Postnatal development of somatostatin 2A (sst2A) receptors expression in the rabbit retina

Developmental Brain Research 123 (2000) 67–80 www.elsevier.com / locate / bres

Research report

Postnatal development of somatostatin 2A (sst2A) receptors expression in the rabbit retina a b a, Gigliola Fontanesi , Claudia Gargini , Paola Bagnoli * b

a Dipartimento di Fisiologia e Biochimica ‘ G. Moruzzi’, Universita’ di Pisa, Via S. Zeno 31, 56127 Pisa, Italy Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Universita’ di Pisa, 56127 Pisa, Italy

Accepted 13 June 2000

Abstract In the retina, somatostatin (SRIF) acts as a neuromodulator by interacting with specific SRIF subtype (sst) receptors. Aim of this investigation was to determine the cellular localization of the sst2A receptor isoform in the postnatal rabbit retina. Receptor immunoreactivity was localized using the antiserum K-230, directed to the C-terminus of the human sst2A receptor. In the postnatal rabbit retina, sst2A receptors were abundantly expressed without significant regional differences. They were localized predominantly to rod bipolar cells, identified with a protein kinase C (PKC) antibody, to amacrine cells, some of which also containing tyrosine hydroxylase (TH), and to presumed rare horizontal cells. Quantitative analysis showed that sst2A-immunoreactive (-IR) bipolar and amacrine cells reached their maximum density and absolute number at the time of eye opening, when the expression pattern of sst2A receptors was similar to that in adult retinas. In the adult retina, 68% of the PKC-IR rod bipolars and 34% of the TH-IR amacrine cells were observed to also express sst2A receptors. The appearance of sst2A receptor immunolabeling prior to eye opening and the developmental profile of sst2A receptor expression are compatible with a role of SRIF in the maturation of retinal circuitries. The partial expression of sst2A receptors in PKC-IR rod bipolar cells and in TH-IR amacrine cells may suggest some type of heterogeneity within these cell populations.  2000 Elsevier Science B.V. All rights reserved. Keywords: Rod bipolar cell; Amacrine cell; Peptide receptor; Development

1. Introduction The peptide somatostatin (SRIF, somatotropin-release inhibiting factor) is widely distributed throughout the nervous system where it plays a variety of biological roles, including neurotransmission, neuromodulation, and growth regulation (for review, see Ref. [16]). In particular, the observed transient expression of SRIF and SRIF receptors in differentiating brain structures [2,46], the transient appearance of SRIF-containing ganglion cells in developing rat retinas [20] together with the reported actions of SRIF on the growth of neuronal processes (for references, see Ref. [49]) suggest that this peptide may be involved in important processes during neural development. The presence of SRIF and of SRIF-containing cells in mammalian retinas has been investigated using radioimmunoassay and immunohistochemical techniques (for references, see Ref. [38]). In addition, SRIF mRNAs have *Corresponding author. Tel.: 139-50-554-074; fax: 139-50-552-183. E-mail address: [email protected] (P. Bagnoli).

been detected in the rat retina by using RNA blot and in situ hybridization techniques [18]. In the rabbit retina, SRIF is expressed by displaced amacrine cells that are sparsely distributed in the ganglion cell layer (GCL) of the ventral retina, and they originate processes that extensively arborize in laminae 1 and 5 (following Cajal [4]) of the inner plexiform layer (IPL) [38,42]. Electrophysiological and pharmacological studies have provided evidence that SRIF influences the activity of retinal neurons [55]. Indeed, the application of the synthetic peptide SRIF-14 to a rabbit eyecup preparation induces a slow-onset, long-lasting decrease in spontaneous firing of retinal ganglion cells and a shift in the ganglion cell receptive field center–surround balance in favor of the center. The biological effects of SRIF are mediated by at least five SRIF subtype (sst) receptors, designated sst1-5 receptors, that have been found to belong to the class of seven transmembrane spanning G-coupled receptors (for review, see Ref. [34]). One of the SRIF receptors, the sst2 receptor, is widely distributed in the nervous system [14]

0165-3806 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0165-3806( 00 )00073-0

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where it exists in two variant forms, sst2A and sst2B which differ at their C-terminus and originate from alternative splicing of the sst2 receptor mRNA [50]. Both the sst2A receptor protein and its mRNAs are predominantly expressed in mouse and rat brain [43,51]. SRIF binding sites have been identified in retinas of mice, rats, sheeps and rabbits [12,17,28,30], while SRIF receptor mRNAs have been identified in the rat retina [27,35], where sst2 receptor mRNA is the most abundantly expressed [35]. In the rat retina, SRIF binding sites are present at high levels during the late prenatal period, when also SRIF mRNAs and the SRIF peptide are detected in large amounts [2,17]. Immediately after birth, the number of SRIF binding sites declines and then it returns to high levels by the time of maximum synapse formation that coincides with eye opening. During the same period, SRIFcontaining retinal neurons complete their morphologic maturation [19,20]. Earlier autoradiographic receptor binding studies do not provide information on either the relative abundance of the different SRIF receptors or their cellular distribution. Polyclonal antibodies directed against the C-terminus of the human, rat or mouse sst2A receptors have been recently generated in sheep and in rabbit [14,25,44,47]. Studies with these antisera have localized sst2A receptors in distinct regions of the adult rat brain [14,15,44] as well as in rabbit [26] and rat [24,27] retinas. In rabbit retinas, sst2A receptors have been reported in rod bipolar cells and in sparsely occurring amacrine cells [26]. In rat retinas, different patterns of sst2A receptor expression have been described. Johnson et al. [27] reported sst2A receptor immunostaining in cone photoreceptors, horizontal cells, rod and cone bipolar cells and large tyrosine hydroxylase (TH)-containing amacrine cells. In contrast, Helboe and Moller [24], while also reporting sst2A expression in cone photoreceptors and in TH-containing amacrines, did not detect sst2A immunostaining either in rod bipolar or in horizontal cells. In the present study, we investigated the cellular expression pattern of sst2A receptors both in adult and in developing rabbit retinas using antiserum K-230. In addition, the identity of retinal neurons expressing sst2A receptors was assessed with double-label experiments using the K-230 antiserum in conjunction with antibodies directed to protein kinase C (PKC) to identify rod bipolar cells (for references, see Ref. [7]) or to TH to identify the population of dopaminergic amacrine cells (for references, see Refs. [5,6]). Preliminary results have been presented in abstract form [21].

