Antonio Flores-Burgess

A Role for Galanin N-Terminal Fragment (1–15) in Anxiety- and Depression-Related Behaviors in Rats

Background: Galanin (GAL) plays a role in mood regulation. In this study we analyzed the action of the active N-terminal fragment [GAL(1–15)] in anxiety- and depression-related behavioral tests in rats. Methods: The effect of GAL(1–15) was analyzed in the forced swimming test, tail suspension test, open field test, and light/dark test. The proximity of GAL1 and GAL2 receptors was examined with the proximity ligation assay (PLA). We tested the GAL receptors involved in GAL(1–15) effects with the GAL2 receptor antagonist M871 and with an in vivo model of siRNA GAL2 receptor knockdown or siRNA GAL1 receptor knockdown rats. The effects of GAL(1–15) were also studied in the cell line RN33B. Results: GAL(1–15) induced strong depression-like and anxiogenic-like effects in all the tests. These effects were stronger than the ones induced by GAL. The involvement of the GAL2 receptor was demonstrated with M871 and with the siRNA GAL2 receptor knockdown rats. The PLA indicated the possible existence of GAL1 and GAL2 heteroreceptor complexes in the dorsal hippocampus and especially in the dorsal raphe nucleus. In the siRNA GAL1 receptor knockdown rats the behavioral actions of GAL(1–15) disappeared, and in the siRNA GAL2 receptor knockdown rats the reductions of the behavioral actions of GAL(1–15) was linked to a disappearance of PLA. In the cell line RN33B, GAL(1–15) decreased 5-HT immunoreactivity more strongly than GAL. Conclusions: 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 GAL1 and GAL2 heteroreceptor complexes in the raphe-limbic system for the treatment of depression and anxiety.

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.

Galanin N-terminal fragment (1–15) [GAL(1–15)] is associated with depression-related and anxiogenic-like effects in rats. In this study, we analyzed the ability of GAL(1–15) to modulate 5-HT1A receptors (5-HT1AR), a key receptor in depression. GAL(1–15) enhanced the antidepressant effects induced by the 5-HT1AR agonist 8-OH-DPAT in the forced swimming test. These effects were stronger than the ones induced by Galanin (GAL). This action involved interactions at receptor level since GAL(1–15) affected the binding characteristics and the mRNA levels of 5-HT1AR in the dorsal hippocampus and dorsal raphe. The involvement of the GALR2 was demonstrated with the GALR2 antagonist M871. Proximity ligation assay experiments indicated that 5-HT1AR are in close proximity with GALR1 and GALR2 in both regions and in raphe RN33B cells. The current results indicate that GAL(1–15) enhances the antidepressant effects induced by 8-OH-DPAT acting on 5-HT1AR operating as postjunctional or as autoreceptors. These results may give the basis for the development of drugs targeting potential GALR1–GALR2–5-HT1AR heteroreceptor complexes linked to the raphe-hippocampal 5-HT neurons for the treatment of depression.

Galanin receptor 2-neuropeptide Y Y1 receptor interactions in the amygdala lead to increased anxiolytic actions

Galanin (GAL) and neuropeptide Y (NPY) are neuropeptides involved in behaviors associated with anxiety. Both neuropeptides interact in several central functions. However, the potential behavioral and cellular interactions between them in anxiety are unknown. GAL was found to act through GAL receptor 2 (GALR2) to enhance NPYY1 receptor (NPYY1R)-mediated anxiolytic behaviors in rats. Using receptor autoradiography, c-Fos expression and in situ proximity ligation assay, the medial paracapsular intercalated nuclei of the amygdala were determined to be a key area in the interaction probably involving the formation of GALR2/NPYY1R heteroreceptor complexes. In cell cultures costimulation of GALR2 and NPYY1R induced changes in the functions of these receptors. The changes involved a potentiation of the decrease in the phosphorylation of CREB induced by NPYY1R and a delay in the internalization of NPYY1R. These results indicate that GALR2/NPYY1R interactions can provide a novel integrative amygdaloid mechanism in anxiety.

Galanin (1-15) enhancement of the behavioral effects of Fluoxetine in the forced swimming test gives a new therapeutic strategy against depression

