Interacciones entre la Galanina (1-15) y los receptores Serotoninérgicos 5HT1A en el Sistema Nervioso Central: Implicación en la depresión

La Galanina (GAL) es un neuropéptido ampliamente distribuido en el Sistema Nervioso Central (SNC), que presenta un fragmento N-terminal de 15 aminoácidos común en todos los mamíferos. La GAL participa en diversas funciones, destacando más su implicación en la depresión. El fragmento N-terminal de GAL 1-15 [GAL(1-15)] posee actividad biológica en el SNC, y además presenta un papel diferencial de la molécula completa de GAL, como en el Control Central Cardiovascular. Los tres subtipos de receptores de GAL (GALR1, GALR2 y GALR3) tienen una mayor afinidad por la molécula de GAL que por la GAL(1-15), sin embargo existen lugares de unión específicos para la GAL(1-15) como es el caso del Hipocampo dorsal y el núcleo del Rafe dorsal, áreas implicadas en la depresión. Estudios recientes han propuesto, que el mecanismo de acción del fragmento GAL(1-15) podría ser a través de un Heterodímero formado por los receptores GALR1/GALR2. La GAL y la Serotonina (5HT) colocalizan en distintos núcleos, destacando el Rafe dorsal, núcleo de gran importancia en la patofisiología de la depresión, además se ha descrito una interacción antagónica entre la GAL y los receptores 5HT1A, tanto a nivel de receptor en preparaciones de membranas límbicas y a nivel del Rafe dorsal como en estudios de conducta animal, en el test de evitación pasiva. Estas interacciones antagónicas se explicarían mediante la existencia de Heterodímeros entre los receptores de GAL y el receptor 5HT1A. Recientemente nuestro grupo de investigación ha demostrado la existencia de heterodímeros GALR1/5HT1A, y además se ha sugerido la existencia de mosaicos de receptores GALR1/GALR2/5HT1A. Por ello el objetivo de este trabajo ha sido analizar la función de la GAL(1-15) en pruebas de comportamiento animal relacionadas con la depresión y ansiedad, así como describir el posible mecanismo de actuación de este fragmento de GAL en el SNC. Además se ha estudiado la modulación de la GAL(1-15) sobre los receptores 5HT1A en pruebas de conducta animal de depresión analizando los mecanismos implicados en dicha interacción. Los resultados de la presente tesis demuestran que la GAL(1-15) presenta un fuerte efecto prodepresivo y ansiogénico en las pruebas de conducta empleadas, que además resulta más potente que el generado por la GAL. Hemos demostrado la implicación del receptor GALR2 en este efecto mediante el uso de antagonistas específicos y animales Knockdown GALR2. La técnica de ligazón por proximidad nos muestra la existencia de posibles complejos heterodímeros GALR1/GALR2 en el hipocampo dorsal y especialmente en el Rafe dorsal, estos complejos GALR1/GALR2 se redujeron notablemente en el hipocampo dorsal y de una forma más intensa en el Rafe dorsal en animales Knockdown GALR2, desapareciendo además los efectos comportamentales de la GAL(1-15) en estos animales. Además, la GAL(1-15) presenta un patrón de internalización diferente a la GAL en células HEK293 que coexpresan los receptores GALR1 y GALR2, siendo principalmente la GAL(1-15) quien induce la internalización de los receptores en estas células. En células derivadas del núcleo del Rafe RN33B, GAL(1-15) produce una disminución de la inmunorrecatividad de la 5HT y C-Fos de una forma más potente que la GAL. Estos resultados sugieren que la GAL(1-15) participa en los mecanismos de control de la ansiedad y depresión disminuyendo la actividad celular y síntesis y almacenamiento de 5HT en el Rafe dorsal, este efecto podría estar realizado a través del heterodímero GALR1-GALR2. En esta tesis también se ha estudiado la interacción entre la GAL(1-15) y los receptores 5HT1A, demostrándose una potenciación de la acción antidepresiva del agonista 5HT1A. Este efecto se bloquea con antagonistas GALR2, demostrando así la implicación de este receptor en la interacción. Esta interacción se observa también a nivel de receptor ya que GAL(1-15) modula las características y la expresión de los receptores 5HT1A, disminuyendo los sitios de unión del 5HT1A en el rafe dorsal y aumentándolos en el hipocampo dorsal. Hemos observado que los receptores GALR1/5HT1A y GALR2/5HT1A se encuentran a una distancia que permite la interacción física entre ellos en estas zonas. En células derivadas del Rafe RN33B la GAL(1-15) inducía una reducción de la expresión del 5HT1A de una más potente que la GAL. Por tanto, estos resultados sugieren la existencia de una interacción entre GAL(1-15) y el 5HT1A en el hipocampo dorsal y el Rafe dorsal que sería responsable de la potenciación del efecto antidepresivo del agonista 5HT1A inducido por GAL(1-15). El mecanismo molecular de esta interacción podría implicar la formación de mosaicos de receptores GALR1/GALR2/5HT1A

