Neuropeptide Y Receptors Y₁ and Y₂ are Present in Neurons and Glial Cells in Rat Retinal Cells in Culture

Abstract

Purpose.: Neuropeptide Y (NPY) is one of the most abundant peptides in the central nervous system (CNS), including the retina. This peptide activates various different G-coupled receptors (NPY Y₁, Y₂, Y₄, and Y₅) that are also present in the retina. However, the localization of NPY receptors in the several types of retinal cells is not completely known. In this study, we have looked at the distribution of NPY Y₁ and Y₂ receptors in rat retinal cells to reveal new perspectives on the role of NPY receptors in retina physiology.

Methods.: Rat retinal neural cell cultures were prepared from newborn Wistar rats (P3–P5) and pure rat Müller cell culture was obtained after treatment of these cells with ascorbic acid. The presence of NPY Y₁ and Y₂ in retinal cell types was studied by immunocytochemistry.

Results.: We show that NPY Y₁ and Y₂ receptors are present on every cell type of rat retinal cell cultures. Neurons, as photoreceptors, bipolar, horizontal, amacrine, and ganglion cells, express these two types of NPY receptors. NPY Y₁ and Y₂ receptors are also located in macroglial cells (Müller cells and astrocytes) and microglial cells.

Conclusions.: We have clarified the presence of the NPY Y₁ and Y₂ receptors in all different cell types that constitute the retina, which we believe will help open new perspectives for studying the physiology and the potential pathophysiologic function of NPY and its receptors in the retina.

Introduction

Neuropeptide Y (NPY) is a member of the NPY or “PP-fold” family. ¹ This 36-amino acid peptide was first isolated from the pig brain in 1982 by Tatemoto. ² NPY is one of the most abundant peptides in the central nervous system (CNS), including the retina, and is involved in numerous physiologic functions, such as feeding, memory processing, and cognition. ^3,4 NPY actions are mediated by G protein-coupled receptors, which have been named NPY Y₁, Y₂, Y₄, Y₅, and y₆. ^3,5  

NPY is expressed in the retina of mammals and nonmammals. ^6–12 In rodent retinas, NPY-immunoreactivity (NPY-ir) is present in the inner retina, it is localized in cell bodies at both inner nuclear layer (INL) and ganglion cell layer (GCL), and is also present in processes located in the inner plexiform layer (IPL). ^11,13 NPY shows one of the highest degrees of phylogenetic preservation, compared with other neuropeptides, and does not show marked differences between species. ^14,16 From a consideration of the phylogenetic preservation, it is expected that the NPY and NPY receptor distribution in the retina does not vary significantly between species. NPY-ir is present in amacrine cells in the majority of species studied (fish, frogs, lizards, rodents, baboons, pigs, cats, chickens, and pigeons). Cats, dogs, dolphins, and humans also show NPY in their ganglion cells, ^9,17–20 while some turtles, lizards, and frogs present NPY-ir in bipolar cells. ^8,21,22 Few studies have analyzed the distribution of NPY receptors either in the retina or in specific retinal cell types. The presence of both Y₁ and Y₂ receptors in the mouse retina has been confirmed by mRNA analysis ^13,23 ; however, it is unclear which cell types express these receptors. Moreover, in primary cultures of Müller cells isolated from the retina of guinea pig, a functional assay of cell proliferation suggested the presence of NPY Y₁ receptor in these glial cells. ²⁴ In human retinal pigment epithelium (RPE), mRNA encoding for NPY Y₁, Y₂, and Y₅ receptors has been detected, while in bovine RPE only mRNAs encoding for NPY Y₁ and Y₂ receptors have been found. ²⁵ Although the presence of NPY receptors in the rat retina has been described, in particular in NPY Y₁, Y₂, Y₄, and Y₅ mRNA expression, ^26,27 little is known about their localization. The NPY Y₁ receptor-ir is localized in horizontal and amacrine cell bodies and involved in their processes, ²⁸ while NPY Y₂ receptor can be found in terminals of rod bipolar cells. ²⁷  

NPY and its receptors play important roles in other parts of CNS. However their role in the retina is little known. Therefore, a deeper investigation into how NPY is distributed in the retinal cells that have specific roles in retina physiology will contribute to a better understanding of the human retina and will open new avenues for application of that knowledge in human retinal diseases. Our group has previously shown that NPY activates different NPY receptors to perform the same effect. More specifically, NPY induces retinal cell proliferation through the activation of NPY Y₁, Y₂, and Y₅ receptors, ²⁹ and also inhibits the [Ca²⁺]_i increase by the activation of NPY Y₁, Y₄, and Y₅ receptors in rat retinal cells. ³⁰  

The involvement of several NPY receptors in the same NPY effect, ^26,29–31 and the shortage of information on the distribution of NPY receptors in the retina have led us to characterize the localization of NPY receptors in the different cell types present in retina neural cell cultures, namely retinal neurons, macroglial and microglial cells. 

