Immunohistochemical C-FOS expression and autoradiography to study galnin/neuropeptide y Y1 receptor-receptor interactions in the amygdala

We have shown Galanin(GAL)/Neuropeptide Y Y1 receptor(Y1) interactions in the nucleus tractus solitarius and the arcuate nucleus. Since both peptides play an important role in mood disorders, the aim of this work was to study GAL/Y1 interactions in the amygdala(AMY), key nucleus for fear, mood, and motivation. We have combined the analysis of the expression of c-Fos immunoreactivity(c-Fos IR) with an autoradiographic study in the AMY. Groups of anaesthetized rats (n=4) received intracerebroventricular injections(icv) of GAL(3nmol) and the Y1 agonist Leu31-Pro34NPY(3nmol) alone or in combination, and were sacrificed 90 minutes after the injections. Immunohistochemical detection of c-Fos protein(1:5000) in AMY sections and stereological analysis were performed in: Basal(BA), lateral(LA), Central [lateral capsular subdivision(CeC), lateral intermediate subdivision(CeI), medial subdivision(CeM)] and the medial paracapsular intercalated(ITC) subnuclei of the AMY. For Autoradiography, rats (n=6) were sacrificed 15 minutes after icv injections of GAL(3nmol) and AMY sections were incubated with Y1 agonist [125I]-Leu31-Pro34-PPY (25 pM). Autoradiograms were analyzed using the NIH image analysis system. Student’s unpaired t- test and ANOVA followed by Newman-Keuls were used, respectively. We observed within the AMY that GAL increased c-Fos IR in ITC and CeC; the Y1 agonist induced both, an increase of c-Fos IR in BA and CeC and a decrease of c-Fos IR in LA and ITC. The coadministration of both peptides induced a specific effect in the ITC, significantly decreasing the c-Fos expression (P<0,05) induced by GAL or the Y1 agonist alone. Moreover, we observed that GAL significantly increased (p<0,05) the Y1 receptor agonist binding [I125]Leu31Pro34-PYY in the AMY. These results demonstrate an interaction between GAL and Y1 at the cellular and receptor level in the AMY and suggest that endogenous GAL and NPY system might interact to regulate emotional behaviours.Spanish CVI6476, TV3-Marató 090130/31/32 and Universidad de Málaga (Campus de Excelencia Internacional Andalucía Tech

Galanin/ Neuropeptide Y Y1 receptor interaction at the cellular and receptor level in the amygdala

Galanin (GAL) interacts functionally with Neuropeptide Y Y1 receptor (Y1) in the nucleus tractus solitarius and the arcuate nucleus. Since GAL and Y1 participate in mood disorders, the aim of this work was to analyze GAL/Y1 interactions in the amygdala (AMY), a key nucleus for fear, mood, and motivation. We have combined an autoradiography study with the analysis of the expression of c-Fos immunoreactivity (c-Fos IR) in the AMY. Rats (n=6) were sacrificed 15 minutes after intracerebroventricular injections (icv) of GAL (3nmol) and AMY sections were incubated with Y1 agonist [125I]-Leu31-Pro34-PPY (25 pM). The autoradiograms were analyzed using the NIH image analysis system. In c-Fos IR experiments, groups of anaesthetized rats (n=4) received icv GAL (3nmol) and the Y1 agonist Leu31- Pro34NPY (3nmol) alone or in combination, and the rats were sacrificed 90 minutes after the injections. AMY sections were stained with immunohistochemical of c-Fos protein (1:5000). Stereological analysis was performed in: Basal (BA), lateral (LA), Central [lateral capsular subdivision (CeC) , lateral intermediate subdivision (CeI), medial subdivision (CeM)] and the medial paracapsular intercalated (ITC) subnuclei of the AMY. Student’s unpaired t-test and ANOVA followed by Newman-Keuls were used in autoradiographic and c-Fos IR studies. We observed that GAL significant increased (p<0,05) the Y1 receptor agonist binding, [I125]Leu31Pro34-PYY in the AMY. Within the AMY, GAL increased c-Fos IR in ITC and CeC; the Y1 agonist induced an increased of c-Fos IR in BA and CeC, while a decrease in c-Fos IR was observed in LA and ITC. However, we only observed a modification of the c-Fos expression after the coadministration of both peptides compared to the effect of GAL or the Y1 agonist alone in the ITC. The coadministration of both peptides significantly decreased the c-Fos expression (P<0,05) induced by GAL or the Y1 agonist alone. These results demonstrate an interaction between GAL and NPY Y1 at the cellular and receptor level in the AMY and suggest that endogenous GAL and NPY system might interact to regulate emotional behaviours.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Marató Tv