2. Materials and methods

2.1. Animals and tissue preparation New Zealand albino rabbits obtained from commercial

sources were used in this study. Care and handling of the animals were approved by the Animal Research Committee of the University of Pisa (Law on Animal Care No. 116 / 1992), in accordance with the European Community Council Directive (EEC / 609 / 86) and in compliance with the National Institutes of Health Standards. Rabbits at various postnatal ages were obtained from matings in our colony and birth usually occurred on embryonic day 31. The day of parturition was designed as postnatal day (PD) 0. Retinas were collected from animals ranging in age from PD0 to PD45. The number of retinas used at each age is as follows (number given in parentheses): PD0 (6), PD4 (6), PD7 (6), PD11 (6), PD18 (5), PD45 (7). Rabbits were deeply anesthetized with 30% chloral hydrate (i.p.), the eyes were removed, the anterior segments were cut away, and the posterior eyecups containing the retinas were immersion fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4, for 2 h. After fixation, retinas were isolated from the eyecup and they were stored overnight in 25% sucrose in 0.1 M PB at 48C. Cryostat retinal sections were cut at 15 mm in a plane either perpendicular or parallel to the vitreal surface. Sections were mounted onto gelatin-coated slides and air dried before immunohistochemical staining. Alternatively, whole mounted retinas were frozen and thawed, treated with 2.3% sodium metaperiodate in distilled water and subsequently with 1% sodium borohydride in 0.25 M Tris buffer before immunohistochemical staining.

2.2. Antibodies and immunohistochemical procedures The sst2A receptor antibody (K-230) was generously provided by Dr M. Schindler (Glaxo Institute of Applied Pharmacology, Cambridge, UK). It is a polyclonal antiserum raised in sheep and directed to the carboxy-terminal sequence of the human sst2A receptor [44]. As reported by Schindler et al. [44], this antibody does not cross-react with sst1 receptor, neurotensin receptor or metabotropic glutamate receptor 4. In addition, Western blotting experiments as well as preadsorption controls and use of pre-immune serum showed high specificity of this antibody for sst2A receptors [44]. Colocalization of sst2A receptor immunoreactivity with neurochemical markers for retinal cells was evaluated by using double-label immunofluorescence. Rod bipolar cells were identified with a monoclonal antibody directed to PKC (clone MC5, Amersham, Buckinghamshire, UK) [37], while the population of dopaminergic amacrine cells was identified with a monoclonal antibody directed to TH (Boerhinger Mannheim, Mannheim, Germany) [40]. Cryostat sections were incubated in the sst2A receptor antiserum diluted in 0.1 M PB containing 5% normal rabbit serum and 0.5% Triton X-100 overnight at 48C. Optimal dilution for this antiserum was 1:200. Sections were washed in 0.1 M PB and incubated in fluorescein isothiocyanate (FITC)-conjugated rabbit anti-sheep IgG

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(1:50; Vector Laboratories, Burlingame, CA) in 0.1 M PB containing 0.5% Triton X-100 for 2 h at room temperature. Subsequently, the sections were washed in 0.1 M PB and coverslipped with Vectashield mounting medium (Vector). Whole mount preparations were transferred to 10% normal rabbit serum with 1% Triton X-100 in 0.25 M Tris buffer for 1 h and then incubated with K-230 antiserum diluted 1:200 in 0.25 M Tris buffer containing 10% normal rabbit serum and 1% Triton X-100 for 3–4 days at 48C. After incubation in biotinylated rabbit anti-sheep IgG (1:50; Vector) for 2 days at 48C and subsequently in an avidin– biotin–peroxidase mixture (Vectastain ABC Kit, Vector) for 2 days at 48C, the retinas were incubated in 3,39diaminobenzidine tetrahydrochloride (DAB; Sigma) for 15 min and then in DAB with 0.03% hydrogen peroxide for an additional 15 min. Retinas were mounted GCL up and air dried on gelatin-coated slides. They were then treated with 0.05% osmium tetroxide in H 2 O for 20–30 s to intensify staining, washed, dehydrated and mounted with Permount. Specificity of the immune reactions was assessed by preadsorbing the primary antiserum with 50 mM synthetic sst2A receptor peptide, overnight at 48C. Further controls included the omission of the primary antibody and the use of the pre-immune serum instead of the primary antibody. No immunostaining was observed in control sections. Cryostat sections used for double-label immunofluorescence were washed in 0.1 M PB and incubated in 0.1 M PB with 0.5% Triton X-100 and 5% normal goat or rabbit serum containing sst2A receptor antiserum (1:200) and PKC (1:50) or TH (1:200) monoclonal antibody, overnight at 48C. Sections were then washed in 0.1 M PB and incubated in the presence of the appropriate affinitypurified secondary IgGs conjugated with FITC or Texas Red (1:50 and 1:200, respectively) in 0.1 M PB containing 0.5% Triton X-100 for 2 h at room temperature. The sections were finally washed in 0.1 M PB and the slides coverslipped with Vectashield mounting medium (Vector). Control experiments were performed to ensure that the primary antibodies did not cross-react when mixed together and that the secondary antibodies reacted only with the appropriate antigen–antibody complex [22]. Control experiments for immunostaining specificity were also performed using the preadsorbed sst2A antiserum (see above) in conjunction with normal mouse serum in place of PKC or TH monoclonal antibodies. No immunostaining was observed in these control conditions.