Materials and Methods

Primary Rat Retina Neural Cell Cultures

Three- to five-day-old Wistar rats were used to prepare primary rat retina neural cell cultures, as previously described. ³² All procedures involving animals were in agreement with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Briefly, rat retinas were dissected under sterile conditions, using a light microscope (Zeiss, Jena, Germany), in Ca²⁺- and Mg²⁺-free Hanks' balanced salt solution (in mM: 137 NaCl, 5.4 KCl, 0.45 KH₂PO₄, 0.34 Na₂HPO₄, 4 NaHCO₃, 5 glucose, pH 7.4), and digested with 0.1% trypsin (wt/vol) for 15 minutes at 37°C. Cells were resuspended in Eagle's Minimum Essential Medium (MEM; Sigma-Aldrich, St. Louis, MO), supplemented with 25 mM HEPES (Sigma-Aldrich), 26 mM NaHCO₃, 10% Fetal bovine serum (FBS; Gibco BRL Life Technologies, Scotland, UK), and penicillin (100 U/mL)/streptomycin (100 mg/mL) (Gibco BRL), plated on glass coverslips coated with poly-D-lysine (0.1 mg/mL; Sigma-Aldrich) at a density of 2 × 10⁶ cells/cm², and cultured for 8 days (37°C, 5% CO₂). 

Primary Rat Müller Cell Culture

The preparation of primary rat Müller glial cell cultures was based on a protocol previously described by Reis and colleagues, ³³ with the following modifications: 8- to 9-day-old primary rat retina neural cell cultures in poly-D-lysine-coated coverslips were treated with ascorbic acid (4 mM; Sigma-Aldrich) for 3 hours and then washed abundantly in order to eliminate all neurons. The Müller glial cell culture was maintained for up to 2 weeks with MEM medium (supplement with 25 mM HEPES, 26 mM NaHCO₃, 10% FBS, and penicillin [100 U/mL]/streptomycin [100 mg/mL]), which was changed every 3 days. Immunocytochemical experiments were performed after that period. 

Immunocytochemistry

Cells cultured on glass coverslips were washed twice with PBS. They were fixed with 4% paraformaldehyde (20 minutes; room temperature), permeabilized with 1% Triton/PBS for 5 minutes, and, to prevent nonspecific binding, were blocked with 3% (wt/vol) fatty acid-free BSA in 0.2% Tween 20/PBS, for 1 hour at room temperature. Cells were then incubated with the selected primary antibodies overnight at 4°C (Table). To identify photoreceptors, rhodopsin antibody was used. Rhodopsin is a photopigment found in rods, the most common type of photoreceptor in the retina. ³⁴ Bipolar cells were identified by Protein Kinase C (PKC) alfa. ^34–36 Different subpopulations of amacrine cells were identified by calcium binding proteins: calbindin, calretinin, and parvalbumin. ^37–40 The same proteins may also be present in ganglion cells; however, a more specific marker of ganglion cells was used, Brn3a. ^41,42 Additionally, calbindin can also identify horizontal cells. Glial Fibrillary Acidic Protein (GFAP) and vimentin were used as macroglial cell markers (astrocytes and Müller cells), while CD11b identifies resting and activated microglial cells. ^43,44 After washing, the cells were incubated for 1 hour at room temperature with the respective secondary antibodies: Alexa 488 anti-sheep Immunoglobulin (IgG), Alexa 488 anti-mouse IgG, Alexa 594 anti-mouse IgG, Alexa 594 anti-rat IgG, Alexa 488 anti-rabbit IgG or Alexa 594 anti-rabbit IgG (1:200; Invitrogen, Eugene, OR). Finally, after 5 minutes of washing, cell nuclei were stained with Hoechst 33342 (1 mg/mL in PBS; Invitrogen) for 5 minutes, and upon rinsing twice with PBS, the coverslips were mounted on glass slides using Dako Fluorescent mounting medium (Dako Cytomation, Glostrup, Denmark). 

Table. 

 