Galanin n-terminal fragment (1-15) induces an anxiety- and depressive-like behaviours in the light/dark and tail suspension tests

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<0.05) and the latency time for entering the dark box by 65% (p<0.05). An increased in the latency time for re-entering the light box was also observed (p<0.05). This pattern of results reflects an anxiogenic-like effects of Gal(1-15) in this test. In the TST, the administration of Gal(1-15) 3nmol significantly increased rat immobility by 16% (p<0.05) indicating a depressive-like effect. These results confirm the depressive- and anxiety-like effects of Gal(1-15) in rats. Future therapeutic strategies based on these results could be developed for depression and anxiety disorders. This work has been supported by the Junta de Andalucia CVI6476 and Spanish Ministry of Economy and Competitiveness (PSI2013-44901-P to L.J.S)Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. This work has been supported by the Junta de Andalucia CVI6476 and Spanish Ministry of Economy and Competitiveness (PSI2013-44901-P to L.J.S)Author A.F-B. holds a ‘FPI’ grant from the Spanish Ministry of Economy and Competitiviness (grant number BES-2014-068426)

Effects of galanin n-terminal fragment (1-15) in the light/dark and tail suspension tests

Galanin N-terminal fragment (1-15) [Gal(1-15)] is involved in mood regulation. The 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. Gal(1-15) (3nmol), effective dose in FST and OF, or artificial cerebrospinal were injected in animals (n=5-8) 15 minutes before the test. Behavioural assessment were conducted with at least one week between tests. Student’s t-test was used for comparision between experimental groups. In the light/dark test we analyzed during 5 min three parametres as indicators of anxiety-like behaviour. Gal(1-15) significantly reduced the time spent in the light compartiment by 52% (p<0.05) and the latency time for entering the dark box by 65% (p<0.05). An increased in the latency time for re-entering the light box was also observed (p<0.05). In the TST, total immobility time was analyzed during 6 min test as parameter indicator of depressive-like behaviour. Gal(1-15) significantly increased rat immobility by 16% (p<0.05). Our results describe that Gal(1-15) exerts strong depressive- and anxiety-like effects in these tests, indicating a potential role of Gal(1-15) in mood disorders. These results may give the basis for the development of novel therapeutic drugs targeting Gal(1-15) for depression and anxiety.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Junta de Andalucia CVI6476 // Spanish Ministry of Economy and Competitiveness (PSI2013-44901-P to L.J.S). Author A.F-B. holds a ‘FPI’ grant from the Spanish Ministry of Economy and Competitiviness (grant number: BES-2014-068426)

A Role For Galanin N-Terminal Fragment (1-15) In Anxiety And Depression in rats

Ponencia InvitadaGalanin (GAL) is involved in several functions including mood regulation. The GAL N-terminal fragment (1-15) [GAL(1-15)] also participates at central level and a differential role of GAL(1-15) compared with GAL has been proposed. In this work we have analysed if GAL(1-15) contributes to depression- and anxiety -related behaviours using the forced swimming test, tail suspension test, open field and light/dark test. We tested the involvement of the GAL receptor 2 (GALR2) in GAL(1-15) effects with the GAL receptor antagonist M871 and with an in vivo model of siRNA GALR2 knockdown rats. The proximity of GALR1 and GALR1 was also examined with the proximity ligation assay (PLA). GAL(1-15) induced strong depression-like and anxiogenic-like effects in all the tests. The involvement of the GALR2 was demonstrated with M871 and with the siRNA GALR2 knockdown rats. The PLA indicated the existence of GALR1-GALR2 heteroreceptor complexes in the dorsal hippocampus and especially in the dorsal raphe nucleus. Our results indicate that GAL(1-15) exerts strong depression-related and anxiogenic-like effects and may give the basis for the development of drugs targeting GALR1-GALR2 heteroreceptor complexes in the raphe-limbic system for the treatment of depression and anxiety. This study was supported by Junta de Andalucía CVI6476.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Galanin receptor 2 modifies neuropeptide Y Y1 receptor internalization and β-Arrestin recruitment

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

GALR1/GALR2 Knockdown rats block the Depression and Anxiogenic effects induced by GAL(1-15): The Heterodimer GALR1/GALR2 as a target of GAL(1-15).