2.3. Conventional fluorescence and confocal microscopy Immunofluorescence was examined with a Leitz Orthoplan fluorescence microscope equipped with a Plan Neofluar 633, 1.4 NA oil objective or a PlanApo 1003, 1.4 NA oil objective. FITC fluorescence was visualized with a Leitz L2 filter cube, and Texas Red fluorescence was observed with a Chroma Texas Red cube. FITC and Texas

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Red were also simultaneously visualized with a Chroma FITC / TRITC dual-band filter. The distribution of sst2A immunostaining and its colocalization with PKC or with TH immunoreactivities were also examined with a Leica laser scanning microscope TCS-NT 1.6.551 (Laser Technik GmbH, Heidelberg, Germany) equipped with a krypton–argon laser (Omnichrome Corp., Chino, CA) and attached to a Leica DMRBE microscope with Plan Neofluar 633, 1.4 NA or 1003, 1.4 NA oil objectives. Generally, ten to 12 optical sections were taken with a z-axis resolution of 1 mm through immunolabeled cells.

2.4. Figure preparation Electronic images from conventional or confocal microscopy were processed using Adobe Photoshop (version 5.0; Adobe Systems, Inc., Mountain View, CA). Processing of images included both adjustment of brightness and contrast levels. Figures were then assembled and labeled. Scaling to final size yielded a final resolution of 300 pixels / inch.

2.5. Quantitative and statistical analysis of whole mount preparations Computer-assisted quantitative analysis was performed on four whole mount preparations processed with K-230 antiserum and treated with the avidin–biotin–peroxidase technique at each postnatal age. The four retinas originated from different animals to account for the variations in the number and in the density of immunostained cells [32]. Quantitative analysis was performed using a computerassisted image analysis system which included a Zeiss Axioplan microscope equipped with a color CCD video camera (JVC TK 1280E, SDS, Cambridge, MA), interfaced with a computer-assisted image analyzer. The images were automatically digitized with FG100AT (Image Technology Inc., Park Coburn, MA) plugged to the bus of a 486 personal computer. Each digitized image consisted of 3203200 pixels. The software package for quantitative image analysis ( OPTIMAS 6.1, Media Cybernetics, Silver Spring, MD) included a routine for automatic counts of immunolabeled profiles and morphometric analysis. No correction for shrinkage was applied, since the whole mounts were attached to the slides before dehydration [48]. Details of the procedure used for quantitative analysis have been published previously [7,9]. Briefly, the analysis was performed in 10 000-mm 2 fields at 20–25 different locations in each of the four retinas at each age (Fig. 1). The overall cell density at each age was expressed as the mean6standard deviation of all cell densities measured in the four retinas and expressed as numbers of sst2A-immunoreactive (-IR) cells per mm 2 of retinal surface. Since immunostained cell densities were found to be relatively homogeneous in all retinal locations, the total number of sst2A-IR cells was determined by multiplying the mean labeled cell density times the area of the retina. Statistical

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retina. Similarly, the percentages of PKC- and of THcontaining cells also expressing sst2A receptors were calculated. To assess for possible differences in the numbers of sst2A-IR cells in retinal sections treated for immunofluorescence versus numbers of immunostained cells in whole mount preparations treated with the avidin–biotin– peroxidase technique, two adult retinas were treated as whole mounts for sst2A receptor immunocytochemistry using the avidin–biotin–peroxidase technique. After the DAB step and before dehydration, the retinas were washed in Tris buffer and cut at 15 mm with a cryostat. About 40 sections were sampled as described above and the different types of sst2A-IR cells were counted. The resulting numbers of sst2A-IR cells were then compared with those of corresponding types of sst2A-IR cells in sections treated for immunofluorescence. No significant differences were observed in the numbers of sst2A-expressing cells in whole mount preparations and in sections treated for immunofluorescence.

3. Results Fig. 1. Schematic reconstructions of one of the four retinas used at each age for quantitative analysis. The solid circles indicate the retinal locations where sst2A-IR cell densities were measured. Retinal locations were similarly sampled in the other retinas. Scale bar: 8 mm.

analysis was performed by using the Sigmastat statistical package (Jandel Scientific, CA). The effect of the independent variable (age) on the dependent variables was assessed by means of the analysis of variance (two-way ANOVA). The Scheffe’s test (0.05–0.001 levels of significance) was used to identify differences between the density and the number of labeled cells as a function of the independent variable.

