f~euron,

Vol. 6, 525-531,

April,

1991, Copyright

0 1991 by Cell Press

Localization of the Inositol 1,4,5=Trisphosphate Receptor in Synaptic Terminals in the Vertebrate- Retina Y.-W. Peng,*+ A. H. Sharp,+ S. H. Snyder,+ and K.-W. Yau*+ *Howard Hughes Medical Institute ‘Department of Neuroscience Johns Hopkins University School of Medicine Haltimore, Maryland 21205

Summary lnositol 1,4,5-trisphosphate (InsP3) mobilizes internal Ca*+ in cells by binding to a receptor protein, which has recently been purified and molecularly cloned. To clarify those neuronal functions that are regulated by InsP3, we have localized this InsP3 receptor protein immunocytochemically in the retina, a neural tissue of well-defined structure and function. Positive staining in neurons is confined almost exclusively to the synaptic layers. Using dissociated retinal neurons, we have further localized the receptor to presynaptic terminals of photoreceptors and bipolar cells, as well as the synaptic processes of amacrine cells. The specific association of InsP, receptors with synaptic terminals suggests a role for lnsPs in synaptic modulation, especially with respect to transmitter release. introduction Signal transduction via phosphoinositide turnover mediates a myriad of cell functions (for review, see Nishizuka, 1984; Berridge, 1987; Berridge and Irvine, ‘1989). This turnover is triggered when certain neurotransmitters, hormones, and growth factors bind to cell surface receptors and activate, via a C protein, a phospholipase (phosphoinositidase) C enzyme that hydrolyzes inositol lipids. One of the key products of this pathway is inositol 1,4,5-trisphosphate (InsP3, a second messenger that mobilizes Ca*+ from internal stores and perhaps also stimulates its entry from the cell exterior (Streb et al., 1984; Putney, 1986; Penner et J., 1988; see Berridge and Irvine, 1989, for review). This Ca*+ mobilization is brought about bythe binding of InsP3 to a receptor protein that functions as a Ca*+ channel when bound to ligand (Kuno and Gardner, ‘1987; Worley et al., 1987a; Ehrlich and Watras, 1988; Meyer et al., 1988; Ferris et al., 1989; Restrepo et al., 1990). Such an lnsPs receptor has recently been puriiied and cloned from the cerebellum, where it is present at high concentration in Purkinje cells (Supattapone et al., 1988; Furuichi et al., 1989). The role of this receptor in neuronal function, however, remains unclear because antibodies against the protein have localized it to all cell regions of the Purkinje cells (Furuichi et al., 1989; Mignery et al., 1989; Ross et al., 1989; Satoh et al., 1990). To examine this question further, we have now mapped InsP3 receptor-like immunore.tctivity in the vertebrate retina. The disposition of

anatomical layers is clear-cut in this tissue, and a rich knowledge of physiology and pharmacology exists, including reports of phosphoinositide turnover induced by light and neurotransmitters (for review, see Anderson and Brown, 1988;Osborneand Ghazi, 1990). Unlikethesituation inthecerebellum,wefindavirtually exclusive association of the InsP3 receptor-like immunoreactivity with synaptic regions of the retina, especially presynaptic terminals. Results The InsPa Receptor Is localized to Synaptic layers in Rat Retina An affinity-purified rabbit polyclonal antibody directed against the InsP3 receptor purified from rat cerebellum (Sharp and Snyder, unpublished data) was used in all immunostaining. In rat retina, there is clear staining of the outer and the inner plexiform layers, which are the synaptic layers (Figure IA). Some positive staining is also present in the pigment epithelium as well as theouter and the inner limiting membranes, the latter two (Figure IA, arrows a and b) being formed by the distal and proximal terminal processes of the Miiller glial cells (see, for example, Dowling, 1987). The outer and inner segments of the photoreceptors show no obvious staining, as do the ganglion cell bodies. In the outer and inner nuclear layers, there are stained varicosities that appear to be associated mostly with Miller cells as well (see below). Thus, as far as retinal neurons are concerned, the antibody seems to label the two plexiform layers exclusively. The immunostaining seems specific for the InsP3 receptor, because it disappears after preadsorbing the purified receptor to the antibody (Figure IB). Furthermore, as shown in Figure IC, an immunoblot prepared from total proteins of the rat neural retina (lane 2) shows a band that corresponds in molecular weight (M, ~260,000) to the InsP, receptor protein purified from rat cerebellum (lane 1). The relatively weak staining of this band probably reflects the restricted presenceof the protein in the retina. The band of M,29,000 (Figure IC, lane 1) corresponds to concanavalin A (Con A), which was used for purifying the receptor (see Experimental Procedures). Finally, Figure IC, lane 3, shows, as another control, a blot of total protein extract from rat cerebellum; it also has a single labeled band at the expected correct molecular weight. Synaptic Localizations in Other Vertebrate Retinas InsP3 receptor-like immunoreactivity was also tested in retinal sections from other vertebrate species. In monkey and salamander retinas, the outer and the inner plexiform layers are again strongly labeled (Figure 2). The darkly stained, knob-like structures at the outer plexiform layer of the salamander retina can be recognized as synaptic terminals of photoreceptors,