Comunicación OralThe 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<0.001) and decreased climbing (p<0.01) induced by GAL(1-15). In the OFT M871 also significantly decreased the number of entries (p<0.001) and time spent in the center (p<0.05) mediated by GAL(1-15). Down-regulation of GALR2 or GALR1 by siRNA was sufficient to block the effect of GAL(1-15) in behavioural tests. Thus, GAL(1-15) 3nmol lacked effect on the immobility, climbing and swimming time in the FST. The same effect was observed in the number of entries and time spent in the central square in the OFT. These results indicated that GALR1 and GALR2 are involved in the GAL(1-15) depression- and anxiogenic-like effects suggesting that GAL(1-15) could act through GALR1/GALR2 heteroreceptor complex. These findings may give the basis for the development of novel therapeutic drugs targeting GAL(1-15) system for treatment of depression and anxiety disorders. This study was supported by Junta de Andalucía CVI6476.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Long-lasting memory deficits in mice withdrawn from cocaine are concomitant with neuroadaptations in hippocampal basal activity, GABAergic interneurons and adult neurogenesis

Cocaine addiction disorder is notably aggravated by concomitant cognitive and emotional pathology that impedes recovery. We studied whether a persistent cognitive/emotional dysregulation in mice withdrawn from cocaine holds a neurobiological correlate within the hippocampus, a limbic region with a key role in anxiety and memory but that has been scarcely investigated in cocaine addiction research. Mice were submitted to a chronic cocaine (20 mg/kg/day for 12 days) or vehicle treatment followed by 44 drug-free days. Some mice were then assessed on a battery of emotional (elevated plus-maze, light/dark box, open field, forced swimming) and cognitive (object and place recognition memory, cocaine-induced conditioned place preference, continuous spontaneous alternation) behavioral tests, while other mice remained in their home cage. Relevant hippocampal features [basal c-Fos activity, GABA+, parvalbumin (PV)+ and neuropeptide Y (NPY)+ interneurons and adult neurogenesis (cell proliferation and immature neurons)] were immunohistochemically assessed 73 days after the chronic cocaine or vehicle protocol. The cocaine-withdrawn mice showed no remarkable exploratory or emotional alterations but were consistently impaired in all the cognitive tasks. All the cocaine-withdrawn groups, independent of whether they were submitted to behavioral assessment or not, showed enhanced basal c-Fos expression and an increased number of GABA+ cells in the dentate gyrus. Moreover, the cocaine-withdrawn mice previously submitted to behavioral training displayed a blunted experience-dependent regulation of PV+ and NPY+ neurons in the dentate gyrus, and neurogenesis in the hippocampus. Results highlight the importance of hippocampal neuroplasticity for the ingrained cognitive deficits present during chronic cocaine withdrawal

Galanin (1-15) increased the neuropétide-Y Y1 receptor bindng in the amygdala of the rats

Galanin (GAL) and N-terminal Galanin fragment (1-15) [GAL(1-15)] interact at membrane level with Neuropeptide Y receptors. In the nucleus of the solitary tract GAL modified the cardiovascular action of the NPYY1 and GAL(1-15) facilitated the NPYY2 agonist effect. Recently, we observed that in the amygdala, GAL increased the anxiolytic effect and the binding of the NPYY1 agonist. The aim of this work was to study if GAL(1-15) interacts as well with NPYY1 receptors in the amygdala. We analyzed the binding of the NPYY1 agonist [125I]-Leu31-Pro34-PPY (25pM) in the presence of GAL(1-15) at different doses. The GAL antagonist M40 was also used. In behavioural experiments, groups of rats (n=8-10) received intracerebroventricular GAL(1- 15)(1nM) and Leu31-Pro34-NPY (0.1nM or 3nM) alone or in combination fifteen minutes before starting 5-minute session in the open field (OF) and in the elevated plus maze (EPM). We analyzed the number of entries and time in the central square of OF and the percentages of entries and time in the open arms of EPM. GAL(1-15) at 1nM increased the NPYY1 agonist binding by 10% (p<00.1) in amygdala, M40 blocked this effect. In the behavioral tests, GAL(1-15) did not change the effect of NPYY1 in the OF or EPM. These results indicate that in the amygdala, GAL(1-15) interacts with NPYY1 receptor at membrane level but not in the unconditioned anxiety-like tests. These results may be of relevance for GAL(1-15) mediated actions in the central nervous system.This work has been supported by the Junta de Andalucia CVI6476.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Galanin decreases NPYY1R internalization and β- Arrestin2 recruitment

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

GALR2/NPYY1R heterodimers interact at receptor level in the dentate gyrus of the hippocampus in rats

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<0,05) and the NPYY1R mRNA expression in the granular layer of DG by 31% (p<0,001). Moreover, PLA-positive red clusters were found specifically in the polymorphic layer and subgranular zone of the DG. No PLA clusters were observed neither in the molecular layer of the DG nor in the corpus callosum, an area that seems to lack of GALR2 receptor. These results demonstrate a novel mechanism of interaction between GAL and NPY1R in the DG at receptor level, probably involving the formation of GALR2/NPYY1R heteroreceptor complexes. Study supported by Junta de Andalucia CVI6476.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Study supported by Junta de Andalucia CVI6476