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Activation of Neuropeptide Y Receptors Modulates Retinal Ganglion Cell Physiology and Exerts Neuroprotective Actions In Vitro.

Martins J, Elvas F, Brudzewsky D, Martins T, Kolomiets B, Tralhão P, Gøtzsche CR, Cavadas C, Castelo-Branco M, Woldbye DP, Picaud S, Santiago AR, Ambrósio AF - ASN Neuro (2015)

Bottom Line: We found that NPY attenuated the increase in the [Ca2+]i triggered by glutamate mainly via Y1 receptor activation.Using in vitro cultures of rat retinal explants exposed to NMDA, we found that NPY pretreatment prevented NMDA-induced cell death.In conclusion, we found modulatory effects of NPY application that for the first time were detected at the level of RGCs.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, 3004-548 Coimbra, Portugal CNC.IBILI, University of Coimbra, 3004-548 Coimbra, Portugal.

No MeSH data available.


Related in: MedlinePlus

NPY reduces NMDA-induced cell death in the GCL of retinal explants via Y1 and Y5 receptor activation. Retinal explants were exposed to 300 µM NMDA for 48 h to induce an excitotoxic insult. The agonists, 1 µM NPY, 1 µM (Leu31, Pro34) − NPY, 300 nM NPY13–36, and 1 µM (Gly1, . . . Aib32)-PP, were applied 24 h and 60 min before NMDA application. All the antagonists: 1 µM BIBP 3226, 1 µM BIIE 0246, and 1 µM L-152,804 were applied 30 min before the agonist. (a) Representative images of TUNEL-positive cells (green) in the GCL of control (aa) or NMDA-treated (ab-af) retinal explants. Nuclei were stained with DAPI (blue). Retinal explants were pretreated with different drugs 24 h before exposure to NMDA. Scale bar: 100 µm. (b) Quantification of TUNEL-positive cells. NMDA exposure induced a significant increase in the number of TUNEL-positive cells in the GCL compared with control. Pretreatment with 1 µM NPY or 1 µM (Leu31, Pro34) − NPY (Y1/Y5 receptor agonist), 24 h and 60 min before NMDA, was able to reduce the increase in the number of TUNEL-positive cells induced by NMDA. Pretreatment with Y1 or Y5 receptor antagonist (1 µM BIBP 3226 or 1 µM L-152,804, respectively), when applied 30 min before NPY, blocked its protective effect. The effect of (Leu31, Pro34) − NPY was blocked by BIBP 3226. (c) NMDA exposure induced a significant increase in the number of PI-positive cells in retinal explants. Data are presented as the ratio between PI-positive cells at DIV4 and DIV2. NPY or (Leu31, Pro34) − NPY was able to reduce the increase in the number of PI-positive cells induced by NMDA. BIBP 3226 or L-152,804 blocked the protective effect of NPY, and BIBP 3226 blocked the protective effect of (Leu31, Pro34) − NPY. The application of 1 µM (Gly1, . . . Aib32)-PP was able to reduce the increase in PI-positive cells induced by NMDA, and this effect was blocked by L-152,804. Together, the results from TUNEL and PI assays suggest that NPY is able to protect retinal cells from an excitotoxic insult through the activation of Y1 and Y5 receptors. Data are presented as mean ± SEM of n = 5 to 11 independent experiments. ***p < .001, compared with control; ##p < .01, ###p < .001, compared with NMDA; §p < .05, §§p < .01, §§§p < .001, compared with NMDA + NPY, or NMDA + (Leu31, Pro34) − NPY, or NMDA + (Gly1, . . . Aib32)-PP. Kruskal-Wallis followed by Dunn’s test.
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fig6-1759091415598292: NPY reduces NMDA-induced cell death in the GCL of retinal explants via Y1 and Y5 receptor activation. Retinal explants were exposed to 300 µM NMDA for 48 h to induce an excitotoxic insult. The agonists, 1 µM NPY, 1 µM (Leu31, Pro34) − NPY, 300 nM NPY13–36, and 1 µM (Gly1, . . . Aib32)-PP, were applied 24 h and 60 min before NMDA application. All the antagonists: 1 µM BIBP 3226, 1 µM BIIE 0246, and 1 µM L-152,804 were applied 30 min before the agonist. (a) Representative images of TUNEL-positive cells (green) in the GCL of control (aa) or NMDA-treated (ab-af) retinal explants. Nuclei were stained with DAPI (blue). Retinal explants were pretreated with different drugs 24 h before exposure to NMDA. Scale bar: 100 µm. (b) Quantification of TUNEL-positive cells. NMDA exposure induced a significant increase in the number of TUNEL-positive cells in the GCL compared with control. Pretreatment with 1 µM NPY or 1 µM (Leu31, Pro34) − NPY (Y1/Y5 receptor agonist), 24 h and 60 min before NMDA, was able to reduce the increase in the number of TUNEL-positive cells induced by NMDA. Pretreatment with Y1 or Y5 receptor antagonist (1 µM BIBP 3226 or 1 µM L-152,804, respectively), when applied 30 min before NPY, blocked its protective effect. The effect of (Leu31, Pro34) − NPY was blocked by BIBP 3226. (c) NMDA exposure induced a significant increase in the number of PI-positive cells in retinal explants. Data are presented as the ratio between PI-positive cells at DIV4 and DIV2. NPY or (Leu31, Pro34) − NPY was able to reduce the increase in the number of PI-positive cells induced by NMDA. BIBP 3226 or L-152,804 blocked the protective effect of NPY, and BIBP 3226 blocked the protective effect of (Leu31, Pro34) − NPY. The application of 1 µM (Gly1, . . . Aib32)-PP was able to reduce the increase in PI-positive cells induced by NMDA, and this effect was blocked by L-152,804. Together, the results from TUNEL and PI assays suggest that NPY is able to protect retinal cells from an excitotoxic insult through the activation of Y1 and Y5 receptors. Data are presented as mean ± SEM of n = 5 to 11 independent experiments. ***p < .001, compared with control; ##p < .01, ###p < .001, compared with NMDA; §p < .05, §§p < .01, §§§p < .001, compared with NMDA + NPY, or NMDA + (Leu31, Pro34) − NPY, or NMDA + (Gly1, . . . Aib32)-PP. Kruskal-Wallis followed by Dunn’s test.