Primary Antibodies

Primary Antibody	Immunolabelled Retinal Cell/Receptors Immunogen	Supplier	Host/Dilution	Specificity/Control
Anti-rhodopsin	Rhodopsin is a photoreceptor marker.34 Antibody raised against amino acids 1–100 mapping at the N-terminus of rhodopsin of human origin.	Santa Cruz Biotechnology, Inc., Santa Cruz, CA	Mouse 1:50	Staining absent when primary antibody was omitted. Western blot analysis of rhodopsin expression in mouse eye tissue extract revealed a single band at 40 kDa (manufacturer statement). The use of this antibody has been previously reported for Western blotting (WB) and immunocytochemistry (ICC).45–47
Anti-PKC alfa	PKC alfa is a retinal bipolar cells marker.34–36	Cell Signaling Technology, Danvers, MA	Rabbit 1:200	Staining absent when primary antibody was omitted. Use of this antibody has been previously reported for WB and ICC.45,48.49
Anti-calbindin	Calbindin is a marker of horizontal cells and subpopulations of amacrine and ganglion cells.37,38 Antibody obtained from recombinant calbindin.	Merck Millipore, Billerica, MA, (AB 1778)	Rabbit 1:100	Staining absent when primary antibody was omitted. This antibody was characterized by Western blotting and Immunofluorescence. It shows no cross reactivity to calretinin by Western blot. The antibody produces specific staining of cerebellum Purkinje cells, molecular layer dendrites, and axonal fibers, stains cell bodies and fibers in neuronal subpopulations (manufacturer statement).Use of this antibody has been previously reported for immunohistochemistry (IHC) and WB.50–60 A primary antibody negative control and preabsorbation control were previously performed.59
Anti-calretinin	Calretinin is an amacrine and ganglion cells marker.39 Antibody obtained from recombinant rat calretinin.	Merck Millipore (MAB1568)	Mouse 1:200	Staining absent when primary antibody was omitted. Use of this antibody has been previously reported for ICC and IHC.61–65
Anti-parvalbumin	Parvalbumin is a marker of amacrine and ganglion cells.37,40 Antibody obtained from parvalbumin purified from frog muscle.	Merck Millipore (MAB 1572)	Mouse 1:200	Staining absent when primary antibody was omitted. By immunoblot it recognizes a protein of 12 kDa. The antibody is directed against an epitope at the first Ca2+-binding site and specifically stains the Ca2+-bound form of parvalbumin. Use of this antibody has been previously reported for ICC and IHC.61,63,65–67
Anti-Brn3a	Brn3a is a ganglion cells marker.41,42 Antibody obtained from amino acids 186-224 of Brn3a fused to the T7 gene 10 protein.	Merck Millipore (MAB 1585)	Mouse 1:25	Staining absent when primary antibody was omitted. This antibody shows no reactivity to Brn3b or Brn3c by Western blot and no reactivity to Brn-3a knock-out mice. Use of this antibody has been reported previously for ICC, IHC, and WB (47 kDa single band).42,52,53,57,68
Anti-GFAP	Glial Fibrillary Acidic Protein (GFAP) is a macroglial cells (astrocytes and Müller cells) marker.43 Immunogen is GFAP, Clone G-A-5.	Sigma-Aldrich	Mouse 1:400	Staining absent when primary antibody was omitted. Use of this antibody has been previously reported for IHC and WB.53,57,69–71
Anti-CD11b	CD11b is a microglial cells marker.44 This antibody was obtained from rat peritoneal macrophages.	AbDSerotec, Kidlington, UK	Mouse 1:200	Staining absent when primary antibody was omitted. This antibody recognizes the rat equivalent of human CD11b, the receptor for the iC3b component of complement. The antigen is expressed on most macrophages, including resident and activated peritoneal macrophages and Kupffer cells and around 35% of alveolar macrophages. The antibody also labels dendritic cells, granulocytes and microglial cells in the brain (manufacturer statement). Use of this antibody has been previously reported for IHC and immunoprecipitation.72–75
Anti-TUJ 1	This antibody identifies neurons76 expressing neuronal class III ß- Tubulin - Clone TUJ 1.	Covance Research Products, Inc., Princeton, NJ	Mouse 1:500	Staining absent when primary antibody was omitted. Antibody highly reactive to neuron specific Class III β-tubulin (βIII). TUJ1 does not identify β-tubulin found in glial cells. Use of this antibody has been previously reported for ICC, fluorescence-activated cell sorting (FACS), and flow cytometry.77–82
Anti-vimentin	Vimentin identifies macroglial cells (astrocytes and Müller cells).83 This antibody was obtained from vimentin clone V9.	Thermo Fisher Scientific, Waltham, MA	Mouse 1:400	Staining absent when primary antibody was omitted. Use of this antibody has been previously reported for ICC, IHC, and WB (48 kDa).84,85
Anti NPY Y1 receptor	Peptide sequence: KQASPVAFKKINNN, a synthetic peptide corresponding to amino acids 365 378 of human NPY1R, conjugated to KLH.	AbDSerotec	Sheep 1:500	Staining absent when primary antibody was omitted. Cross reactivity with rat. Use of this antibody has been previously reported.86,87 This antibody detects a band of approximately 55 kDa in human brain, 40 kDa in dentate gyrus neurosphere-derived cultures,86 and murine N9 microglial cell line.87
Anti-NPY Y2 receptor	Peptide sequence CEQRLDAIHSEVSMTFKAK, corresponding to amino acid residues 346-364 of mouse NPY2R.	Alomone Labs, Jerusalem, Israel	Rabbit 1:100	Staining absent when primary antibody was omitted. Use of this antibody has been previously reported.86 Western blot analysis of rat hippocampus, rat whole brain, and dentate gyrus neurosphere-derived cultures reveals a 50 kDa single band.86 The preabsortion control has shown no immunostaining.

