Pre- and Postsynaptic Sites of Action of mGluR8a in the Mammalian Retina

Abstract

purpose. The distribution of the most recently identified metabotropic glutamate receptor type 8a (mGluR8a) in the mammalian retina is unknown, but it is known to function as a presynaptic autoreceptor in rod photoreceptors. In this study, the localization of mGluR8a in the adult rat, mouse, and rabbit retina and during postnatal development of the rat retina was analyzed.

methods. mGluR8a immunoreactivity was detected using immunocytochemistry and light and electron microscopy.

results. mGluR8a expression was found in both synaptic layers and in extrasynaptic locations, predominantly on the somata of ganglion, amacrine, and horizontal cells. This distribution pattern is different from the localization patterns of the other members of group III mGluRs in the mammalian retina, which are restricted to either the outer plexiform layer (OPL) or the inner plexiform layer (IPL). Analysis of the expression of mGluR8a at the ultrastructural level in rat retina showed that the receptor is localized pre- and postsynaptically in the OPL and postsynaptically in the IPL. During postnatal development, mGluR8a was expressed at synapses in parallel with synapse formation, but appeared earlier at extrasynaptic sites.

conclusions. These results suggest that mGluR8a is involved in synaptic processing in both plexiform layers and in both the scotopic and photopic pathways in the mammalian retina. The authors propose that, depending on its localization—pre- versus postsynaptic—mGluR8a modulates the release of l-glutamate by photoreceptors and the responses of retinal neurons to inhibitory and excitatory neurotransmitters, respectively. Furthermore, mGluR8a may have regulatory functions during neuronal development of the mammalian retina.

The major excitatory neurotransmitter in the mammalian retina is l-glutamate. Glutamatergic neurotransmission is the basis of the vertical pathway for processing visual information in the photoreceptors that contact bipolar cells in the outer plexiform layer (OPL) and the bipolar cells that make synaptic contact with ganglion cells in the inner plexiform layer (IPL). In addition, l-glutamate is used by photoreceptor and bipolar cells to provide input into the horizontal pathways made by horizontal cells in the OPL and amacrine cells in the IPL. ¹ These laterally connecting inhibitory interneurons predominantly use γ-aminobutyric acid (GABA) or glycine as their neurotransmitters to modulate and process the vertical signal pathway. ^{2 3 4 5} As in other parts of the central nervous system (CNS), two classes of receptors, ionotropic glutamate receptors (iGluRs) and metabotropic glutamate receptors (mGluRs) mediate the effects of l-glutamate in the retina. Whereas iGluRs mediate fast excitatory synaptic transmission, ^{6 7 8} mGluRs influence different intracellular second-messenger systems through G-proteins and thereby modulate neuronal activity. ⁹ To date, eight mGluRs, some with isoforms and splice variants, have been identified, and they are subdivided into three groups (I–III), according to sequence homologies, pharmacological properties, and second-messenger systems to which they preferentially couple. ^{9 10 11} Depending on the second-messenger pathway to which mGluRs are coupled and on their synaptic localization, they can exert excitatory or inhibitory effects in neurons. ^{9 12}  

In several studies, the functional localization of mGluRs has been defined in the mammalian retina ^{13 14} and vertebrate retina. ¹⁵ The most recently described member of the mGluR family, mGluR8, was identified in a mouse retina cDNA library ¹⁶ and subsequently in the human genome as potentially relevant to a form of retinitis pigmentosa. ¹⁷ Even though sequence similarities led to the categorization of mGluR8 as a group III mGluR, the pharmacological profile of the rat mGluR8 shows mixed behavior, with properties of both group II and III mGluRs. ¹⁸ In addition to the originally cloned receptor, two variants generated by alternative splicing have been described: mGluR8b, present in rat cerebral cortex and hippocampus, shows differences from mGluR8a in the C terminus, but has an identical pharmacological profile. ^{19 20} mGluR8c, identified in a human fetal brain cDNA library, does not have the transmembrane domains and could therefore function as a secreted receptor isoform. ²⁰ In the mammalian retina, messenger RNA for mGluR8 has been found in all nuclear layers and at increased levels during ontogenesis by in situ hybridization ¹⁶ and in ganglion cells by single-cell RT-PCR. ²¹ We recently described mGluR8a as the first glutamate receptor in terminals of photoreceptors in the mammalian retina. ²² On activation, mGluR8a mediates a decrease in the intracellular concentration of Ca²⁺ in photoreceptors and provides an inhibitory feedback loop at photoreceptor synapses in the mammalian retina, possibly modulating and fine-tuning synaptic strength. ²²  