Galanin(1-15) enhanced the antidepressant-like effects of escitalopram in the olfactory bulbectomy rats in the forced swimming test through 5-HT1A receptors

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<0.05) and increasing the swimming time (p<0.05). M871 blocked the behavioural effects of GAL(1-15) in the immobility time (p<0.001) and in the swimming time (p<0.05) in the FST. Moreover, the decrease in 5-HT1AR was sufficient to block GAL(1-15) enhancement of the antidepressant-like effects mediated by ESC. Our results indicate a potent effect of the combination GAL(1-15) with SSRIs in reversed depressive symptoms in the animal model of chronic depression OBX. The results open up the possibility of using GAL(1-15) in combination with SSRIs as a novel strategy for treating depression.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. PID2020-114392RB-I00, UMA18-FEDERJA-008 and P20_00026, PI-0083-2019

Galanin (1-15) enhances the behavioural effects of escitalopram in the forced swimming test in rats

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<0.05), while lacking effects over immobility time. The threshold dose of GAL(1-15) 1nmol enhanced the antidepressant-like effects mediated by ESC 7,5mg/Kg, decreasing the immobility (p<0.05) and increasing the swimming time (p<0.05) in the FST. In TST, no differences were found between the treatments. Our results indicate an interaction between GAL(1-15) and ESC in the FST and open up the possibility to use GAL(1-15) for a combination therapy with SSRIs as a depression treatment.This study was supported by Spanish SAF2016-79008-P, PI-0083-2019 and UMA18-FEDERJA-008 Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Galanin (1-15) potentiates the antidepres-sant-like effects induced by Escitalopram in a rat model of depression

Selective 5-HT reuptake inhibitor antidepressants (SSRIs) are the first choice in major depressive disorder (MDD), but 50% of affected patients do not show improvement. Galanin(1-15) [GAL(1-15)] enhanced Fluoxetine antidepressant-like effects in an animal model of depression, the olfactory bulbectomy (OBX); however, further detailed analysis of GAL(1-15) effects as augmentation treatment in OBX rats are needed. In OBX rats, we analysed the effect of GAL(1–15) on Escitalopram (ESC)-mediated responses in behavioural tests related to despair. We studied whether GAL(1–15) effects involved 5-HT1AR using an in vivo model siRNA 5-HT1A knockdown rats. Moreover, we analysed by immunohistochemistry the expression of the immediate-early gene c-Fos (c-Fos IR) after the administration of GAL(1-15)+ESC in OBX rats in several nuclei involved in MDD. GAL(1-15) enhances the antidepressant-like effects of ESC, and the GALR2 antagonist M871 blocked GAL(1-15) mediated actions. The downregulation of 5-HT1AR by siRNA was sufficient to block GAL(1-15) effects. Our immunohistochemistry and principal component analysis (PCA) analysis suggest that two functional networks are involved in these effects; one includes the lateral (LHb) and medial (mHb) habenula, dorsal raphe (DR) and ventral tegmental area (VTA), and the other consists of the dentate gyrus (DG), and prefrontal cortex (PFC). The results open up the possibility of using GAL(1-15) in combination with SSRIs as a novel strategy for treating MDD

Galanin (1-15) enhancement of the behavioral effects of a 5-HT1AR agonist and fluoxetine in the forced swimming test gives a new therapeutic strategy against depression: possible role of GALR1-GALR2-5-HT1AR heteroreceptor complexes

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 administration of Galanin N-Terminal fragment (1-15) induces depressant- and anxiogenic effects in rats.