ABSTRACT The pharmacological treatment of major depression is mainly based on drugs elevating serotonergic (5‐HT) activity. Specifically, selective 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 serotoninergic system and neuropeptides signaling could be a key aspect. We examined the ability of the neuropeptide Galanin(1–15) [GAL(1–15)] to modulate the behavioral effects of FLX in the forced swimming test (FST) and studied feasible molecular mechanisms. The data show that GAL(1–15) enhances the antidepressant‐like effects induced by FLX in the FST, and we demonstrate the involvement of GALR1/GALR2 heteroreceptor complex in the GAL(1–15)‐mediated effect using in vivo rat models for siRNA GALR1 or GALR2 knockdown. Importantly, 5‐HT1A receptors (5HT1A‐R) also participate in the GAL(1–15)/FLX interactions since the 5HT1AR antagonist WAY100635 blocked the behavioral effects in the FST induced by the coadministration of GAL(1–15) and FLX. The mechanism underlying GAL(1–15)/FLX interactions affected the binding characteristics as well as the mRNA levels of 5‐HT1A‐R specifically in the dorsal hippocampus while leaving unaffected mRNA levels and affinity and binding sites of this receptor in the dorsal raphe. The results open up the possibility to use GAL(1–15) as for a combination therapy with FLX as a novel strategy for treatment of depression. HIGHLIGHTSGAL(1–15) enhances the antidepressant‐like effects induced by Fluoxetine.5‐HT1A receptors participate in the GAL(1–15)/FLX interactions.GAL(1–15)/FLX interactions affected binding and 5‐HT1AR mRNA levels in hippocampus.

Galanin receptor 2-neuropeptide Y Y1 receptor interactions in the dentate gyrus are related with antidepressant-like effects

Galanin (GAL) and the NPYY1 agonist play a role in mood regulation and both neuropeptides interact in several central functions. The present study examined the interaction between Galanin receptor 2 (GALR2) and Neuropeptide Y Y1 receptor (NPYY1R) in the dentate gyrus (DG) of the Hippocampus in relation to depression-like behavior. Using receptor autoradiography, in situ hybridization and in situ proximity ligation assay an interaction between GALR and NPYY1R was demonstrated in the DG probably involving the formation of GALR2-NPYY1R heteroreceptor complexes. These complexes were specifically observed in the polymorphic and subgranular subregions of the DG, where both receptors were found to colocalize. Moreover, this GALR2/NPYY1R interaction was linked to an enhancement of the antidepressive-like behavior mediated by NPYY1R in the forced swimming test. Specific cells populations within DG subregions may be involved in this behavioral effect since the coactivation of GALR2 and NPYY1R enhances the NPYY1R-mediated reduction in the number of c-Fos immunoreactive nuclei in the polymorphic region. These results indicate that GALR2/NPYY1R interactions can provide a novel integrative mechanism in DG in depression-related behavior and may give the basis for the development of drugs targeting GALR2/NPYY1R heteroreceptor complexes in the DG of the hippocampus for the treatment of depression.

The neuropeptides Galanin and Galanin(1–15) in depression-like behaviours

Galanin is a 29 amino acid neuropeptide widely distributed in neurons within the central nervous system. Galanin exerts its biological activities through three different G protein-receptors and participates in a number of functions, including mood regulation. Not only Galanin but also Galanin N-terminal fragments like Galanin(1-15) are active at the central level. In this work, we review the latest findings in studies on Galanin and Galanin(1-15) in depression-related behaviours. Our focus is on animal models for depression, and we pay some attention to research data obtained in human studies. Since Serotonin (5-HT), especially through 5-HT1A, and Galanin receptors interact at both pre-and postsynaptic level, the development of drugs targeting potential GAL1-GAL2-5-HT1A heteroreceptor complexes linked to the raphe-hippocampal 5-HT neurons may represent new treatment strategies in depression.

Galanin receptor/neuropeptide y receptor interactions in the central nervous system.

The presence of Galanin and Neuropeptide Y and/or their receptors in several areas of the brain involved in memory, mood, cardiovascular control and food intake indicates that Galanin, and Neuropeptide Y could equilibrate the physiological actions of each other. There is evidence for the existence of interactions between Galanin Receptor and Neuropeptide Y Receptor in the nucleus of the solitarii tract (NTS), hypothalamus and dorsal raphe nucleus probably taking place with the formation of heteromers between Galanin Receptor and Neuropeptide Y Y1 Receptor. The galanin fragment (Gal 1-15) preferring receptors may instead be formed by the GalR1-GalR2 heteromer which in the NTS may interact with Neuropeptide Y Y2 receptors. These receptor heteromers may be one key molecular mechanism for Galanin and its N-terminal fragment (Galanin 1-15) to modulate the function of different types of glia-neuronal networks in the CNS, especially the emotional, metabolic and cardiovascular networks.

Central administration of galanin N-terminal fragment 1-15 decreases the voluntary alcohol intake in rats: Galanin (1-15) and alcohol

Alcohol consumption is considered a major risk factor for disease and mortality worldwide. In the absence of effective treatments in alcohol use disorders, it is important to find new biological targets that could modulate alcohol consumption. We tested the role of the N‐terminal galanin 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 the whole molecule of GAL. We describe for the first time that GAL(1–15), via central mechanisms, induces a strong reduction in preference and ethanol consumption in rats. These effects were significantly different than GAL. GAL receptor (GALR) 2 was involved in these effects, because the specific GALR2 antagonist M871 blocked GAL(1–15) mediated actions in preference and ethanol intake. Importantly, the mechanism of this action involves changes in GALR expression and also in immediate‐early gene C‐Fos and receptors‐internalization‐related gene Rab5 in the striatum. The relevance of the striatum as a target for GAL(1–15) was supported by the effect of GAL(1–15) on the locomotor activity of rats after ethanol administration. These results may give the basis for the development of novel therapeutics strategies using GAL(1–15) analogues for the treatment of alcohol use disorders in humans.