Sequential Immunocytochemistry

In the case of double labeling with primary antibodies made with the same species, a sequential immunolabeling was performed. For that, the first primary antibody (Table) was incubated overnight at 4°C, followed by the respective secondary antibody (1 hour at room temperature). The cells were then blocked to prevent all nonspecific binding (1 hour at room temperature). In the second part of the immunolabeling, cells were incubated with the second primary antibody (overnight at 4°C) followed by the respective secondary antibody (1 hour at room temperature). Between the various incubations, cells were washed three times with PBS 1×. Cell nuclei were detected by Hoechst 33342 (as previously referred) and coverslips were mounted. 

Tissue Preparation

Adult Wistar rats eyes were removed and fixed in 4% paraformaldehyde solution (overnight at 4°C), transferred into 30% sucrose in PBS, embedded in Tissue Tek OCT Compound (Sakura Finetek Europe BV, AV Alphen aan den Rijn, The Netherlands) and frozen. Eyes were then cryosected in a cryostat (CM3050-S; Leica, Wetzlar, Germany) in 7-μm sections. 

Immunohistochemistry

Eye sections were fixed with acetone at −20°C for 20 minutes, blocked in PBS containing 10% newborn goat serum (NGS; Gibco) and 0.3% Triton X-100 (Sigma-Aldrich) and incubated in primary antibody overnight at 4°C. Primary antibody (rabbit anti-NPY Y₁R, Alomone, or rabbit anti- NPY Y₁R, 1:500, Alomone) were diluted in 2% NGS and 0.3% Triton X-100 in PBS. After washing, slices were incubated with Alexa 488 anti-sheep IgG and Alexa 488 anti-rabbit IgG (1:200, Invitrogen) for 1 hour at room temperature. Finally, after 5 minutes of washing, cell nuclei were stained with Hoechst 33342 (1 mg/mL in PBS; Invitrogen) for 10 minutes, and upon rinsing twice with PBS, the slides were mounted using Dako Fluorescent mounting medium (Dako Cytomation). 

Cells and retinal slices were visualized using a fluorescence microscope (Zeiss Axioshop 2 Plus) coupled to a digital camera (AxiocamHRc) and a laser scanning confocal microscope LSM 510 META (all; Zeiss, Jena, Germany). Images were analyzed using Adobe Photoshop (Adobe; San Jose, CA) or Image J (National Institutes of Health, Bethesda, MD). Negative controls were performed for each individual experiment by staining the cells without the primary antibodies. Each antibody was tested separately to determine its correct dilution. 

Results

Our group has previously shown that mRNAs coding for NPY Y₁, Y₂, Y₄, and Y₅ receptors are expressed in primary rat retina neural cell cultures. ²⁶ In the present study, we have used immunocytochemistry to analyze the distribution and localization of NPY Y₁ and Y₂ receptors in the different cell types present in the rat retina neural cell culture. 

Cultured retinal cells were immunoreactive for NPY Y₁ and Y₂ receptors (Fig. 1). NPY Y₁ receptor immunoreactivity (-ir) and NPY Y₂ receptors-ir were localized in cells with distinct morphologies, specifically neurons (small cell bodies and long processes), astroglial and microglial cells (Figs. 1A, 1B). However, some cells were not double labeled for both NPY Y₁ and Y₂ receptors-ir (Fig. 1C). 

Figure 1. 

 

The immunoreactivity (green fluorescence) for NPY Y₁ and Y₂ receptors in primary rat retinal neural cell cultures: (A) NPY Y₁ receptor was detected (green) with an anti-NPY Y₁ antibody; (B) NPY Y₂ receptor was identified (green) with an anti-NPY Y_2. Filled arrow represents cells with neuron-like shape. Head arrow shows cells with microglia-like shape. (C) Double immunolabeling of rat retinal cells with NPY Y₁ (green) and NPY Y₂ receptor (red). No double-labeled cell (top image) and double-labeled cells (bottom image) were magnified on right column. Cell nuclei were stained with Hoechst 33342 (blue). Negative controls were stained without primary antibodies. The images are representative of three independent cell cultures. Scale bar: 50 μm.

We carried out immunohistochemistry studies in adult rat retina slices (Fig. 2) to confirm the NPY Y₁ and Y₂ immunoreactivity localization in rat retina cells. NPY Y₁ and Y₂ receptor immunoreactivities were detected in RPE, photoreceptor outer and inner segments (P), in cell processes of outer plexiform layer (OPL), INL, IPL, and in GCL. To study the distribution of the NPY receptors in retinal neurons, and, specifically, those in distinct subtypes of neurons, different markers were used for double immunostaining (Figs. 3–5). NPY Y₁ and Y₂ receptor-ir were detected in cells which were immunoreactive for TUJ1, a neuronal marker (Figs. 3A, 3B). The immunoreactivity of NPY receptors in retinal neurons was found to be localized mainly in the neuronal cell bodies, but was also seen to be distributed along the cell processes (Fig. 3). In this retinal neural cell culture, we detected: rhodopsin-positive cells (photoreceptors), anti-PKC α-positive cells (bipolar cells), calbindin-positive cells (horizontal cells), Brn3a-positive cells (ganglion cells), parvalbumin-, calretinin-, and calbindin-positive cells (subsets of amacrine cells and ganglion cells). As can be seen from Figures 4 and 5, some of the cells were immunoreactive for rhodopsin, suggesting that some photoreceptors are present in our model of rat retinal neural cells. Furthermore, in some cases, the immunostaining results resembled the shape of photoreceptor outer segments. PKC-α immunoreactivity (bipolar cells) was also present in several neurons. Calbindin and calretinin immunoreactivity was detected in a number of cells, and the staining was particularly evident in cell bodies, although some processes were also positive for both calcium binding proteins. In contrast, the other calcium binding protein, parvalbumin, was much less expressed in this culture, and was only identified in a small number of neurons. Brn3a was also present in a few neuronal cells. NPY Y₁ and Y₂ receptors-ir were detected in all types of neurons present in these cell culture, specifically in rhodopsin-, PKC α-, calbindin-, parvalbumin-, calretinin-, and Brn3a-positive cells (Figs. 4, 5). 