However, except for the expression of mGluR8a in terminals of photoreceptors, the exact distribution of the receptor in the retina and its possible function in retinal synaptic circuitry is still unknown. Therefore, we examined the cellular and subcellular distribution of mGluR8a in the retina at the light- and electron microscopic–level. We show distribution patterns of mGluR8a in adult rat, mouse, and rabbit retinas and during postnatal development of the rat retina, by immunocytochemistry and with use of an mGluR8a-specific antiserum. The unique distribution of mGluR8a, compared with other mGluRs in the retina, suggests that the receptor functions at both pre- and postsynaptic sites and may play an important role in adult retinal physiology, as well as during the development of retinal neurons. 

Methods

All experiments were performed in compliance with the guidelines for the welfare of experimental animals issued by the National Institutes of Health and the Max Planck Society and in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

Antiserum against mGluR8a

An affinity-purified polyclonal antiserum against mGluR8a raised in rabbit against a peptide corresponding to the C-terminal amino acid sequence of mouse mGluR8a (residues 890–908: ETNTSSTKTTYISYSDHSI) was used in the present study. The specificity of this antiserum has been characterized previously. ²² The epitope labeling by the antiserum could be specifically blocked by preadsorption of the antiserum with its antigen (Figs. 1C 1F 1I) . The antiserum was used at a concentration of 0.1 μg protein/mL. 

Animals and Tissue Preparation

Retinas of adult albino mice and rats, as well as of rats of different postnatal stages, 1 to 30 days old, were investigated. In addition, eyes of adult rabbits were obtained from experimental animals that had been used in experiments unrelated to the present study, involving techniques that did not affect the retina or the CNS. For the developmental studies, only retinas from the same littermates and only retinal pieces with the same eccentricity were compared. Mice and rats were anesthetized deeply with halothane and decapitated. A detailed description of the preparation of the retinal tissue for light and electron microscopic immunocytochemistry is given in Brandstätter et al. ²³ Briefly, the eyes were opened and immersion fixed in 4% (wt/vol) paraformaldehyde (PA) in phosphate buffer (PB; 0.1 M, pH 7.4) for 15 to 30 minutes. After fixation, the retinas were dissected from the eyecup, cryoprotected in a graded series of sucrose in PB (10%, 20%, and 30%) and sectioned vertically at 12-μm thickness on a cryostat. For electron microscopy, the fixation was in 0.05% glutaraldehyde, 4% (wt/vol) PA in PB for 10 minutes, followed by incubation in 4% (wt/vol) PA in PB for 40 minutes. After fixation for electron microscopy, the retinas were washed in PB and cryoprotected, as described for light microscopy. 

Light Microscopic Immunocytochemistry

Immunocytochemical labeling was performed by the indirect fluorescence method, as described previously. ²⁴ The mGluR8a antiserum was used at a concentration of 0.1 μg protein/mL, and sections were incubated in the primary antiserum overnight at room temperature. The binding sites of the primary antiserum were revealed by a secondary antiserum, goat anti-rabbit IgG coupled to a fluorescent dye (Alexa 594; Molecular Probes, Eugene, OR) diluted 1:500. In control experiments, either the primary or secondary antiserum was omitted or the primary antiserum was incubated with the antigenic peptide against which the antiserum had been raised (10-fold excess of the peptide, wt/wt) for 1 hour before application to the sections. Labeled sections were examined and photographed with a fluorescence photomicroscope (Axiophot; Carl Zeiss, Oberkochen, Germany). 