2.6. Quantitative analysis of double labeled material Percentages of sst2A-IR cells that were also labeled with PKC or with TH antibodies were determined in sections cut perpendicular to the vitreal surface and deriving from at least two retinas at each age. Each retina was cut in serial sections at a thickness of 15 mm. In newborn and in PD4 retinas, one every 15th section was selected for analysis. In PD7, PD11, PD18 and in adult retinas, one every 20th, 25th, 30th and 40th section, respectively, was selected for analysis. This procedure resulted in about 40 sections analyzed for each retina. In each selected section, the different types of sst2A-IR cells as well as PKC- or TH-IR cells were counted separately. The percentages of sst2A-IR cells also expressing PKC or TH immunoreactivity were calculated from the numbers of immunostained cells in all selected sections deriving from one

3.1. sst2 A immunostaining patterns At all ages, sst2A immunoreactivity was detected in different types of retinal cells that were uniformly distributed to all retinal regions. Immunolabeling was mainly associated with the cell plasma membrane. In adult retinas (Fig. 2), sst2A immunostaining was present in numerous cell bodies located in the distal inner nuclear layer (INL; Fig. 2A,B,D) and in several somata localized to the proximal INL (Fig. 2A,C,D). The sst2A-IR cells in the distal INL resembled bipolar and / or horizontal cells. Those in the proximal INL were characterized by either round- or oval-shaped somata and were mostly localized to the INL adjacent to the IPL. sst2A-IR fibers could be observed in the outer plexiform layer (OPL) and in laminae 1 and 5 of the IPL. Some immunolabeled processes could also be seen in lamina 3 of the IPL. In addition, sst2A immunostaining was also observed in presumed bipolar cell axons directed towards the IPL, where they seemed to arborize in lamina 5 forming large terminal profiles (Fig. 2A,B). Overall, the sst2A immunostaining pattern observed in the adult rabbit retina was consistent with the expression of sst2A receptors in bipolar cells, in some amacrine cells and, possibly, in rare horizontal cells. The developmental expression of sst2A receptors is summarized in Fig. 3. In newborn retinas, some sst2A-IR cell bodies were located in the distal INL adjacent to the OPL and in the proximal INL adjacent to the IPL (Fig. 3A). The immunolabeled somata in the distal INL were likely to belong to rare horizontal and / or bipolar cells, whereas those in the proximal INL were likely to be

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Fig. 2. One mm thick confocal images showing the localization of sst2A receptor immunoreactivity in transverse sections of the adult rabbit retina. The localization of sst2A immunostaining suggests a close association of sst2A immunolabeling with the cell plasma membrane both in the cell body and in the processes. (A) sst2A immunostaining pattern in the adult rabbit retina. (B–D) Higher power photomicrographs showing sst2A-IR cells with the morphology of bipolar cells (B), sst2A-IR amacrine cells (arrows in C, D), and sst2A-IR presumed horizontal cells (arrowhead in D). Open arrowheads in (A) and in (B) point to large immunostained boutons in the inner plexiform layer (IPL) likely representing terminal arbors of bipolar cells. OPL, outer plexiform layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar: 45 mm in A; 30 mm in B–D.

amacrine cell bodies (see below). A dense band of immunostaining was observed in the OPL. In addition, fine sst2A-IR processes resembling bipolar cell axons were observed to originate from some of the immunostained somata in the distal INL and to run vertically through the INL. sst2A-IR fibers originating from sst2A-IR somata in the proximal INL were also detected in the IPL adjacent to the INL. A similar pattern of immunostaining was observed at PD4 (Fig. 3B). At PD7 (Fig. 3C), a higher number of sst2A-IR cell bodies was detected in the distal INL, and

most of these immunolabeled somata originated a process similar to a bipolar cell axon. sst2A-IR processes could also be seen in IPL subdivisions best corresponding to laminae 1, 3 and 5 of the adult IPL. At PD11 (around the time of eye opening, Fig. 3D), rare immunolabeled somata of large size were clearly detected in the distal INL. Similar to PD7, many sst2A-IR profiles with the morphology of bipolar cells and immunolabeled somata in the proximal INL were also observed. In the IPL, immunostained processes were detected in laminae 1, 3 and 5. In addition, large immunostained profiles resem-

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cells per mm 2 of retinal surface) and calculations of total cell numbers (immunolabeled cell density times retinal area) were performed in selected regions of whole mount preparations treated with the avidin–biotin–peroxidase technique (Fig. 1). Values for the populations of sst2A-IR presumed bipolar and amacrine cells were obtained. Putative sst2A-IR bipolar cells were counted keeping the focus of the microscope into the distal portion of the INL and excluding all immunolabeled somata displaying morphological characteristics similar to those of horizontal cells. Immunolabeled cells in the proximal INL were counted keeping the focus of the microscope into the proximal portion of the INL. In this analysis, no differences in immunolabeled cell density were recorded in different retinal regions at any ages examined.

Fig. 3. Confocal images of sst2A-IR cells in transverse sections of rabbit retinas at different postnatal ages. At birth (A), some sst2A-IR somata were observed in the distal INL. They included presumed bipolar cells (arrows) and sparse horizontal-like cells (arrowhead). sst2A-IR cell bodies were also present in the INL adjacent to the IPL. sst2A immunoreactivity was also seen in processes both in the OPL and in the IPL adjacent to the INL. A comparable pattern of immunostaining was observed at PD4 (B). At PD7 (C), more sst2A-IR bipolar cells were detected, and immunopositive processes were localized to IPL subdivisions best corresponding to laminae 1, 3 and 5 of the adult IPL. Similar to PD7, at the time of eye opening (PD11, D) sst2A immunoreactivity was associated to bipolar cells, to rare horizontal-like cells (arrowhead in D) and to some amacrine cells. sst2A-IR processes ramified in laminae 1, 3 and 5 of the IPL. No changes were observed in sst2A immunostaining patterns from PD11 to adult ages. See Fig. 2 for abbreviations. Scale bar: 37 mm.

bling rod bipolar cell terminal boutons were observed in the IPL adjacent to the GCL.