Mentions: NPY exerts neuroprotective actions in various brain regions, and also in the retina (Alvaro et al., 2008b; Santos-Carvalho et al., 2013a), suggesting that NPY receptors might be possible therapeutic targets for retinal degenerative diseases such as glaucoma. Therefore, in addition to the NPY modulatory effect on RGC activity detected in ex vivo retinas, we also evaluated whether NPY could be able to prevent retinal cell death induced by an excitotoxic insult. The excitotoxic insult was induced in retinal explants (cultured for four DIV) exposed to 300 µM NMDA for 48 h. Apoptotic retinal cell death was assessed by TUNEL assay, and necrotic or late apoptotic cell death was assessed by PI incorporation assay (Figure 6). Different drug treatments were applied to assess the potential protective effect of NPY receptor activation. Thus, NPY (1 µM, n = 6 retinas for TUNEL, n = 9 retinas for PI), the Y1/Y5 agonist (Leu31, Pro34) − NPY (1 µM, n = 6 for TUNEL, n = 7 for PI), the Y2 agonist NPY13–36, (300 nM, n = 6 for TUNEL, n = 11 for PI), the Y5 agonist (Gly1,…Aib32)-PP (1 µM, n = 6 for TUNEL, n = 6 for PI), or a drug-free solution (control, n = 11 for TUNEL, n = 10 for PI), were applied at DIV1 and DIV2, respectively 24 h and 60 min before NMDA treatment.Figure 6.