PosterGalanin (GAL) is involved in several functions including mood regulation. Converging evidence implicates GAL and GAL receptors in anxiety- and depression-related behaviours. Intracerebroventricular (icv) GAL in animals results in enhanced depression-like behaviour in the forced swim test and in anxiolytic-like effects under stress conditions in the elevated plus maze. 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 four tests: forced swimming test (FST), elevated plus maze (EPM), open field (OF) and passive avoidance task (PA). In the first set of experiments four groups of rats (n=8-10) were treated three different times with icv GAL(1-15) 1, 3 and 6nmol or vehicle 15 minutes before the tests. Behavioural assessments were conducted with at least 1 week between tests. In the EPM the percentages of entries and time in open arms during 300 Sec were scored. In the OF test, the time and entries in the central square during 300 Sec were scored. The distance and spent were also determined. In the FST two sessions were conducted: a 15 min pre-test followed 24 h later by a 5 min test. The total duration of immobility and active climbing was recorded during the second, 5 min test. In the second set the PA was performed. Rats (n=8-10) received icv GAL(1-15) 1, 3 and 6nmol or vehicle 15 minutes prior the training session. Retention latency was tested 24 h after training during 600 sec. In the OF test the administration of GAL(1-15) 3 and 6nmol significantly decreased the number of entries (p<0.01) and time spent (p<0.05) in the central square by 42% and 39% respectively. However, in the EPM GAL(1-15) did not change any of the parameters analysed. Gal(1-15) didn’t change the Locomotor Activity in any test. In the FST GAL(1-15) 3 and 6nmol significantly increased immobility (p<0.01) and decreased the climbing behaviour (p<0.01) by 44% and 46% respectively. In the PA GAL(1-15) 6nmol significantly decreased the retention latency (p<0.05). These results indicate that GAL(1-15) produces anxiogenic-like effects in the OF and depression-like behaviour in the FST, GAL(1-15) may also have an effect on emotional memory in PA. These results may give the basis for the development of novel therapeutic drugs targeting GAL(1-15) system for treatment of depression and anxiety disorders.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. This study was supported by Junta de Andalucía CVI6476 and TV3- Marató 090130/31/32

New augmentation strategy in depression: Galanin (1-15) enhances the behavioral effects of Fluoxetine in the olfactory bulbectomy rat.

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<0.05) and increases the swimming time by 30% (p<0.01) compared with FLX in the FST, and in the SPT reversed the effects of the OBX procedure increasing the sucrose intake (p<0.05) and preference (p<0.05). The coadministration of GAL(1-15)(1nmol)+FLX(10mg/kg) also reduced the OBX-increased corticosterone levels by approximately 50% (p<0.05). In conclusion, these novelty results suggest using GAL(1-15) in combination with FLX as a novel strategy fortreating depression.Supported by Spanish Ministry of Economy PID2020-114392RB-I00, PDC2021-121566-I00; Junta de Andalucia P20_00026, PI-0083-2019 and UMA18-FEDERJA-008. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

The Combination of Galanin (1–15) and Escitalopram in Rats Suggests a New Strategy for Alcohol Use Disorder Comorbidity with Depression

Alcohol use disorder (AUD) is highly prevalent, and over 50% of AUD patients also suffer major depressive disorders. Selective 5-HT reuptake inhibitors (SSRIs) can reduce rodent ethanol drinking but exert modest clinical efficacy in alcoholic individuals. Finding new pharmacological strategies that could modulate alcohol consumption and depression is necessary. We have analyzed the effect of Galanin (1–15) [GAL(1–15)] on escitalopram (ESC)-mediated effect in alcohol consumption using the alcohol self-administration test, the nuclei involved in the effect, and whether GAL(1–15) + ESC modulated the response in despair or anxiety tests in animals under chronic alcohol intake. GAL(1–15) + ESC combination substantially reduced alcohol intake in the alcohol self-administration test and, moreover, enhanced the reduction of reward capacity of ESC on different reinforcers such as sucrose or saccharine. GAL(1–15) + ESC coadministration significantly decreases the number of C-Fos-IR TH cell bodies in the VTA, and PCA analysis suggests that one functional network, including VTA, RMTg and DR, is involved in these effects. Significantly in rats with chronic alcohol consumption, GAL(1–15) reversed adverse ESC-mediated effects in the depression-related behavioural test and forced swimming test. The results open up the possibility of using GAL(1–15) in combination with the SSRI Escitalopram as a novel strategy in AUD comorbidity with depression