3.2. Quantitative analysis Measurements of sst2A-IR cell density (immunolabeled

3.2.1. Bipolar cells Immunostained bipolar cell bodies at the INL / OPL border in whole mounted retinas of different ages are shown in Fig. 4A–C. In addition to sst2A-IR cells displaying the morphological characteristics of bipolar cells, large immunolabeled somata were occasionally observed in the outer portion of the INL (Fig. 4C). Their morphological features reminded those of horizontal cells. At all ages examined, no significant differences in sst2A-IR bipolar cell density were observed among different retinal regions. As shown in the diagram of Fig. 4D, the density of sst2A-IR bipolar cells increased significantly from birth to PD11 (P,0.001). At PD7, the density value was about two times higher than that found both at birth and PD4. From birth to PD11, the overall increase in the density of sst2A-immunoreactive bipolar cells was about 78%. The density of sst2A-IR bipolar cells gradually and steadily decreased from PD11 to adulthood (P,0.001 between PD11 and PD18 and between PD18 and PD45). From PD11 to adulthood, the overall decrease in the density of sst2A-immunoreactive bipolar cells was about 55%. As shown in the diagram of Fig. 4E, the absolute number of sst2A-IR bipolar cells significantly increased from birth to PD11 (P,0.001). From PD11 to adulthood, no significant changes in the total number of sst2A-IR bipolar cells were observed, indicating that their final number was established by PD11. 3.2.2. Amacrine cells Immunolabeled profiles in the proximal INL and presumably representing sst2A-IR amacrine cells were observed at all ages examined. Immunostained amacrine cells in whole mounted retinas of different ages are shown in Fig. 5A–C. At PD0, PD4 and PD7, sst2A-IR amacrine cell somata were small and usually round-oval in shape. At PD11 (Fig. 5B) and in the adult (Fig. 5C), an extensive network of sst2A-IR processes was seen to originate from immunolabeled somata. As shown in the diagrams of Fig. 5D,E, both the density and the total number of sst2A-IR amacrine cells did not significantly change from birth to PD7. At PD11, both the density and the absolute number

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Fig. 4. Whole mounted retinas at birth (A), at PD11 (B) and at adult age (C) immunolabeled with K-230 antiserum and treated with the avidin–biotin–peroxidase technique. The focus of the microscope was kept into the distal portion of the INL to detect sst2A-IR bipolar and presumed horizontal cell somata. In addition to sst2A-IR cells displaying the morphological characteristics of bipolar cells, large immunolabeled somata were occasionally observed in the outer INL at all ages examined (arrow in C). Scale bar: 30 mm. (D) The histograms represent sst2A-IR bipolar cell densities measured at different ages from birth to adulthood. Mean density values (6standard deviations) increased significantly from birth to PD11 (P,0.001) and then significantly decreased from PD11 to adulthood (P,0.001). (E) Histograms representing mean absolute numbers (6standard deviations) of sst2A-IR bipolar cells from birth to adulthood. This number significantly increased from birth to PD11 (P,0.001), with no further changes until adulthood.

of immunolabeled cells were significantly higher than at PD7 (P,0.001), and no significant changes in cell density or number were observed from PD11 to adulthood, indicating that the final density and number of these cells were established by PD11.

3.3. Double-labeling experiments The general pattern of sst2A immunostaining suggests that sst2A receptors are expressed by bipolar, amacrine and perhaps horizontal cells uniformly distributed in all retinal regions. Using double-label immunofluorescence, we tested the possibility that sst2A receptors were expressed by rod bipolar cells or by TH-IR amacrine cells.

3.3.1. Rod bipolar cells The morphological features of sst2A-IR presumed bipo-

lar cells reminded those of rod bipolar cells. Rod bipolar cells were identified using an antibody directed to PKC. As shown in Fig. 6A–C, coexpression of sst2A and PKC immunoreactivities was observed starting at PD4. Percentages of double-labeled cells are represented in the histograms of Fig. 6D. Specific PKC immunolabeling could first be observed at PD4 in a few somata located in the distal INL adjacent to the OPL (Fig. 6A). At this age, 2% of the PKC-containing cell bodies also expressed sst2A immunoreactivity, while 7% of the sst2A-IR bipolar cell somata also displayed PKC immunostaining. At PD7, PKC immunolabeling was found in several bipolar cell bodies, axons, and axon terminals (Fig. 6B). Many (46%) of these cells were also labeled by the K-230 antiserum, while 31% of the sst2A-IR bipolars also expressed PKC immunoreactivity. At PD11 (Fig. 6C), 73% of the PKC-IR rod bipolars also expressed sst2A receptors. In addition, 71%

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Fig. 5. Whole mounted retinas at birth (A), at PD11 (B) and at adult age (C) immunolabeled with K-230 antiserum and treated with the avidin–biotin–peroxidase technique. The focus of the microscope was kept into the proximal portion of the INL to detect sst2A-IR amacrine cell somata. Scale bar: 60 mm. (D) Means (6standard deviations) of density values of sst2A-IR amacrine cells. Density values increased significantly from birth to PD11 (P,0.001) and then did not change significantly from PD11 to adulthood. (E) Means (6standard deviations) of absolute numbers of sst2A-IR amacrine cells. Absolute numbers of sst2A-IR amacrine cells at PD11 were significantly higher than at previous ages (P,0.001), while no significant differences were observed from PD11 to adulthood.

of the sst2A-IR bipolar cells were found to also contain PKC immunostaining. In adult retinas, 68% of the PKC-IR rod bipolar cells also expressed sst2A receptors, while 73% of the sst2A-IR bipolar cells were found to also contain PKC immunostaining. Overall, the pattern of PKC / sst2A coexpression was established by PD11, and no substantial changes were observed from PD11 to adulthood.

