [5] were involved in these effects [4, 6] and demonstrated also in cellular models. In the present study, we have analyzed the ability of GAL(1-15) to modulate 5-HT1AR located at postjunctional sites and at the soma-dendritic level in rats. We have analyzed the effect of GAL(1-15) on the 5-HT1AR-mediated response in a behavioral test of depression and the involvement of the GALR2 in these effects. GAL(1-15) enhanced the antidepressant effects induced by the 5-HT1AR agonist 8-OH-DPAT in the forced swimming test [7]. These effects were stronger than the ones induced by GAL. The mechanism of this action involved interactions at the receptor level in the plasma membrane with changes also at the transcriptional level. Thus, GAL(1-15) affected the binding characteristics as well as the mRNA level of 5-HT1AR in the dorsal hippocampus and DR. GALR2 was involved in these effects, since the specific GALR2 antagonist M871 blocked GAL(1-15) mediated actions at the behavioral and receptor level [7]. Furthermore, the results on the proximity ligation assay (PLA) in this work suggest the existence of GALR1-GALR2-5-HT1AR heteroreceptor complexes since positive PLA were obtained for both GALR1-5-HT1AR and GALR2-5-HT1AR complexes in the DR and hippocampus. Moreover the studies on RN33B cells, where GALR1, GALR2 and 5-HT1AR exist [4], also showed PLA-positive clusters indicating the existence of GALR1-5-HT1AR and GALR2-5-HT1AR complexes in these cells [7]. In conclusion, our results indicate that GAL(1–15) enhances the antidepressant effects induced by the 5-HT1AR agonist 8-OH-DPAT probably acting on GALR1-GALR2-5-HT1AR heteroreceptor located at postjunctional sites and at the soma-dendritic level. The development of new drugs specifically targeting these heteroreceptor complexes may offer a novel strategy for treatment of depression. This work has been supported by Junta de Andalucia CVI646 1. Borroto-Escuela, D.O., et al., Galanin receptor-1 modulates 5-hydroxtryptamine-1A signaling via heterodimerization. Biochem Biophys Res Commun, 2010. 393(4): p. 767-72. 2. Hedlund, P.B. and K. Fuxe, Galanin and 5-HT1A receptor interactions as an integrative mechanism in 5-HT neurotransmission in the brain. Ann N Y Acad Sci, 1996. 780: p. 193-212. 3. Diaz-Cabiale, Z., et al., Neurochemical modulation of central cardiovascular control: the integrative role of galanin. EXS, 2010. 102: p. 113-31. 4. Millon, C., et al., A role for galanin N-terminal fragment (1-15) in anxiety- and depression-related behaviors in rats. Int J Neuropsychopharmacol, 2015. 18(3). 5. Hedlund, P.B., N. Yanaihara, and K. Fuxe, Evidence for specific N-terminal galanin fragment binding sites in the rat brain. Eur J Pharmacol, 1992. 224(2-3): p. 203-5. 6. Borroto-Escuela, D.O., et al., Preferential activation by galanin 1-15 fragment of the GalR1 protomer of a GalR1-GalR2 heteroreceptor complex. Biochem Biophys Res Commun, 2014. 452(3): p. 347-53. 7. Millon, C., et al., Galanin (1-15) enhances the antidepressant effects of the 5-HT1A receptor agonist 8-OH-DPAT: involvement of the raphe-hippocampal 5-HT neuron system. Brain Struct Funct, 2016. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Poster en Congreso We have described that Galanin N-terminal fragment (1-15) [GAL(1-15)] is associated with depressive effects and also modulates the antidepressant effects induced by the 5-HT1A receptor (5-HT1AR) agonist 8-OH-DPAT. The aim of this study is to analyze the ability of GAL(1-15) to modulate 5-HT1AR at the autoreceptor and postsynaptic receptor level in rats by using quantitative autoradiography. We analyzed the effect of intracerebroventricular GAL(1-15)-3nmol (n=6) or aCSF (n=6), 10 minutes, 2 and 5 hours after the injection, on the binding characteristics of the 5-HT1AR agonist [H3]-8-OH-DPAT in sections of the Dorsal Raphe (DR) and Dorsal Hippocampus, specifically CA1 and Dentate Gyrus (DG). Student’s t-test was used to compare the experimental groups. GAL(1-15) produced a time-dependent effect on the binding of [H3]-8-OH-DPAT. In CA1 and DG, a significant increase in the KD and Bmax was observed, by 90%(p

