GAL(1-15) induces a depression and anxiogenic effect trough GALR1/GALR2 heteroreceptor: siRNA GALR1/GALR2 knockdown rats

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<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

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

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

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

Galanin N-Terminal fragment (1-15) enhances the antidepressant effects of the 5-HT1A receptor agonist 8-OH-DPAT in the Forced Swimming Test.

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<0.05) and climbing (p<0.05) and increased the swimming (p<0.01) behaviour induced by an effective dose of 8-OH-DPAT (0.25mg/Kg) in FST. Moreover, after coadministration of GAL(1-15) and threshold dose of 8-OH-DPAT (0.125mg/Kg) a significant decreased appeared in immobility (p<0.01) and climbing (p<0.01) and increased the swimming behavior (p<0.001) vs 8-OH-DPAT group. Moreover, M871 blocked completely this interaction. These results indicate that GAL(1-15) enhances the antidepressant effects induced by 8-OH-DPAT in the FST. These findings may give the basis for the development of novel therapeutic drugs. This study was supported by Junta de Andalucía CVI6476.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. This study was supported by Junta de Andalucía CVI6476

Aggressive behavior of Merkel cell carcinoma of the skin may be related to tumor microvasculature density

First primary MCC is a disease of elderly people. It is only sporadically reported before age 50; then, its overall age-adjusted incidence rate starts increasing gradually, from age 50 to age 65, then progressively in both males and females This study was undertaken to: (1) assess Langerhans cell population, that of their precursor, stromal microvasculature density, and epidermal proliferative index in Merkel Cell Carcinoma (MCC) and (2) to evaluate the prognostic value of these parameters in its metastasis occurrence. This is a retrospective study investigating twenty-five subjects aged between 20 and 90 (mean 70) years with biopsy-proven MCC. Patients were distributed into several groups according to the occurrence of metastasis. Diagnosis was based on the positive identification of a specific immunohistochemical profile including CK 20, NSE and Chromogranin. Langerhans cells were identified using an antibody directed against membrane CD1a while their precursors were characterized by human CD34 positive immunostaining. Proliferation index was quantified using MIB-1 antibody. As expected, surface density of CD1a+ cells was significantly reduced in epidermis overlaying the tumor, as compared with normal skin.. On the other hand, proliferation index of MCC cells and overlaying keratinocytes was very high (41% and 13%, respectively). No correlation was found between these parameters and the clinical outcome of patients. Immunohistochemical staining labeling with CD34 also showed a higher number of intratumoral blood vessels compared with other skin malignancies such as melanoma among others. The median observation period was 38 months. Together, these results indicate that intratumoral microvasculature density may be the one of these quantitative parameters that correlates with metastasis occurrence.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

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

Comunicación OralThe presence of Galanin and Neuropeptide Y and/or their receptors in many relevant brain regions related to learning and memory, mood, cardiovascular control and food intake implies that Galanin, and Neuropeptide Y may balance the physiological actions of one another. We will describe the evidence for the existence of Galanin Receptor/ Neuropeptide Y Receptor interactions in several nucleus including the nucleus of the solitarii tract, hypothalamus, dorsal raphe nucleus and amygdala, probably taking place in Galanin Receptor-Neuropeptide Y Y1 Receptor heteromers. These receptor heteromers may be one key molecular mechanism for Galanin to modulate the function of different types of glia–neuronal networks in the CNS, especially the emotional, metabolic and cardiovascular networks.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Galanin N-Terminal fragment (1-15) induces a stronger effect than Galanin in depression- and anxiety- like related behavioural tests in rats.

Galanin (GAL) and GAL Receptors are implicated in anxiety- and depression- related behaviours. The GAL N-terminal fragment (1-15) [GAL(1-15)] also induces depressant- and anxiogenic- like actions. In this work, we have compared the effects of GAL and GAL(1-15) in depression- and anxiety- related behaviours using the Forced Swimming Test (FST) and the Open Field Test (OFT). In the first set of experiments, rats (n=8-10) were injected with GAL 3nmol, GAL(1-15) 3nmol or vehicle 15min before the FST. Two sessions were conducted: a 15min. pre-test followed 24h later by 5min. test. The duration of immobility and climbing were recorded during the test. In the second set, rats (n=6-10) were injected with GAL 3nmol, GAL(1-15) 3nmol or vehicle 15min before the OFT. Time and entries in the central square during 300 Sec. were scored. In the FST, the increase in the immobility induced by GAL(1-15) was significantly higher (p<0.01) than the one induced by GAL. In the climbing response GAL(1-15) also induced a significantly stronger decrease (p<0.05) in climbing compared with GAL. In the OFT, GAL(1-15) significantly decrease the number of entries (p<0.01) and time (p<0.05) in centre while GAL lacks of effect. These results indicate that GAL(1-15) has more powerful effects than GAL in depression-like behaviour in the FST and anxiogenic-like effects in the OFT. 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.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

NPYY1 /Galanin 2 heterodimers in the amygdala: a proximity ligation assay study

We have shown that Galanin(GAL) interacts with Neuropeptide Y Y1 receptor(Y1) at behavioural, cellular and receptor levels in the Amygdala. Since both peptides play an important role in mood disorders, the aim of this work was to study the GALR2/Y1 heterodimer formation within the amygdala(AMY), key nucleus for fear, mood, and motivation. We performed in situ proximity ligation assay(PLA) in the Amygdala and also in HEK-293 cells. Amygdala sections were incubated with anti-GALR2 Rabbit(1:100) and anti- NPYY1R Goat(1:200). In transiently transfected HEK293T cells coexpressing Y1R(3xHA) and GALR2 mouse anti-HA(1:5000) and rabbit anti-GALR2(1:500) were used. In both cases 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). PLA-positive red clusters were found in large number of cells of the Basolateral, Intercalated and Central nuclei of the Amygdala. No PLA clusters were observed in lateral corpus callosum, an area that seems to lack of GALR2 receptor. In HEK293T cells we reproduced the results obtained in the Amygdala, showing PLA-positive red clusters in large number of cells. The fact that we found PLA-positive red clusters in a large number of cells not only in the Amygdala but also in the HEK293T cells line give indications that GALR2 and NPYY1R are forming GALR2/NPYY1R heterodimers, providing a novel mechanism with implications in behavior, particularly in the anxiety response.Universidad de Málaga, Campus de Excelencia Internacional Andalucía Tech. Spanish CVI647