3.3.2. TH-IR amacrine cells In addition to rod bipolar cells, amacrine cells were found to express sst2A receptors both in the developing and in the adult rabbit retina. The morphological features of many of these cells could remind those of dopaminergic amacrine cells. Dopaminergic amacrine cells were identified using antibodies directed to TH. Coexpression of sst2A and TH immunoreactivities was detected at all ages

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Fig. 6. Confocal images of transverse retinal sections at different postnatal ages double-labeled with sst2A and PKC antibodies (A–C). sst2A (green) and PKC (red) immunoreactivities were simultaneously visualized in the same preparation using a dual-band filter. Double-labeled profiles display a yellow-orange color. Specific PKC immunolabeling could first be observed at PD4 (A) in a few somata located in the distal INL. The percentage of double-labeled bipolar cells increased at PD7 (B) and at PD11 (C). Arrows indicate double-labeled profiles, while arrowheads and open arrowheads point to, respectively, only PKC- and only sst2A-IR bipolar cells. See Fig. 2 for abbreviations. (D) Graph showing the percentages of double-labeled sst2A- and PKC-expressing bipolar cells at the tested postnatal ages. Confocal images of transverse retinal sections at PD4 (E), PD7 (F) and at PD11 (G) and of a horizontal retinal section at PD11 (H) double-labeled with sst2A and TH antibodies. sst2A (green) and TH (red) immunoreactivities were simultaneously visualized in the same preparation using a dual-band filter. Double-labeled profiles display a yellow-orange color. Scale bar: 55 mm in A–C; 40 mm in E–G; 60 mm in H. (I) Graph showing the percentages of double-labeled sst2A- and TH-expressing amacrine cells at the tested postnatal ages.

(Fig. 6E–H), and percentages of double-labeled cells are represented in the histograms of Fig. 6I. In particular, at PD4 (Fig. 6E), 12% of the TH-containing cells also expressed sst2A receptors, while 18% of the sst2A-IR cells also displayed TH immunostaining. At PD7 (Fig. 6F), 18%

of the TH-immunostained cells also expressed sst2A receptors, while 24% of sst2A-IR cells also displayed TH immunostaining. At PD11 (Fig. 6G,H), 27% of the TH-IR cells also expressed sst2A receptors, while 30% of the sst2A-IR cells also contained TH immunoreactivity. In the

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adult retina, 30% of the TH-IR cells also expressed sst2A receptors, while 34% of the sst2A-IR cells also contained TH immunoreactivity. Overall, the percentages of both the TH-IR amacrines also expressing sst2A receptors and the sst2A-IR cells also containing TH immunoreactivity almost doubled from birth to adulthood.

4. Discussion The polyclonal antibody directed to sst2A receptors employed in the present study has been previously used to localize sst2A receptors in the rat brain and spinal cord [44]. Our results show that sst2A receptors are abundantly expressed in rabbit retinas throughout postnatal development. They are localized predominantly to rod bipolar cells and to amacrine cells, including some TH-IR amacrine cells. Rare, putative horizontal cells in the distal INL may also express sst2A receptors. At the time of eye opening, the pattern of expression of sst2A receptors is similar to that observed in adult retinas. It is well established that SRIF acts as a neuromodulator in the rabbit retina [55]. In the adult rabbit retina, SRIF is expressed by displaced amacrine cells that are sparsely distributed in the GCL and largely confined to the ventral retina [38,42]. They give rise to processes that are widely distributed to all retinal regions and presumably influence several retinal cell types by acting at multiple levels of the retinal circuitry [11]. Indeed, our findings show that sst2A receptors are expressed by a variety of cell populations in all retinal regions. Given the widespread distribution of SRIF-IR processes, SRIF-containing terminals may establish direct contacts with the cells expressing sst2A receptors. On the other hand, SRIF may also interact with its receptors via paracrine mechanisms, as also suggested for other transmitter systems in mammalian retinas. For instance, there is a significant mismatch between the location of neurokinin 1- and tachykinin-IR processes in the rabbit retina, suggesting a paracrine action of tachykinin peptides [8]. In addition, dopamine has also been suggested to act in a paracrine fashion at some distance from its release sites in vertebrate retinas [13,56].