Preembedding Immunoelectron Microscopy

The labeling for preembedding immunoelectron microscopy was performed as described in detail previously. ²³ Briefly, after dissection and cryoprotection, retinas were frozen and thawed repeatedly to enhance tissue penetration by the antiserum. Small pieces of retina were embedded in agar, and vertical sections (50-μm-thick) were cut with a vibratome. The primary antiserum was used at the same concentration and diluted in the same medium, but without Triton X-100, as used for light microscopy. Tissue sections were incubated in primary antiserum for 4 days at 4°C. Binding sites of the primary antiserum were visualized with a biotinylated goat anti-rabbit IgG secondary antiserum diluted 1:100 (Vector Laboratories, Burlingame, CA) and a peroxidase-based enzymatic detection system (Vectastain Elite ABC kit; Vector Laboratories). The reaction product was silver intensified and gold toned. Control experiments were performed as described for light microscopic immunocytochemistry. The microscopic analysis was performed with an electron microscope (model EM10; Carl Zeiss). Cell types were identified according to well established anatomical criteria, such as the position of processes in defined sublayers of the IPL, the size and morphology of neuronal processes, the presence of electron-dense material, and the presence and quantity of synaptic vesicles. The presence of single versus multiple ribbon synapses in photoreceptor terminals was used to distinguish rod from cone photoreceptor terminals. The lager size of cone photoreceptor terminals in the rat retina was used as an additional parameter to distinguish the two types of photoreceptor terminals. Similarly, cone bipolar cell terminals were distinguished from rod bipolar cell terminals by their difference in size, the presence of multiple ribbon synapses and the absence of postsynaptic ganglion cell processes at rod bipolar cell terminals. 

Results

Expression of mGluR8a in the Mammalian Retina

In all three species investigated (mouse, Figs. 1A 1B 1C ; rat, Figs. 1D 1E 1F ; and rabbit, Figs. 1G 1H 1I ), mGluR8a immunoreactivity was present in both plexiform layers of the retina and in the GCL and the INL of mouse and rat retina (Figs. 1B 1E) . In the rabbit retina, faint staining was found in the INL, predominantly on the somata of horizontal cells (Fig. 1H) . When the anti-mGluR8a antiserum was preadsorbed with the antigenic peptide before application to retinal sections, no specific staining was detected (Figs. 1C 1F 1I) . Punctate staining was observed in the IPL of all three species. This is indicative of clustering of mGluR8a at synapses. mGluR8a immunolabel was also found in the OPL and on somata of horizontal cells that are in proximity to the OPL. In mouse and rat retina (Figs. 1B 1E) in addition to the staining of the plexiform layers and of horizontal cells, somata of amacrine cells in the INL close to the IPL and of ganglion cells in the GCL were stained (somata of horizontal cells are identified by arrows in Fig. 1 ). The somata of these neurons were immunoreactive for mGluR8a to varying degrees, with ganglion cells showing the most intense staining. In the rabbit retina mGluR8a immunoreactivity was concentrated in the plexiform layers, and only faint immunolabel was associated with horizontal cell somata in the outer part of the INL close to the OPL (Fig. 1H , arrows). The differences in mGluR8a staining observed in mouse, rat, and rabbit retina most likely reflect species differences in the expression of mGluR8a or in the antibody–antigen interaction after tissue fixation. 

For the ultrastructural analysis of mGluR8a distribution we chose the rat retina, because most of the data on the distribution of mGluRs and iGluRs are available from studies on rat retina. ^{13 14}  

Expression of mGluR8a in the OPL of the Rat Retina

Because of the epitope specificity of the antiserum (C terminus, see the Materials and Methods section) the reaction product of the mGluR8a immunostaining was found intracellularly. To detect mGluR8a immunoreactivity at the subcellular level, a very sensitive immunocytochemical method combining peroxidase staining with silver intensification and gold toning of the label was used. Because of the diffusion of the 3,3′-diaminobenzidine (DAB) reaction product, the spatial resolution is lower than would be obtained with gold-coupled secondary antibodies. However, the current method enabled us to localize low concentrations of antigen with an antiserum that exhibits a high sensitivity to alterations in its antigen due to fixation and embedding procedures. 

Previously, we reported that mGluR8a immunoreactivity is present presynaptically in the terminals of rod photoreceptors ²² (Fig. 2A) . In the current study mGluR8a was also found presynaptically in the terminals of cone photoreceptors, with aggregates of mGluR8a immunolabel associated with the ribbon synaptic complex (Fig. 2B) . The presence of multiple versus single ribbon synapses in photoreceptor terminals and the lager size of cone photoreceptor terminals were used to distinguish cone from rod photoreceptor terminals. Presynaptic expression of mGluR8a was less frequently found in cone synaptic terminals than in rod synaptic terminals. Typically, 25% of the cone synaptic terminals were labeled, whereas nearly all rod synaptic terminals were mGluR8a immunoreactive. This indicates that only a subset of cone photoreceptors expresses mGluR8a at detectable levels. We never found mGluR8a immunoreactivity presynaptically at basal contacts made between the cone synaptic terminals and the dendrites of OFF-cone bipolar cells. The dendrites of OFF-cone bipolar cells were identified as flat, noninvaginating processes contacting the cone photoreceptor synaptic terminals, whereas the dendrites of ON-cone bipolar cells form the central invaginating element of the ribbon synaptic complex. 