Receptor–Receptor Interactions in Multiple 5-HT1A Heteroreceptor Complexes in Raphe-Hippocampal 5-HT Transmission and Their Relevance for Depression and Its Treatment

Due to the binding to a number of proteins to the receptor protomers in receptor heteromers in the brain, the term “heteroreceptor complexes” was introduced. A number of serotonin 5-HT1A heteroreceptor complexes were recently found to be linked to the ascending 5-HT pathways known to have a significant role in depression. The 5-HT1A–FGFR1 heteroreceptor complexes were involved in synergistically enhancing neuroplasticity in the hippocampus and in the dorsal raphe 5-HT nerve cells. The 5-HT1A protomer significantly increased FGFR1 protomer signaling in wild-type rats. Disturbances in the 5-HT1A–FGFR1 heteroreceptor complexes in the raphe-hippocampal 5-HT system were found in a genetic rat model of depression (Flinders sensitive line (FSL) rats). Deficits in FSL rats were observed in the ability of combined FGFR1 and 5-HT1A agonist cotreatment to produce antidepressant-like effects. It may in part reflect a failure of FGFR1 treatment to uncouple the 5-HT1A postjunctional receptors and autoreceptors from the hippocampal and dorsal raphe GIRK channels, respectively. This may result in maintained inhibition of hippocampal pyramidal nerve cell and dorsal raphe 5-HT nerve cell firing. Also, 5-HT1A–5-HT2A isoreceptor complexes were recently demonstrated to exist in the hippocampus and limbic cortex. They may play a role in depression through an ability of 5-HT2A protomer signaling to inhibit the 5-HT1A protomer recognition and signaling. Finally, galanin (1–15) was reported to enhance the antidepressant effects of fluoxetine through the putative formation of GalR1–GalR2–5-HT1A heteroreceptor complexes. Taken together, these novel 5-HT1A receptor complexes offer new targets for treatment of depression.

A Novel Integrative Mechanism in Anxiolytic Behavior Induced by Galanin 2/Neuropeptide Y Y1 Receptor Interactions on Medial Paracapsular Intercalated Amygdala in Rats

Anxiety is evoked by a threatening situation and display adaptive or defensive behaviors, found similarly in animals and humans. Neuropeptide Y (NPY) Y1 receptor (NPYY1R) and Galanin (GAL) receptor 2 (GALR2) interact in several regions of the limbic system, including the amygdala. In a previous study, GALR2 enhanced NPYY1R mediated anxiolytic actions on spatiotemporal parameters in the open field and elevated plus maze, involving the formation of GALR2/NPYY1R heteroreceptor complexes in the amygdala. Moreover, the inclusion of complementary ethological parameters provides a more comprehensive profile on the anxiolytic effects of a treatment. The purpose of the current study is to evaluate the anxiolytic effects and circuit activity modifications caused by coactivation of GALR2 and NPYY1R. Ethological measurements were performed in the open field, the elevated plus-maze and the light-dark box, together with immediate early gene expression analysis within the amygdala-hypothalamus-periaqueductal gray (PAG) axis, as well as in situ proximity ligation assay (PLA) to demonstrate the formation of GALR2/NPYY1R heteroreceptor complexes. GALR2 and NPYY1R coactivation resulted in anxiolytic behaviors such as increased rearing and head-dipping, reduced stretch attend postures and freezing compared to single agonist or aCSF injection. Neuronal activity indicated by cFos expression was decreased in the dorsolateral paracapsular intercalated (ITCp-dl) subregion of the amygdala, ventromedial hypothalamic (VMH) nucleus and ventrolateral part of the periaqueductal gray (vlPAG), while increased in the perifornical nucleus of the hypothalamus (PFX) following coactivation of GALR2 and NPYY1R. Moreover, an increased density of GALR2/NPYY1R heteroreceptor complexes was explicitly observed in ITCp-dl, following GALR2 and NPYY1R coactivation. Besides, knockdown of GALR2 was found to reduce the density of complexes in ITCp-dl. Taken together, these results open up the possibility that the increased anxiolytic activity demonstrated upon coactivation of NPYY1R and GALR2 receptor was related to actions on the ITCp-dl. GALR2-NPYY1R heteroreceptor complexes may inhibit neuronal activity, by also modifying the neuronal networks of the hypothalamus and the PAG. These results indicate that GALR2/NPYY1R interactions in medial paracapsular intercalated amygdala can provide a novel integrative mechanism in anxiolytic behavior and the basis for the development of heterobivalent agonist drugs targeting GALR2/NPYY1R heteromers, especially in the ITCp-dl of the amygdala for the treatment of anxiety.