4.1. SRIF and its receptors in the developing retina SRIF-expressing cells appear at late prenatal ages in the rabbit retina, and postnatally, they follow a pattern of maturation [3,39] similar to that characterizing the postnatal development of different components of the rod pathway, including dopaminergic [5,6] and rod bipolar cells [7]. The present study represents the first attempt to determine the cellular expression pattern of sst2A receptors in the rabbit retina during postnatal development. sst2A

receptors are expressed by different cell types at early postnatal ages, and the possibility exists that sst2A receptors, similar to SRIF, are already expressed in the late prenatal retina. At these early times, there is a poor synaptic specialization of the IPL and photoreceptor outer segments are not developed [31,33], indicating absence of visual information processing. The early expression of both SRIF and its receptors suggests possible roles played by SRIF in the processes of maturation of retinal neurons. The ontogeny of SRIF receptors has been investigated in the rat retina, where high prenatal levels of SRIF binding sites have been localized to the IPL and to the GCL. These levels decline immediately after birth and SRIF binding sites become localized to the IPL and to the OPL. Maximal levels are then reported around the time of eye opening in the IPL and in the OPL [17]. This developmental pattern of SRIF binding sites reminds that of the sst2A-expressing cells as shown by the present results. Although the evaluation of SRIF binding sites is comprehensive of all SRIF receptors and our data are not indicative of the quantity of sst2A receptors, the density and the total number of the cells expressing this receptor also reach maximum values at the time of eye opening, in coincidence with the reported maximum values of binding site levels. Eye opening coincides with major morphofunctional rearrangements in retinal circuitries [31,33], and represents a crucial period in the maturation of different retinal cell populations identified by specific markers [5– 7,9]. We can therefore hypothesize an involvement of SRIF neuromodulation subserving the morphogenetic events taking place at this time.

4.2. Quantitative analysis The quantitative analysis provided in the present study allows a comparison between the developmental pattern of the different types of sst2A-expressing cells and those of identified populations of bipolar and amacrine cells. Over the postnatal period, sst2A-expressing bipolar cells display a developmental profile similar to that of rod bipolar cells. As previously reported for PKC-IR rod bipolars [7], also sst2A-expressing bipolar cells reach their maximum density at PD11 followed by a decrease likely due to retinal growth. In contrast, the total number of these cells increases drastically from birth to PD11 and it remains almost unchanged until adulthood. As also proposed for PKC-IR rod bipolars, the steady increase in sst2A-expressing bipolar cells up to PD11 can be explained by the expression of these receptors in existing cells that are committed to this particular phenotype. Indeed, neurogenesis in the rabbit INL is virtually ceased at PD6 [45]. The observed developmental pattern of cell densities and numbers of sst2A-IR amacrine cells is somewhat different from those reported for specific amacrine cell types.

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Similar to previously studied amacrine cell populations [6,9], sst2A-expressing amacrines show a significant increase in both cell density and number during the first postnatal week. In contrast, while amacrine cell densities decrease after the first postnatal week due to increase of retinal area, such a decrease is not observed in the case of the sst2A-expressing amacrine cells. This discrepancy is likely to be due to amacrine cells that start expressing sst2A receptors at late postnatal ages. This possibility is supported by our observations of increasing percentages, throughout the postnatal period, of TH-containing amacrines that also express sst2A receptors.

4.3. sst2 A- and PKC-IR bipolar cells Most sst2A-IR cells with somata in the distal INL are rod bipolar cells, based on their appearance, size, distribution, and observed colocalization of sst2A and PKC immunostainings. At all ages, we have observed that a considerable percentage of sst2A-containing bipolars do not express PKC immunostaining (27% in adult retinas), and not all PKC-containing rod bipolars also express sst2A receptors (32% in adult retinas). In contrast, previous results of the adult rabbit retina reported a 100% colocalization of sst2A- and PKC-immunostainings [26]. Regarding the subset of sst2A-IR bipolar cells that are not PKC-IR, it must be considered that, given the abundance of previous studies reporting PKC expression by all rod bipolar cells, it is unlikely that sst2A receptors are expressed by rod bipolars that do not contain PKC. Therefore, it is likely that, as in rat retinas [27], sst2A receptors are also expressed by some types of cone bipolar cells. Regarding the observed lack of sst2A expression in a group of PKC-IR rod bipolar cells, the possibility exists that the use of the K-230 antiserum results in an underestimation of rod bipolars that may express sst2A receptors at levels that are not detectable by this antiserum. Indeed, the developmental pattern of sst2A-expressing bipolar cell density and total number is similar to that of developing PKC-IR rod bipolar cells [7]. The appearance of PKC immunostaining, as observed in the present study, is slightly earlier than previously reported [7]. This may be due to variable PKC labeling around PDs4–6 that may have resulted in missing PKCexpressing cells at PD4 in previous studies. On the other hand, the developmental pattern of PKC-IR bipolars observed in the present study is similar to that reported in the postnatal retinas of rats and rabbits [7,57].

4.4. Amacrine cells expressing sst2 A receptors The present study shows that sst2A receptors in the rabbit retina are expressed by a subset of TH-IR amacrine cells. The postnatal development of TH-containing amac-

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rine cells observed in the present study is similar to that reported previously [5,6]. The percentage of sst2A-IR amacrine cells also expressing TH immunoreactivity increases during retinal development and, in adult retinas, 34% of sst2A-IR amacrines is represented by TH-IR amacrine cells, indicating that dopaminergic amacrines constitute an important target of SRIF acting at sst2A receptors. Similarly, the percentage of TH-containing amacrines that express sst2A receptors gradually increases during postnatal development and reaches 30% in adult retinas. In the rat retina, sst2A-immunostaining was recently localized to all TH-containing amacrines [24] and to other, small-diameter amacrine cells [27]. In the adult rabbit retina, recent data also reported the localization of sst2A receptors in amacrine cells. However, none of these cells were found to be TH-IR [26]. In addition, the sst2A-IR amacrines of Johnson et al. [26] were observed to ramify in laminae 2 and 4 of the IPL, while the sst2A- and TH-IR amacrines observed in the present study correctly arborize in laminae 1, 3 and 5 of the IPL. This discrepancy can be ascribed to the use of different sst2A receptor antibodies. In particular, only sst2A receptors expressed by non-THIR amacrine cells may be immunolabeled by the antibody used by Johnson et al. [26]. At the functional level, there is evidence suggesting that dopamine has stimulatory effects on somatostatinergic transmission in different brain regions [53]. In contrast, little is known on the influence of SRIF on dopaminergic cells. Some evidence supports the possibility that SRIF exerts an inhibitory role on striatal dopaminergic neurons [36]. In addition, SRIF has been recently shown to increase dopamine release in the rat striatum, presumably through an increased excitatory amino acid release [23]. Finally, alterations of SRIF metabolism have been associated to brain diseases that also involve alterations of dopaminergic systems [41].