1

200~-

c;) 97x0

r;

68-

2- a29---

::-

Figure

1. InsPg Receptor

lmmunoreactivity

in the Rat Retina

(A) lmmunostaining of a cross section of the rat retina with an anti-lnsP4 receptor antibody. Nomarski differential interference contrast optics. Frozen section (2 Frn thick). The anatomical layers are as follows. RPE, retinal pigment epithelium; PRL, photoreceptor layer (containing the outer and inner segments of the receptor cells); ONL, outer nuclear layer (containing the cell bodies of the photoreceptors); OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; CCL, ganglion cell layer (with the unstained ganglion cell bodies clearly visible). The arrows indicate the outer limiting membrane (a) and the inner limiting membrane (b). (B) Absence of staining of the rat retina after adsorbing the lnsPs receptor purified from rat cerebellum to the antibody prior to immunostaining. The arrows again indicate the positions of the outer (a) and inner (b) limiting membranes. (C)An immunoblot obtained from SDS-extracted total proteins of the rat neural retina (i.e., less pigment epithelium). Lane 1 shows a purified lnsPs receptor preparation. The band at about M, 260,000 corresponds to the lnsPp receptor; that at M, 29,000 corresponds to Con A, which was used for purifying the receptor (see Experimental Procedures). Lane 2 shows the total retinal protein extract; a single band comigrating with the InsPs receptor is visible. Lane 3 shows, as a control, total protein extracted from rat cerebellum, where the InsP, receptor is present at high concentration. Again, a single band of the appropriate molecular weight is visible. The amount of protein was 0.1 ug in lane 1, 250 pg in lane 2, and 4 wg in lane 3. The three lanes were separated by blank lanes to prevent spillover.

which is confirmed by studies on dissociated cells to be described later. In addition to the two plexiform layers, theouter limiting membrane in the salamander retina is also stained (Figure 26); in the monkey retina some bipolar cell bodies in the inner nuclear layer are stained (Figure 2A, arrow). Similar staining of the two plexiform layers was observed in retinal sectionsfrom mouse, turtle,goldfish, and chicken. An enlarged view of the inner plexiform layer of the turtle retina, which is particularly thick and well developed among different species, is shown in Figure 3. The darkly stained varicosities in this region (Figure 3, white arrows) can be traced under the microscope to the inner nuclear layer and represent the synaptic terminals of bipolar cells. These stained terminals seem to be arranged in several layers,

though not all of these layers are clearly recognizable in every retinal section. The most prominent and consistent of these layers is indicated by arrow a in Figure 3. From its relative location in the inner plexiform layer, this layer of terminals most likely comes from OFF-bipolar cells (Marchiafava and Weiler, 1980; Weiler, 1981; see also Famiglietti and Kolb, 1976; Famiglietti et al., 1977; Nelson et al., 1978). Since stained terminals are also present in other layers of the inner plexiform layer, however, it seems possible that ON-bipolar cells have the immunoreactivity as well (see above references). Dissociated Retinal Cells Clearly Reveal Receptor in Synaptic Terminals To examine more closely the localization receptor, we performed immunostaining