Figure 2. 

 

NPY Y₁ and Y₂ receptors immunoreactivity in rat retinal slices: Rat retinal slices were immunostained against NPY Y₁ (A) and Y₂ (B) receptors antibodies. (A) NPY Y₁ receptor immunoreactivity is particularly evident in the inner retinal layers, mainly in inner nuclear and plexiform layers as well as in retinal GCL. NPY Y₁R immunoreactivity is also observed in RPE, photoreceptors (P), and in outer layers: ONL and OPL. (B) NPY Y₂R immunoreactivity is mainly detected in inner and outer plexiform layers and GCL. NPY Y₂ receptors immunoreactivity is also identified in RPE, photoreceptors, and nuclear layers (INL and ONL).White arrows highlight NPY Y₁ and Y₂ receptors immunostaining. Cell nuclei were stained by Hoechst 33342 (blue). Negative controls were performed without respective primary antibody (a, b). Scale bar: 50 μm.

Figure 3. 

 

NPY Y₁ and Y₂ receptors are present in rat retinal neurons. Retinal neurons were double-labeled with the TUJ1 antibody (green) and the different antibodies against NPY receptors (NPY Y₁ and Y₂, red). A representative double-stained neuron is highlighted in a small square and presented on the right, magnification of double-labeled neurons. (A) TUJ 1-positive neuron expressing NPY Y₁ receptors; (B) TUJ 1-positive neuron expressing NPY Y₂ receptors. Cell nuclei were identified by Hoechst 33342 staining (blue). The images were representative of three independent cell cultures. Scale bar: 50 μm.

Figure 4. 

 

NPY Y₁ receptors are present in several types of cultured retinal neurons. Retinal cells were immunolabeled using an antibody against NPY Y₁ receptor (A–F). Photoreceptors were identified with anti-rhodopsin (A); bipolar cells (B) were recognized using anti-PKC α; horizontal cells were marked using anti-calbindin (C); amacrine cells subpopulations were identified using anti-parvalbumin (D) and anti-calretinin (E); ganglion cells were identified using anti-Brn3a (F). Double immunolabeling of NPY Y₁ receptor and each cell type marker was highlighted in a square, and presented on the right, magnification of double labeled neurons. Cell nuclei were identified by Hoechst 33342 (blue). The images are representative of three independent cell cultures. Scale bar: 50 μm.

Figure 5. 

 

NPY Y₂ receptors are present in several types of cultured retinal neurons. Retinal cells were immunolabeled using an antibody against NPY Y₂ receptor (A–F). Photoreceptors were identified with anti-rhodopsin (A); bipolar cells (B) were identified using anti-PKC α; horizontal cells were identified using anti-calbindin (C); amacrine cells sub-populations were labeled using anti-parvalbumin (D), and anti-calretinin (E); ganglion cells were identified using anti-Brn3a (F). Double immunolabeling of NPY Y₂ receptor and each cell type marker was highlighted in a square, and presented on the right, magnification of double labeled neurons. Cell nuclei were identified by Hoechst 33342 (blue). The images are representative of three independent cell cultures. Scale bar: 50 μm.

The presence of NPY Y₁ (Fig. 6) and Y₂ (Fig. 7) receptors was also assessed by double-immunolabeling both in macroglial (Müller cells and astrocytes) and microglial cells. The presence of NPY Y₁ and Y₂ receptors was also observed in purified rat Müller cell cultures (Figs. 6, 7). 

Figure 6. 

 

NPY Y₁ receptors are present in rat retina microglial and macroglial cells. An antibody against NPY Y₁ receptor was used to identify the expression of this receptor in microglial and/or macroglial cells present either in retinal neural cell cultures [A–C]; red) or purified Müller cell cultures [D]; red). Retinal microglial cells were immunolabeled with an antibody against CD11b [A]; green), and macroglial cells (astrocytes and Müller cells) were identified by anti-GFAP [B]; green) and anti-vimentin [C]; green) specific antibodies. Cells/cell co-expressing NPY Y₂ receptor and each specific cell type marker were highlighted in the right column. Cell nuclei were identified by Hoechst 33342 (blue). The images are representative of three independent cell cultures. Scale bar: 50 μm.