4.5. Partial expression of sst2 A receptors in bipolar and amacrine cell populations At all developmental ages, both rod bipolar and TH-IR amacrine cells have been found to express sst2A receptors, however only a part of each of these cell types actually express the sst2A receptor. This partial expression in adult retinas likely results in retinal distribution of sst2A-IR cells that is different from that of PKC-IR, rod bipolar cells. Indeed, bipolar cells expressing sst2A receptors are uniformly distributed in all retinal regions while PKC-immunolabeled bipolars are known from previous studies to display a peak in cell density in correspondence of the visual streak in adult retinas [54]. Previous studies have shown that both PKC and TH immunostainings identify single morphologically distinct populations of bipolar and amacrine cells, respectively (for

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references, see Refs. [5–7]), although an additional, rare type of TH-IR amacrine cell has been reported in the rabbit retina [5,6]. The expression of sst2A receptors in only a limited number of PKC- and TH-IR cells is unlikely to be caused by poor antibody penetration. Therefore, we may assume that the presence of sst2A receptors identifies two distinct subclasses (sst2A-expressing and -non-expressing) of both rod bipolar and TH-IR amacrine cells. On the other hand, these two putative subclasses do not seem to be distinguishable on the basis of their morphology or retinal location. Alternatively, as discussed above, the K-230 antiserum may be incapable of labeling sst2A receptors expressed at low levels, or some rod bipolar and TH-IR cells may express a form of the sst2A receptor that is not recognized by the antibody used in this study. The possibility of multiple different forms of sst2A receptor expressed in the rabbit retina is also supported by the observation that a different antibody directed to the sst2A receptor labels all rod bipolar cells, while it does not label TH-containing amacrines [26] in rabbit retinas. In addition, recent studies of the rat retina using two different antibodies directed to sst2A receptors reported different expression patterns of these receptors [24,27]. Hints about a certain degree of heterogeneity in cell populations that are considered as composed by a unique cell type on the basis of anatomical, histochemical and functional data are present in the literature. For instance, in the tiger salamander retina about 96% of SRIF-IR cells also express GABA [52], while 18% of SRIF-IR amacrine cells were reported to also exhibit high-affinity uptake of [ 3 H]GABA in the chick retina [29]. In addition, only 71% of the large TH-IR amacrine cell type of the rat retina has been reported to express neurokinin 1 receptors [8] and 75% of type II TH-IR amacrine cells of the primate retina express TrkB receptors [10]. Although the possibility of differentiating subgroups of rod bipolar and dopaminergic amacrine cells on the basis of differential peptide receptor expression is intriguing, it is difficult to speculate about possible functional consequences. For instance, SRIF may regulate the physiological activity of rod bipolar cells by inducing a slow hyperpolarization and amplification of the light response, as reported by studies of eyecup preparations of the rabbit retina [55]. Investigations of isolated rod bipolar cells of goldfish retina indicate that this SRIF action is probably mediated by closure of cation channels leading to suppression of L-type Ca 21 current [1]. sst2A receptors are candidate receptors to mediated SRIF effects on cation channels. However, the physiologic effects of SRIF on rod bipolars as detected in eyecup preparations [55] may rely on such mechanism only in part. Indeed, rod bipolars lacking sst2A receptors would either be insensitive to SRIF regulation or be provided with other SRIF receptor subtypes mediating SRIF effects. In this latter case, the observed functional effects of SRIF on the physiology of the rod bipolar cell population as a whole would be the

result of more variegated and more complex mechanisms than it could appear by only considering the involvement of sst2A receptors alone. In conclusion, consistent with the reported long-lasting effects of SRIF on ganglion cell physiology, the present study suggests that SRIF may indirectly influence ganglion cell function by activating sst2A receptors located at two levels (the rod bipolars and the dopaminergic amacrines) of the rod pathway. In addition, the appearance of sst2A receptor immunolabeling prior to eye opening and the developmental profile of sst2A receptor expression are compatible with a role of SRIF in the maturation of retinal circuitries. Together with previous observations from colocalization studies in vertebrate retinas, the partial expression of sst2A receptors by rod bipolars and dopaminergic amacrines observed in the present investigation could indicate a certain degree of heterogeneity in gene expression patterns within established retinal cell populations.

Acknowledgements We wish to thank the Institute of Neurophysiology of the Italian Research Council for technical facilities. We are also mostly grateful to Paolo Gualtieri for his help in quantitative analysis, to Giovanni Casini and Enrica Strettoi for their helpful suggestions and valuable comments on the manuscript and to Marco Nuti for figure preparation. This work was supported by the Italian Board of Education, grant number: F06 / PB / RS40% and the European Community, grant number: QLG3-1999-00908.

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