In addition to presynaptic staining for mGluR8a in photoreceptor terminals, the receptor was also found in postsynaptic processes at the photoreceptor ribbon synapses. Processes of horizontal cells postsynaptic to both rod and cone photoreceptor ribbon synapses were most frequently labeled. They are the two lateral invaginating elements of the ribbon synaptic complex (Figs. 2C 2D) . Typically, only one of the two lateral horizontal cell processes at the photoreceptor cell ribbon synapses was stained for mGluR8a. Very rarely, in less than 10% of all synapses investigated, a putative dendrite of an OFF-cone bipolar cell making a flat, noninvaginating synaptic contact at a cone synaptic terminal was labeled for mGluR8a (Fig. 2D) . The invaginating dendrites of rod bipolar cells and of ON-cone bipolar cells, the central elements of the photoreceptor ribbon synapse, were not labeled for mGluR8a (Fig. 2) . 

Localization of mGluR8a in the IPL of the Rat Retina

In the INL and the GCL, mGluR8a immunoreactivity was associated with somata of amacrine and ganglion cells and was found on processes of these cells postsynaptic to bipolar cell ribbon synapses in the IPL (Figs. 1 3) . mGluR8a immunoreactivity was detected on amacrine and ganglion cell processes postsynaptic to OFF-cone bipolar cell terminals (Fig. 3A) , ON-cone bipolar cell terminals (Fig. 3B) , and rod bipolar cell terminals (Figs. 3C 3D) . Typically, only one of the two postsynaptic processes at the bipolar cell ribbon synapses was labeled for mGluR8a (Fig. 3) . Because of poor tissue preservation, we were not able to tell whether the ganglion cell process or the amacrine cell process was stained at a given synapse. At cone bipolar cell synapses the two postsynaptic elements are typically a process of an amacrine cell and a dendrite of a ganglion cell. ²⁵ At rod bipolar cell synapses, both postsynaptic elements belong to amacrine cells. ^{26 27}  

Postnatal Development of mGluR8a Expression in Rat Retina

mGluR8a immunoreactivity was first detected on approximately postnatal day (P)5 in rat retina (Fig. 4 , top left). Immunolabel was confined to the somata of neurons in the GCL and the inner neuroblast layer (NBL). At ∼P10, after differentiation of the outer retinal layers from the NBL, the mGluR8a labeling intensity in the somata increased and comprised ganglion cell somata in the GCL and amacrine and horizontal cell somata in the INL (Fig. 4 , top right). Despite the development of this first weak mGluR8a immunoreactivity in the OPL at ∼P10, label in the IPL was not detectable. mGluR8a labeling in the IPL first appeared at P12. It showed some aggregation of mGluR8a immunoreactive puncta, indicative of developing synaptic contacts at which mGluR8a is clustered (Fig. 4 , bottom left). The number of mGluR8a puncta, the intensity of the immunofluorescence signal, and the degree of stratification increased in the IPL during the second and third postnatal weeks. During the same period, the staining in the OPL increased. The adult labeling pattern, characterized by the intense labeling of ganglion cell somata in the GCL, the labeling of amacrine and horizontal cell somata in the INL, and the receptor staining in the two plexiform layers of the retina, was reached at P19 (Fig. 4 , bottom right). 

Discussion

Since their discovery, the cellular and subcellular distributions of mGluRs have been studied in detail in the mammalian retina. ^{13 14} The most recently discovered member of the mGluR family, mGluR8a, ¹⁶ has been characterized by us previously as the second presynaptic mGluR in the retina, but the first to be present presynaptically in photoreceptor terminals. ^{22 23} Our previous study suggested that mGluR8a, once activated by glutamate that has been released from photoreceptors, decreases the activity of L-type calcium channels in photoreceptor terminals, thereby downregulating Ca²⁺-dependent glutamate release. ²² These findings were in agreement with studies that identified group III mGluRs as mediators of presynaptic autoreceptor inhibition of neurotransmitter release at glutamatergic synapses. ^{10 12 28 29 30}  