Figure 7. 

 

NPY Y₂ receptors are present in rat retina microglial and macroglial cells. An antibody against NPY Y₂ receptor was used to identify the expression of this receptor in microglial and/or macroglial cells present either in retinal neural cell cultures [A–C]; red) or purified Müller cell cultures [D]; red). Retinal microglial cells were immunolabeled with an antibody against CD11b [A]; green), and macroglial cells (astrocytes and Müller cells) were identified by anti-GFAP [B]; green) and anti-vimentin [C]; green) specific antibodies. Cells/cell co-expressing NPY Y₂ receptor and each specific cell type marker were highlighted in the right column. Cell nuclei were identified by Hoechst 33342 (blue). The images are representative of three independent cell cultures. Scale bar: 50 μm.

In retinal primary cell cultures, microglial cells were identified using an antibody against CD11b, and macroglial cells (astrocytes and Müller cells) were characterized using anti-GFAP and anti-vimentin antibodies. CD11b-positive cells presented a positive labeling for each NPY receptor subtype analyzed (Figs. 6, 7), indicating that both NPY Y₁ and Y₂ were distributed in microglial cells. GFAP- and vimentin-positive cells were immunoreactive for NPY Y₁ and Y₂ receptor subtypes (Figs. 6, 7). The presence of NPY receptor subtypes in Müller cells was confirmed by double staining with antibodies for each NPY subtype receptor and vimentin in purified Müller cell cultures. 

In summary, our results show that two types of NPY receptors (NPY Y₁ and Y₂) are present in neurons, macroglial, and microglial cells. 

Discussion

It is well known that NPY and NPY receptors are present in the retina of a variety of species. ^6–9,11,12 However, to the best of our knowledge, no previous studies have provided a full characterization of the localization and distribution of NPY and NPY receptors in the retina. The high degree of phylogenetic preservation of NPY and its receptors ^14–16 make their distribution similar between species. In fact, among the species for which data are available, the retina of fish, frogs, rodents, baboons, pigs, cats, chickens, and pigeons show NPY-ir in amacrine cells and displaced amacrine cells (INL and cell processes in IPL). ^{6,11,13,88,89} In addition, NPY-ir in cat, dog, dolphin, and human retinas is also localized in ganglion cells at GCL. ^9,17,20 Furthermore, the bovine and human RPE show NPY-ir. ²⁵ Our group has previously shown that NPY-ir is present in macroglial cells (Müller cells) and microglial cells in rat retinal neural cell cultures. ²⁶ In relation to fish retinas, NPY-ir is detected in amacrine cells, which processes originate distinct sublayers in the IPL. ^6,12 By contrast, the NPY-ir of amacrine cells of river lamprey is weakly positive ⁹⁰ and even absent in squid retina. ⁹¹ Lizard retina show NPY- immunoreactivity in amacrine cells in INL, and are sporadically displaced at GCL. ⁹² Turtle retina shows NPY-ir in bipolar cells and amacrine cells, evenly distributed in the retina (in INL, IPL, and GCL and located in peripheral retina). ^21,93 Furthermore, frogs have the highest concentration of NPY-ir levels, which are characterized by seasonal variations ⁷ ; NPY-immunoreactivity is localized in a small population of amacrine cell bodies in INL, ^6,12,22,94 in bipolar-like cell bodies of INL, in GCL, in Müller cells within in INL ²² and in various processes in IPL. ⁶ Our group has previously shown the presence of mRNA encoding for different NPY receptor subtypes in the retina and in the same retinal cell culture model used in this study. ²⁶ In the present study, through immunocytochemistry techniques, we have shown that NPY Y₁ and Y₂ receptors are present in different subtypes of rat retinal neurons. In addition, this study is the first to report that cultured cells expressing rhodopsin also express the NPY Y₁ and Y₂ receptors studied. Furthermore, bipolar, horizontal, amacrine, and ganglion cells express NPY Y₁ and Y₂ receptors. PKC α-positive cells (bipolar cells) were frequently distributed in our cell culture model and also expressed the Y₁ and Y₂ receptors. This finding is consistent with a previous study, which has localized the NPY Y₂ receptor in rod bipolar cell terminals in a rat retinal culture. ²⁷  