Activation of Neuropeptide Y Receptors Modulates Retinal Ganglion Cell Physiology and Exerts Neuroprotective Actions In Vitro.

Martins J, Elvas F, Brudzewsky D, Martins T, Kolomiets B, Tralhão P, Gøtzsche CR, Cavadas C, Castelo-Branco M, Woldbye DP, Picaud S, Santiago AR, Ambrósio AF - ASN Neuro (2015)

NPY reduces NMDA-induced cell death in the GCL of retinal explants via Y1 and Y5 receptor activation. Retinal explants were exposed to 300 µM NMDA for 48 h to induce an excitotoxic insult. The agonists, 1 µM NPY, 1 µM (Leu31, Pro34) − NPY, 300 nM NPY13–36, and 1 µM (Gly1, . . . Aib32)-PP, were applied 24 h and 60 min before NMDA application. All the antagonists: 1 µM BIBP 3226, 1 µM BIIE 0246, and 1 µM L-152,804 were applied 30 min before the agonist. (a) Representative images of TUNEL-positive cells (green) in the GCL of control (aa) or NMDA-treated (ab-af) retinal explants. Nuclei were stained with DAPI (blue). Retinal explants were pretreated with different drugs 24 h before exposure to NMDA. Scale bar: 100 µm. (b) Quantification of TUNEL-positive cells. NMDA exposure induced a significant increase in the number of TUNEL-positive cells in the GCL compared with control. Pretreatment with 1 µM NPY or 1 µM (Leu31, Pro34) − NPY (Y1/Y5 receptor agonist), 24 h and 60 min before NMDA, was able to reduce the increase in the number of TUNEL-positive cells induced by NMDA. Pretreatment with Y1 or Y5 receptor antagonist (1 µM BIBP 3226 or 1 µM L-152,804, respectively), when applied 30 min before NPY, blocked its protective effect. The effect of (Leu31, Pro34) − NPY was blocked by BIBP 3226. (c) NMDA exposure induced a significant increase in the number of PI-positive cells in retinal explants. Data are presented as the ratio between PI-positive cells at DIV4 and DIV2. NPY or (Leu31, Pro34) − NPY was able to reduce the increase in the number of PI-positive cells induced by NMDA. BIBP 3226 or L-152,804 blocked the protective effect of NPY, and BIBP 3226 blocked the protective effect of (Leu31, Pro34) − NPY. The application of 1 µM (Gly1, . . . Aib32)-PP was able to reduce the increase in PI-positive cells induced by NMDA, and this effect was blocked by L-152,804. Together, the results from TUNEL and PI assays suggest that NPY is able to protect retinal cells from an excitotoxic insult through the activation of Y1 and Y5 receptors. Data are presented as mean ± SEM of n = 5 to 11 independent experiments. ***p < .001, compared with control; ##p < .01, ###p < .001, compared with NMDA; §p < .05, §§p < .01, §§§p < .001, compared with NMDA + NPY, or NMDA + (Leu31, Pro34) − NPY, or NMDA + (Gly1, . . . Aib32)-PP. Kruskal-Wallis followed by Dunn’s test.
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fig6-1759091415598292: NPY reduces NMDA-induced cell death in the GCL of retinal explants via Y1 and Y5 receptor activation. Retinal explants were exposed to 300 µM NMDA for 48 h to induce an excitotoxic insult. The agonists, 1 µM NPY, 1 µM (Leu31, Pro34) − NPY, 300 nM NPY13–36, and 1 µM (Gly1, . . . Aib32)-PP, were applied 24 h and 60 min before NMDA application. All the antagonists: 1 µM BIBP 3226, 1 µM BIIE 0246, and 1 µM L-152,804 were applied 30 min before the agonist. (a) Representative images of TUNEL-positive cells (green) in the GCL of control (aa) or NMDA-treated (ab-af) retinal explants. Nuclei were stained with DAPI (blue). Retinal explants were pretreated with different drugs 24 h before exposure to NMDA. Scale bar: 100 µm. (b) Quantification