the

InsPI

of the InsPs on enzymat-

InsP, !,I27

Receptor

in Retina

ically dissociated retinal neurons. The cells from salamander are exceptionally large, so the staining can be most easily resolved (Figure 4). The rod and cone receptors (Figures 4A and 4B) show strong and localized staining at their synaptic terminals, which are situated at the outer plexiform layer, as well as weak staining in the ellipsoid region of the inner segment. The staining at theellipsoid, which contains predominantly mitochondria, is not obvious in the retinal section (see Figure 2B), possibly because of the weakness of the staining together with the thinness (2 j.rm) of the section. This staining may reflect some metabolic role of the receptor. It is, however, absent in the ellipsoids of dissociated photoreceptors from rat and ,nonkey. The bipolar cells likewise show localized staining at the synaptic terminal, plus some weak staining at the idistal appendage of the cell body called the Landolt club. The latter structure is situated near the level of the outer limiting membrane in the retinal section (Hare et al., 1986), but its function is unknown. The dendritic processes, on the other hand, are not stained (Figure 4C). The amacrine cells show uniform staining of their profuse processes (Figure 4E). These processes, like the bipolar synaptic terminal, are situated at the inner plexiform layer and are both presynaptic and postsynaptic in nature (see, for example, Dowling, 1987). All the rods, cones, and amacrine cells in our dissociated retinal cell preparations show the immunostaining as described above. A small fraction of bipolar cells (~20%), however, do not appear to show the staining. Among the stained bipolar cells, nonetheless, we have identified each of the three morphological classes previously described in the salamander retina (Hare et al., 1986). The significance of the unstained cells are therefore uncertain at present. Interestingly, neither the horizontal cells (Figure 4D) nor the ganglion cells (Figure 4F) are stained. Finally, the Miller glial cells (Figure 4G) show intense immunoreactivity at their distal end, which constitutes the staining seen ,at the outer limiting membrane of the retinal section ‘seeFigure2B).Thus,theoverallfindingsfromdissociated cells are consistent with those from retinal sections. Dissociated cells from rat and monkey retinas give a ibroadly similar picture. Rat bipolar cells display some immunoreactivity in the region just distal to the cell oody in addition to the synaptic terminal (Figure 4H). in rat Mtiller cells (Figure 4l), staining is apparent in the cell body and the two ends of the cell; in addition, there are localized regions of staining in the cell body and the primary processes that can also be observed 1s varicosities in retinal sections (see Figure IA). Discussion To summarize, the neural retina shows InsP3 receptor-like immunoreactivity predominantly at the two synaptic layers, a consistent finding among different

vertebrate species. From the immunoblot and preadsorption data, it appears that the staining in the retina indeed comes from an InsP3 receptor protein homologous to that in the cerebellum. Previous immunocytochemical studies on cerebellar Purkinje cells have identified the InsP3 receptor throughout the cell, including the cell body, dendrites, axon, and axon terminals (Furuichi et al., 1989; Mignery et al., 1989; Ross et al., 1989; Satoh et al., 1990). This diffuse distribution makes it difficult to assess the specific functions of lnsPs in neurons. In the retina, on the other hand, the specific association of the receptor protein with the synaptic layers implicates a role for lnsPs in synaptic transmission. In particular, the preponderance of the receptor in presynaptic terminals suggests its involvement in the modulation of transmitter release, a process that requires Ca 2+. It would be interesting to determine whether this observation can be extended to areas elsewhere in the brain. Retinal photoreceptor terminals are known to receive inputs from other photoreceptors as well as feedback inputs from horizontal cells; bipolar cell terminals also receive feedback inputs from amacrine cells (see, for example, Kaneko, 1979; Sterling, 1983; Dowling, 1987, for review). It is possible that the InsP3 receptor is coupled to one or more of these inputs to modulate transmitter release from these terminals. These synaptic terminals are tonically active, which may make them particularly suitable for such a modulation. The receptor may have a similar function in the synaptic processes of amacrine cells, which also have an abundance of reciprocal synapses of both bipolaramacrine and amacrine-amacrine varieties (see, for example, Dowling, 1987). Several neurotransmitters that activate phosphoinositide turnover in the retina, such as glutamate and acetylcholine (see Anderson and Brown, 1988; Osborne and Ghazi, 1990, for review), are known to be released by photoreceptors and amacrine cells (see, for review, Brecha, 1983; MasseyandRedburn,1987;Dawetal.,1989).Thus,examining the location of the InsP3 receptor in relation to the receptors for these transmitters may shed light on these issues. Previous work has indicated a light-activated phosphoinositide turnover in rod outer segments and horizontal cells (Anderson and Hollyfield, 1981,1984; Anderson et al., 1983; Ghalayini and Anderson, 1984; Brown et al., 1987; see, for review, Anderson and Brown, 1988). However, we have not observed any InsP3 receptor-like immunoreactivity in these places. It is possible that an InsP3 receptor isoform unrecognized by our antibody is present. However, the important signal in these locations is more probably diacylglycerol, the other second messenger produced by phosphoinositide turnover (Nishizuka, 1984; Berridge, 1987), rather than InsP3. For instance, protein kinase C activity, which is triggered by diacylglycerol, has been demonstrated in rod outer segments (Kapoor and Chader, 1984; Kelleher and Johnson, 1985; Kelleher, 1986; Kapoor et al., 1987; Binder et al., 1989).