In rat retinal slices, NPY Y₁ and Y₂ receptor immunoreactivities were detected in: photoreceptor outer segment; processes of OPL, layer composed by synapses between photoreceptors, bipolar cells, and horizontal cells; INL, layer composed by horizontal cells, bipolar cells, and amacrine cells; cell processes localized in IPL, layer composed by synapses between bipolar cells, amacrine cells, and ganglion cells; and also in the GCL. The localization of the immunoreactivity in retinal slices is comparable to the immunocytochemistry in the rat retinal cell culture model. In retinal cell cultures, cells are not arranged in organized layers as in the retina, and many cells may not achieve full maturity. ⁴⁰ It is difficult to identify in culture all the neuronal cell types that are normally present in the retina compared with retina slices. Calbindin, one of the calcium binding proteins present in the retina is a common marker used to detect horizontal cells. ⁴⁰ However, subsets of amacrine and ganglion cells may also express calbindin. ³⁷ From the same group of calcium-binding proteins, parvalbumin, and calretinin are more common in specific subpopulations of amacrine cells and some ganglion cells. ^37,40 We observed that rat retinal cells are immunoreactive for all these calcium binding proteins and also for NPY Y₁ and Y₂ receptors. Additionally, we confirmed that retinal ganglion cells are present in the studied culture, through positive immunostaining for Brn3a, a transcription factor expressed only by ganglion cells in the retina. ^41,42 Overall, our observations suggest the presence of horizontal cells, subsets of amacrine cells and ganglion cells expressing NPY Y₁ and Y₂ receptors in this rat retinal neural cell culture model. Our results, in retina slices and retinal cell culture, are in agreement with a previous study showing that NPY Y₁ receptor-ir is mainly localized in horizontal cell bodies in the INL and in cell processes in the OPL, in cholinergic amacrine cell processes in the IPL and in all calbindin horizontal cells in rat retina. ³⁰ Other groups have reported that NPY Y₁, Y₂, and Y₅ receptors are present in bovine and/or human RPE. ²⁵ In this study, NPY Y₁ and Y₂ receptors immunoreactivity was also detected in adult rat retina slices. However, RPE cells are not present in our cell culture. 

NPY and its receptors play a number of different and important roles in CNS that could also occur in the retina, such as neuroprotection, neurogenesis, and neuromodulation regulation. In fact, previous studies have shown that NPY has a neuroprotective role in the hippocampus and striatum. ^5,95,96 In rat and mouse organotypic hippocampal cultures, the involvement of NPY Y₁, Y₂, and/or Y₅ receptors have been shown in the protection against cell death induced by glutamate. ^95,97,98 In contrast, we have shown that NPY has a protective role against 3,4-methylenedioxy-N-methylamphetamine (MDMA)-induced toxicity in rat retinal cells. ³¹ However, the NPY receptor subtypes involved in this neuroprotective effect have not yet been identified. In this study, we show that NPY Y₁ and Y₂ receptors are present in different types of neurons in rat retinal neural cell cultures, suggesting that they may putatively mediate the neuroprotective effect of NPY. We, therefore, speculate that NPY Y₁ and/or Y₂ receptor agonists might be viewed as putative therapeutic drugs against neural cell degeneration in retinal degenerative diseases. Other studies suggest that NPY might have an important role in progenitor cell proliferation and/or differentiation in nervous tissue. ^86,99,100 Our group has demonstrated, using the same model of cultured rat retinal cells, that NPY stimulates the proliferation of neuronal progenitor cells (BrdU+/nestin+ cells). ²⁹ Additionally, NPY, through NPY Y₁ and Y₅ receptor activation, has the potential to maintain human embryonic stem cell (hESC) self-renewal and pluripotency. ¹⁰¹ Consequently, NPY system is a putative target to develop new strategies to increase retinal progenitor cell proliferation. 

Both we and other groups have shown that NPY modulates the intracellular calcium concentration ([Ca²⁺]_i) in the rat retinal neurons. ²⁷ In fact, NPY inhibits the depolarization-evoked Ca²⁺ influx into rod bipolar cells through the activation of NPY Y₂ receptors. ²⁷ We have also demonstrated that NPY inhibits the KCl-evoked increase in [Ca²⁺]_i in cultured rat retinal neurons through the activation of NPY Y₁, Y₄, and Y₅ receptors. ³⁰ The localization of NPY Y₁ and Y₂ receptors in retinal neurons described in the present study are in agreement, at least in part, with those studies. Furthermore, we have shown that amacrine cells expressing NPY are involved in tuning ganglion cells to low spatial frequencies/large spatial patterns. ¹⁰² This provides another possibility for NPY Y₁ and Y₂ receptor function in retinal cells. The visual processing is severely dependent on fine tuning neurotransmission between different retinal neurons (photoreceptors, and bipolar, ganglion, horizontal, and amacrine cells), which depend on [Ca²⁺]_i regulation and may result from the action of neuromodulators as the NPY. NPY modulates the release of neurotransmitters in some areas of CNS. ³ In the retina, at least in chickens and rabbits, this peptide plays a similar role when applied exogenously. ¹⁰³ Therefore, as has previously been suggested, ^11,28 the localization of NPY in several types of retinal cells may indicate a neuromodulatory role of this peptide in the retina. 