In the present study, we analyzed the localization pattern of mGluR8a in the mammalian retina to determine other possible sites of action of this receptor. This is especially important because of the profound effects that agonists and antagonists of group III mGluRs have on retinal physiology and development. ^{31 32 33 34 35 36 37} The genomic organization of mGluR8 suggests an involvement of the receptor in the pathogenesis of a form of retinitis pigmentosa. ¹⁷ In addition, the distinct pharmacological profile that distinguishes mGluR8 from other group III mGluRs, makes the receptor a promising candidate for the targeting of receptor-specific drugs. ^{18 38 39 40} Our results indicate that mGluR8a may be involved in the regulation of horizontal, amacrine, and ganglion cell activity in addition to its function in presynaptic inhibition of neurotransmitter release at photoreceptor terminals. We confirmed our previous findings of mGluR8a expression presynaptically at rod photoreceptor terminals, ²² and we identified mGluR8a in a subset of approximately 25% of the cone photoreceptor terminals. The absence of mGluR8a immunoreactivity from presynaptic sites of flat, noninvaginating synaptic contacts between OFF-cone bipolar cells and cone photoreceptors indicates that mGluR8a may play a predominant role as a presynaptic inhibitor of neurotransmitter release at photoreceptor terminals contacting ON bipolar cells. The presence of mGluR8a at postsynaptic sites in the OPL is the second finding of an L-AP4-sensitive mGluR that is involved in synaptic transmission at photoreceptor cell synapses. The first group III mGluR that was found postsynaptically at photoreceptor cell synapses was mGluR6. ⁴¹ mGluR6 is expressed on the dendritic tips of all ON bipolar cells, ⁴² and an mGluR6 knockout mouse shows a complete loss of the b-wave in the electroretinogram. ⁴³ mGluR8a is not expressed on dendrites of ON bipolar cells, but is found on processes of horizontal cells, the second postsynaptic partner at the photoreceptor synaptic complex. Group III mGluRs have been shown to modulate the voltage-gated sustained calcium current in isolated teleost cone horizontal cells, ³⁵ suggesting a possible role for mGluR8a in this process. In rat retina, only one type of horizontal cell, the axon-bearing B-type horizontal cell, is found. ⁴⁴ Our results that mGluR8a was expressed by only one of the two lateral horizontal cell processes at the photoreceptor synapses suggest a molecular heterogeneity of rat retinal horizontal cells at the level of expression of neurotransmitter receptors. A similar result was found for the expression of kainate receptor subunits in horizontal cell processes of rat retina. ⁴⁵  

The postsynaptic expression of mGluR8a at synapses in the OPL and the IPL indicates that the receptor may be involved in the regulation of the activity of interneurons, as described for group III mGluRs in other parts of the CNS. ^{10 12 28 29 30 46 47 48 49} Mechanisms that may influence the activity and neurotransmitter release of these neurons are the inactivation of voltage-gated calcium channels through G-proteins, modulation of potassium conductance, a direct regulation of presynaptic receptors or the coupling to adenylate cyclase and subsequent activation of PKA. ^{50 51 52} The expression of mGluR8a by only one of the two postsynaptic elements at the rod bipolar and the OFF- and ON-cone bipolar cell synapses in the IPL indicates that binding of glutamate released from the bipolar cell by mGluR8a present on amacrine cell processes may mediate a decrease of transmitter release from the amacrine cell, as proposed for neurons of the olfactory bulb. ⁵³ This mechanism could lead to a modulation of the activity of bipolar, ganglion, and amacrine cells receiving synaptic input from mGluR8a-expressing amacrine cells. ³⁴ Similarly, the activity of ganglion cells may be indirectly regulated by activation of mGluR8a. ³⁶ The recent development of mGluR8-specific agonists and antagonists will allow a more detailed study of its functions in retinal neurons. ^{18 38 39 40 54}  

In addition to its synaptic localization, the extrasynaptic expression of mGluR8a in the adult retina and during postnatal development indicate additional functions of the receptor. Several studies describe extrasynaptic expression of mGluRs and their potential role in regulating neuronal activity. ^{55 56} During postnatal development, the expression of mGluR8a showed different temporal and spatial patterns. Unlike other mGluRs in the retina, ¹³ mGluR8a is expressed in neuronal somata well before the onset of synaptic development, and expression in the IPL parallels synaptic development of the IPL. ^{57 58} The early expression of mGluR8a in the developing OPL indicates a possible function both presynaptically and postsynaptically in maturing horizontal and photoreceptor cells after the initial appearance of synaptic proteins but before the full maturation of glutamatergic synapses in the OPL. ⁵⁹ Several studies show that the concentrations of second-messenger substances, especially of free intracellular Ca²⁺, influence neuronal development. ^{60 61 62} The early expression of mGluR8a (mRNA level ¹⁶ ; protein level, this study) indicates that the receptor may play an important role during early retinal ontogenesis and in the final consolidation of synapses. ⁶³  

 Supported by a University of North Texas Health Science Center Faculty Research grant (PK) and Deutsche Forschungsgemeinschaft Grant SFB269/B4 and a Heisenberg Fellowship (JHB).