Neuron 528

If iPl \r

InsP, Receptor 529

in Retina

described (Supattapone et al., 1988; Ferns et al., 1989). Briefly, the membranes were solubilized with 1% Triton X-lOOor CHAPS, and the InsP, receptor was then purified by heparin-agarose affinity chromatography followed by Con A-Sepharose affinity chromatography. The Con A-Sepharose eluate (purified InsP, receptor) was concentrated l&fold by ultrafiltration with a 100 kd cutoff membrane (Amicon), a step that also reduced the amount of Con A contamination of the lnsPs receptor due to leaching from the Sepharose column. The concentrated eluate was then rediluted with 1 M a-methylmannoside in 50 mM TrisHCI (pH 7.4), 1 mM EDTA, 0.1% Triton X-100. This concentration/ redilution step was repeated once more before the receptor protein was finally concentrated for immunization purposes. The InsP, receptor used for preadsorption of antibody (Figure IB) was purified by the same method, except that the Con A-Sepharose was cross-linked with glutaraldehyde before use to eliminate leaching of Con A from the column, and the ultrafiltration step was omitted. Con A-Sepharose was cross-linked by treatment with 0.25% glutaraldehyde in 100 mM sodium phosphate (pH 7.0) for 1 min at room temperature followed by 1 hr on ice with stirring. The reaction was terminated by extensive washing of this Con A-Sepharose.

+a

“igure zross

3. Enlarged View of the Inner Section from Turtle

Plexiform

Layer of a Retinal

\lomarskioptics.Thesection is6um thick. Whitearrowsindicate ;ome of the immunostained bipolar cell terminals. These tend ‘0 be arranged in layers, the most prominent and consistent of ,Nhich is indicated by arrow a.

tn the brain, a previous study has also suggested that one or the other of these two branches of the phosphoinositide signaling pathway may be biased in a given location (Worley et al., 1987b). Finally, while our focus is on neurons, InsP3 receptor-like immunoreactivity is obviously present in Muller glial cells as well. The lnsPs receptor has so far not been reported in brain glial cells, but a closer examination may be worthwhile. Experimental

Procedures

Purification of the InsP, Receptor TheInsP,receptorforimmunizationof was purified from crude cerebellar

Figure

Production of Antibodies New Zealand White rabbits were initially injected subcutaneouslywith 100 ug of the purified lnsPJ receptor protein in Freund’s complete adjuvant. The rabbits were boosted every 3-4 weeks with 60-300 Rg of the receptor protein in Freund’s incomplete adjuvant. After4 months of immunization, the rabbits were bled every2-4weeks,and theantiserawerestored at -20°C until use. To decrease the concentration of nonspecific antibodies, the antiserum was adsorbed by overnight batch incubation at 4OC with an affinity matrix consisting of cerebellar membrane extracts that had been treated with heparin-agarose (i.e., with the InsP3 receptor already removed) and coupled to cyanogen bromide-activated Sepharose. This process was repeated one or more times before the anti-InsP, receptor antibodies were purified from the treated serum with an affinity matrix consisting of purified InsP, receptor immobilized on cyanogen bromideactivated Sepharose (InsP, receptor-Sepharose). The antiserum was incubated batchwise overnight at 4OC with the InsP, receptor-Sepharose, then poured into a column and washed extensively. Specific antibodies were eluted from the column with 50 mM glycine (pH 2.5) and immediately neutralized by addition of Tris base. After addition of bovine serum albumin (2-10 mglml), the eluted antibodies were dialyzed overnight against 100 mM NaCI, 50 mM HEPES (pH 7.4). The affinity-purified antibodies were stored at 4OC after addition of sodium azide (0.05%). Affinity-purified antibodies were used in all experiments described.

2. lmmunostaining

rabbitsand membranes

of Primate

and

Tissue Preparation and lmmunoqtochemistry For rat (Sprague-Dawley) and salamander (Ambystoma tigrinum), animals were decapitated and the eyes were immediately enucleated. The same procedure applied to mouse (Mus musculus),

immunoblots as previously

Salamander

Retinal

Sections

for the

InsP,

Receptor

Nomarski optics. The section is 2 urn thick in both cases. For the primate M. fascicularis (A), staining is predominantly confined to the outer plexiform layer (opl) and the inner plexiform layer (ipl). In addition, certain bipolar cell bodies (examples indicated by arrows) and their primary processes show immunoreactivity. Study of dissociated neurons indicated that only a fraction (perhaps one-half or iess) of the bipolar cells showed staining of their cell bodies. On the other hand, all bipolar cells showed strong staining at their presynaptic terminals. For the salamander A. tigrinum (B), the outer limiting membrane (arrow) is also stained. Note the darkly stained, knob-like structures at the outer plexiform layer. These represent the synaptic terminals of the rod and cone receptors (see Figure 4). The weakly stained cells with dark pigment above the photoreceptor outer segments are the retinal pigment epithelial cells. Figure