Our results have shown the presence of NPY Y₁ and Y₂ receptors in the different types of retinal cells. Therefore, since both NPY and its receptors are present in the retinal cells, this suggests an NPY autocrine effect that might play a variety of roles in the retina. These include homeostasis, modulation, and even protection of retinal cells against toxic effects. However, additional studies are necessary to unravel the role(s) of NPY Y₁ and Y₂ receptors in retinal physiology and pathophysiology. Following the evidence of previous studies suggesting that NPY and its receptors are not restricted to neurons in the retina, ^22,24,26,104 we have also found that NPY Y₁ and Y₂ receptors, can be expressed in glial cells, specifically astrocytes and Müller cells. In fact, GFAP- or vimentin-positive cells were immunolabeled for the two NPY receptors analyzed. These findings are supported by the presence of NPY Y₁ and Y₂ receptors in the purified primary culture of Müller cells. Previous studies have reported the presence of Y₁ receptors in Müller cells of guinea pigs and diseased human retina, ^24,104 and the authors suggested that the overexpression of NPY Y₁ receptors in glial cells could be involved in retinal glial (Müller) cells proliferation and the development of proliferative vitreoretinopathy (PVR). ²⁴ Other groups have previously shown that NPY effects on neural cell proliferation and differentiation in different parts of CNS (such as, olfactory epithelium, subventricular zone [SVZ], and subgranular zone [SGZ] of dentate gyrus, rostral migratory stream, striatum, and the olfatory bulb) were mediated by the NPY Y₁-receptor activation. ^105–110 The involvement of NPY Y₂ receptor is controversial. ^106,110 However, to the best of our knowledge, there are no studies that have demonstrated the participation of the NPY Y₂ receptor in the physiology of Müller cells. Further studies are needed to dissect the role of NPY receptors in Müller cells. 

We have also demonstrated the presence of NPY Y₁ and Y₂ receptors in retinal microglial cells (CD11b-positive cells). Microglial cells are involved in neuronal homeostasis and innate immune defense in the retina and are considered to mediate several pathogenic mechanisms in a variety of retinal degenerative diseases. ^111,112 In contrast, some studies have suggested that NPY is a key modulator of the crosstalk between the brain and the immune system in both health and disease. ^113,114 Further, in a microglial cell line, it was shown that NPY modulates microglial cell responses, regulating phagocytosis under inflammatory conditions through the Y₁ receptor. ^87,114,115 In addition, NPY, through NPY Y₁ receptor activation, inhibits the release of nitric oxide (NO) and IL-1β by microglial cells exposed to lipopolysaccharide (LPS). Hence, the presence of NPY Y₁ and Y₂ receptors in microglial cells suggests the involvement of these receptors in the immune responses occurring in the retina under pro-inflammatory conditions found in retinal degenerative diseases. In our study, the NPY Y₁ receptor immunoreactivity is distributed in the microglia cell while the NPY Y₂ receptor is also highly expressed close to the nucleus of these cells. Some studies have shown nuclear and perinuclear localization of G protein-coupled receptors. ^116,117 One specific group reported the localization of a NPY receptor in the nucleus membrane. However, in their studies the NPY Y₁ receptor was localized in the nuclear envelope of 20-week-old human fetal endocardial endothelial cells and cells of adult rat hearts. ^116,118  

We have previously shown the presence of mRNA encoding for NPY Y₁, Y₂, Y₄, and Y₅ receptor subtypes in the retina. ²⁶ However, in spite of our findings regarding Y₁ and Y₂ NPY receptors, further studies are needed to characterize the localization of the remaining NPY receptors in rat retinal cells. 

Other studies developed by us have shown that different NPY receptors are activated to perform the same effect, such as retinal progenitor cell proliferation ²⁹ and protection against retina degeneration. ³⁰ In this study, we show that rat retinal cell cultures present cells that do not express simultaneously the NPY Y₁ and Y₂ receptors. Therefore, the fact that not all retinal cells present all NPY receptors simultaneously indicates that different receptors may have similar roles. In fact, we have previously shown that various NPY receptors are able to prevent the increase of intracellular calcium concentration induced by the depolarizing agent KCl. ³⁰  

The main technique used in our study was immunocytochemistry, as an identification and characterization method. The main limitations of this technique involve the possibility of some nonselective binding of the antibodies used and that some antibodies identify more than one retinal cell type. The use of positive and negative controls may not completely abolish these limitations. However, in immunocytochemistry the cell morphology is preserved. Therefore, this technique is a powerful method to visualize and analyze specific biochemical structures (proteins, such as receptors) and cellular compartments (cell body, axons, and dendrites) of the cell. In conclusion, we have demonstrated that NPY Y₁ and Y₂ receptors are expressed in the different retinal cell types, although not necessary simultaneously. These observations suggest that NPY and NPY receptors have important functions in the retinal physiology, and that alterations in their expression and/or function may also be implicated in the pathogenesis of retinal degenerative diseases. 

Acknowledgments

The authors thank Luisa Cortes for microscopy assistance. 

Footnotes

 Supported by grants from the Portuguese Foundation for Science and Technology, Fundo Europeu de Desenvolvimento Regional (FEDER), and Programa Operacional Temático Factores de Competitividade (COMPETE) (PTDC/SAU-NEU/73119/2006, PTDC/SAU-NEU/099075/2008, SFRH/BD/45311/2008).

Footnotes

 Disclosure: A. Santos-Carvalho, None; C.A. Aveleira, None; F. Elvas, None; A.F. Ambrósio, None; C. Cavadas, None