of TUNEL-positive cells. NMDA exposure induced a significant increase in the number of TUNEL-positive cells in the GCL compared with control. Pretreatment with 1 µM NPY or 1 µM (Leu31, Pro34) − NPY (Y1/Y5 receptor agonist), 24 h and 60 min before NMDA, was able to reduce the increase in the number of TUNEL-positive cells induced by NMDA. Pretreatment with Y1 or Y5 receptor antagonist (1 µM BIBP 3226 or 1 µM L-152,804, respectively), when applied 30 min before NPY, blocked its protective effect. The effect of (Leu31, Pro34) − NPY was blocked by BIBP 3226. (c) NMDA exposure induced a significant increase in the number of PI-positive cells in retinal explants. Data are presented as the ratio between PI-positive cells at DIV4 and DIV2. NPY or (Leu31, Pro34) − NPY was able to reduce the increase in the number of PI-positive cells induced by NMDA. BIBP 3226 or L-152,804 blocked the protective effect of NPY, and BIBP 3226 blocked the protective effect of (Leu31, Pro34) − NPY. The application of 1 µM (Gly1, . . . Aib32)-PP was able to reduce the increase in PI-positive cells induced by NMDA, and this effect was blocked by L-152,804. Together, the results from TUNEL and PI assays suggest that NPY is able to protect retinal cells from an excitotoxic insult through the activation of Y1 and Y5 receptors. Data are presented as mean ± SEM of n = 5 to 11 independent experiments. ***p < .001, compared with control; ##p < .01, ###p < .001, compared with NMDA; §p < .05, §§p < .01, §§§p < .001, compared with NMDA + NPY, or NMDA + (Leu31, Pro34) − NPY, or NMDA + (Gly1, . . . Aib32)-PP. Kruskal-Wallis followed by Dunn’s test.
Mentions: NPY exerts neuroprotective actions in various brain regions, and also in the retina (Alvaro et al., 2008b; Santos-Carvalho et al., 2013a), suggesting that NPY receptors might be possible therapeutic targets for retinal degenerative diseases such as glaucoma. Therefore, in addition to the NPY modulatory effect on RGC activity detected in ex vivo retinas, we also evaluated whether NPY could be able to prevent retinal cell death induced by an excitotoxic insult. The excitotoxic insult was induced in retinal explants (cultured for four DIV) exposed to 300 µM NMDA for 48 h. Apoptotic retinal cell death was assessed by TUNEL assay, and necrotic or late apoptotic cell death was assessed by PI incorporation assay (Figure 6). Different drug treatments were applied to assess the potential protective effect of NPY receptor activation. Thus, NPY (1 µM, n = 6 retinas for TUNEL, n = 9 retinas for PI), the Y1/Y5 agonist (Leu31, Pro34) − NPY (1 µM, n = 6 for TUNEL, n = 7 for PI), the Y2 agonist NPY13–36, (300 nM, n = 6 for TUNEL, n = 11 for PI), the Y5 agonist (Gly1,…Aib32)-PP (1 µM, n = 6 for TUNEL, n = 6 for PI), or a drug-free solution (control, n = 11 for TUNEL, n = 10 for PI), were applied at DIV1 and DIV2, respectively 24 h and 60 min before NMDA treatment.Figure 6.

Bottom Line: We found that NPY attenuated the increase in the [Ca2+]i triggered by glutamate mainly via Y1 receptor activation.Using in vitro cultures of rat retinal explants exposed to NMDA, we found that NPY pretreatment prevented NMDA-induced cell death.In conclusion, we found modulatory effects of NPY application that for the first time were detected at the level of RGCs.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, 3004-548 Coimbra, Portugal CNC.IBILI, University of Coimbra, 3004-548 Coimbra, Portugal.

No MeSH data available.


Related in: MedlinePlus