We have recently described a Galanin receptor 2(GALR2) and Neuropeptide Y Y1 receptor(NPYY1R) interaction at behavioural, cellular and receptor levels through GALR2/NPYY1R heterodimers. The aim of this work was to study if GALR2 and NPYY1R costimulation modified NPYY1R internalization and β-Arrestin recruitment after in HEK293T cells. HEK293T cells were transfected with NPYY1REGFPor β-Arrestin2GFP2 cloned with standard molecular biology techniques employing PCR and fragment replacement strategies. NPYY1REGFP/GALR2 and NPYY1R/GALR2 with β- Arrestin2GFP2 HEK293T coexpressing cells were incubated with NPY 1μM and/or GAL1μM, at different times. Antagonist studies were performed 15 min prior to the addition of agonist with NPYY1R antagonist BIBP3226 10μM or GALR2 antagonist M871 10 μM. Timed-interval images of NPYY1REGFP or β-Arrestin2GFP2 endosomes in different cell groups were acquired using a confocal microscope following agonist addition. Percentage of internalization was determined by Leica software analysis of total membrane fluorescence compared to total internal compartment fluorescence at the various time points. We observed that addition of NPY induced a rapid decrease in the cell surface expression of NPYY1REGFP and a redistribution of β-Arrestin2GFP2. In fact, we observed a maximum of internalization of 80% three minutes after the NPY stimulation. However, combined treatment with GAL and NPY induced a delay in the internalization of NPYY1REGFP, with a maximum of internalization thirty minutes after the co-stimulation. Moreover, a delay in the β-Arrestin2GFP2 redistribution was observed. The specific GALR2 antagonist M871 abolished these delays in internalization of NPYY1REGFP and β-Arrestin2GFP2 redistribution, suggesting that this effect was mediated through the coactivation of GALR2 and NPYY1R. These results demonstrate that costimulation with GAL and NPY delays the internalization of  NPYY1REGFP by decreasing recruitment of β-Arrestin2GFP2 and probably could change intracellular signaling. This study was supported by Junta de Andalucia CVI6476. Junta de Andalucia CVI6476.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.

The Galanin N-terminal fragment (1-15) [GAL(1-15)] induces depressant- and anxiogenic- like actions. In this work, we have studied the role of GALR2 and GALR1 on the effects of GAL(1-15) in the Forced Swimming Test (FST) and Open Field Test (OFT) using siRNA GALR2 and GALR1 knockdown rats. Rats (n=6-14) were injected with GAL(1-15) 3nmol, GALR2 antagonist M871 3nmol in combination or alone 15 before the FST or OFT. The time of immobility, climbing and swimming were recorded during 5 min FST and Time and entries in the central square during 5min were scored in the OFT. In other experiment, rats (n=6-14) were injected Intracerebroventricular (icv) with siRNA-GALR2 or siRNA-GALR1 to generate the GALR knockdown rats. These knockdown rats were used in the OFT and in the FST after receiving icv GAL(1-15) 3nmol 15 min before the test. Vehicle was used as control. In the FST, M871 significantly blocked the increased immobility (p

We have recently described a Galanin receptor 2(GALR2) and Neuropeptide Y Y1 receptor(NPYY1R) interaction at behavioural, cellular and receptor levels through GALR2/NPYY1R heterodimers. The aim of this work was to study if GALR2 and NPYY1R costimulation modified NPYY1R internalization and β-Arrestin recruitment after in HEK293T cells. HEK293T cells were transfected with NPYY1REGFPor β-Arrestin2GFP2 cloned with standard molecular biology techniques employing PCR and fragment replacement strategies. NPYY1REGFP/GALR2 and NPYY1R/GALR2 with β- Arrestin2GFP2 HEK293T coexpressing cells were incubated with NPY 1μM and/or GAL1μM, at different times. Antagonist studies were performed 15 min prior to the addition of agonist with NPYY1R antagonist BIBP3226 10μM or GALR2 antagonist M871 10 μM. Timed-interval images of NPYY1REGFP or β-Arrestin2GFP2 endosomes in different cell groups were acquired using a confocal microscope following agonist addition. Percentage of internalization was determined by Leica software analysis of total membrane fluorescence compared to total internal compartment fluorescence at the various time points. We observed that addition of NPY induced a rapid decrease in the cell surface expression of NPYY1REGFP and a redistribution of β-Arrestin2GFP2. In fact, we observed a maximum of internalization of 80% three minutes after the NPY stimulation. However, combined treatment with GAL and NPY induced a delay in the internalization of NPYY1REGFP, with a maximum of internalization thirty minutes after the co-stimulation. Moreover, a delay in the β-Arrestin2GFP2 redistribution was observed. The specific GALR2 antagonist M871 abolished these delays in internalization of NPYY1REGFP and β-Arrestin2GFP2 redistribution, suggesting that this effect was mediated through the coactivation of GALR2 and NPYY1R. These results demonstrate that costimulation with GAL and NPY delays the internalization of  NPYY1REGFP by decreasing recruitment of β-Arrestin2GFP2 and probably could change intracellular signaling. This study was supported by Junta de Andalucia CVI6476. Junta de Andalucia CVI6476.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.

Galanin N-terminal fragment (1-15) [Gal(1-15)] is involved in mood regulation. We have shown that intracerebroventricular (icv) administration of Gal(1-15) produces a depressive-like behaviour in the forced swim test (FST) and an anxiety-like behaviour in the open field test (OF) in rats. In this work we analyze the effect of Gal(1-15) in two more behavioural tests, the tail suspension test (TLT) and the light/dark test. In light/dark test we studied during 5 min the latency time for entering the dark box, time spent in the light compartiment, and the latency time for re-entering the light box as parameters indicators of anxiety-like behaviour. In TLT total immobility time was analyzed during 6 min test as parameter indicator of depressive-like behaviour. Groups of rats (n=5-8) were injected icv with Gal(1-15) 3nmol, a dose effective in FST and OF, or artificial cerebrospinal fluid 15 minutes before the test. Behavioural assessment were conducted with at least one week between tests. Student’s t-test was used for comparation between experimental groups. In the light/dark test Gal(1-15) 3nmol significantly reduced the time spent in the light compartiment by 52% (p