 Submitted for publication November 8, 2001; revised January 8, 2002; accepted January 25, 2002.

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Figure 1.

 

Micrographs of vertical cryostat sections through mouse (A–C), rat (D–F), and rabbit (G–I) retinas. Retina sections were immunostained with the antiserum against mGluR8a. Immunofluorescence was found in both synaptic layers (IPL and OPL) and in the somata of ganglion, amacrine, and horizontal cells in the GCL and the INL (B, E, H). Whereas in the mouse (B) and rat (E) retinas, the mGluR8a staining of somata was strong, only very weak somatic staining was found in the rabbit retina (H). Besides the extrasynaptic staining of somata in the nuclear layers, intense mGluR8a immunoreactivity was present in the IPL and weaker staining in the OPL of all three species investigated (B, E, H). Arrows: labeled somata of mGluR8a-immunoreactive horizontal cells in the outer INL. Control experiments (preadsorption of the anti-mGluR8a antiserum with the antigen) resulted in a complete loss of specific immunoreactivity and are shown for the three species in (C), (F), and (I). Differential interference contrast micrographs show the retinal layers (A, D, G). IS, inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar, 50 μm.

Figure 2.

 

Electron micrographs showing the pre- and postsynaptic localization of mGluR8a immunoreactivity at photoreceptor terminals, rod spherules, and cone pedicles in the OPL of rat retina. (A, B) mGluR8a immunoreactivity was present presynaptically at the ribbon synaptic complex of a rod spherule (A) and a cone pedicle (B). mGluR8a was presynaptically expressed at only one of the two ribbon synaptic complexes in the cone pedicle (B). (C, D) mGluR8a immunoreactivity was also present in horizontal cell processes postsynaptic at rod (C) and cone (D) photoreceptor ribbon synapses. Very rarely, mGluR8a staining was found in dendrites of putative OFF-cone bipolar cells, making flat, noninvaginating contacts at the cone pedicle base (D). Arrowheads: presynaptic ribbons in the photoreceptor terminals. hc, horizontal cell; bc, bipolar cell. Scale bars, 0.2 μm.

Figure 3.

 

Electron micrographs showing the postsynaptic localization of mGluR8a immunoreactivity at glutamatergic bipolar cell ribbon synapses in the IPL of rat retina. Arrowhead: the presynaptic ribbon in the bipolar cell axon terminal of an OFF-cone (A), an ON-cone (B), and rod bipolar cells (C, D). ( ) The two postsynaptic elements at the bipolar cell dyad. mGluR8a immunoreactivity, represented by the electron-dense precipitates close to the active zone of the ribbon synapse, was always confined to one of the two postsynaptic elements. Scale bars, 0.2 μm.

Figure 4.

 

Vertical cryostat sections through rat retinas showing the postnatal development of mGluR8a immunoreactivity. First, mGluR8a labeling of the somata of ganglion cells and of cells in the inner neuroblast layer (NBL) was observed at P5. At P10 the number of mGluR8a-positive cells had increased in the inner NBL, which had developed into the INL. In addition, first mGluR8a immunoreactivity was observed in the developing OPL associated with developing horizontal cells and photoreceptor cells. The first mGluR8a label in the IPL was identified at P12. During the second and third postnatal weeks, mGluR8a immunoreactivity developed further, reaching its adult pattern comprising labeled somata in the GCL and the INL and staining of the two plexiform layers at ∼P19. The retinal layers are shown with differential interference contrast optics accompanying each micrograph on the right (abbreviations as in Fig. 1 ; NBL, neuroblast layer). Scale bar, 50 μm.

The authors thank Anja Hildebrand, Walter Hofer, Gong-Sun Nam, and Margaret and Sara Koulen for excellent technical assistance, and Rainer Kuhn (Novartis Pharma AG, Nervous System Research, CH-4002 Basel, Switzerland) for the kind gift of mGluR8a antiserum.