4. Dissociated

Cells

from

Salamander

and

Rat Retina

Stained

with

the Anti-lnsPp

Receptor

Antibody

(A-C) Salamander retina. (H and I) Rat retina. Nomarski optics. (A) Rod receptor. a, outer segment; b, ellipsoid of inner segment; d, cell body; e, synaptic terminal. (B) Cone receptor. a, outer segment; b, ellipsoid; c, inner segment; d, cell terminal. (C) Bipolar cell. a, dendrites; b, Landolt club; c, cell body; d, presynaptic sites. (D) Horizontal cell. (E) Amacrine cell. a, dendrites; b, axon. (C) Miiller cell. a, distal (scleral) end; b, proximal (vitreal) end. (H) Bipolar cell. a, dendrites; b, :, an adjacent rod cell. (I) Miller cell.

segment; c, inner body; e, synaptic cell. (F) Ganglion synaptic terminal;

Neuron 530

turtle (Pseudemys scripta elegans), and goldfish (Carassius auratus). For chicken (Callus gallus) and monkey (Macaca fascicularis), the eyes were removed under deep anesthesia. In all cases, after removal of the anterior segment, the posterior eyecup was fixed overnight in 4% paraformaldehyde in 100 mM sodium phosphate buffer (pH 7.3) at 4°C. It was then transferred sequentially into 5% and 30% sucrose in the same buffer, each at 4OC and for a period of about 12 hr to allow full penetration of the tissue. The eyecup was then frozen in isopentane pre-cooled in liquid nitrogen and embedded in O.C.T. embedding medium. The whole block was frozen again in liquid nitrogen. Retinal sections, typically 2 pm thick, were made on a Hacker cryostat and mounted on gelatin-coated slides. Each slide had two to four sections. For immunostaining, the sections were first incubated with 5% normal goat serum (Vector Laboratories, CA) in PBS for 1 hr at room temperature to reduce background staining. They were thenincubatedwiththeaffinity-purifiedantibody(1:100dilution) overnight at 4OC, followed by two washes in PBS for 30 min. Triton X-100 (0.3%) was added to all incubation and wash buffers. Next, the sections were incubated with a biotin-conjugated goat anti-rabbit secondary antibody (I:200 dilution; Vector Laboratories) for 2 hr at room temperature, washed twice in PBS for 30 min, and incubated for 1 hr with an avidin-biotin-peroxidase complex (I:100 dilution; Vector Laboratories) in PBS. After two more washes in PBS for 30 min, the stain was developed with a substrate solution consisting of 20 ml of PBS, 0.1 ml of 3% Hz02, and IOmgof diaminobenzidine. The staining reaction was terminated by washing with PBS, and the sections were coverslipped with 50% glycerol in PBS. SDS-PAGE and lmmunoblotting Rats were dark-adapted for 1 hr before decapitation and enucleation. The retinas were carefully separated from the pigment epithelium, then homogenized in 5% SDS, 1 mM benzamidine, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM EDTA in Trisbuffered saline (TBS: 50 mM Tris-HCI [pH 7.41, 150 mM NaCI). Insoluble material was removed by centrifugation at 13,000 x g for 10 min. Proteins were assayed using the Pierce BCA reagent. Parallel procedures were also carried out on rat cerebellar tissue for comparison. SDS-PAGE was performed using the system of Laemmli (1970) on 1.5 mm thick, 5%-16% gradient polyacryamide gels, and the separated proteins were transferred to Immobilon-P membranes according to Towbin et al. (1979). The blots were blocked with 10% non-fat dry milk in TBS for 2 hr before an overnight incubation at 4OC with the affinity-purified antibodies in 3% bovine serum albumin in TBS at 1:200dilution. Afterward, they were washed three times for IO min each in 5% non-fat dry milk in TBS, then incubated with a horseradish peroxidase-linked goat anti-rabbit secondary antibody (I:1500 dilution; Boehringer Mannheim) for 1 hr at room temperature. After three washes of 10 min each in TBS, the blots were developed using 4-chloro-I-naphthol as the substrate. Preparation of Dissociated Retinal Cells Eyes from dark-adapted animals were also used in these experiments in order to facilitate the separation of the retina from the pigment epithelium. The dissociation of the retina into individual cells was performed in room light. The isolated retina was first incubated for 45 min at 20°C, with gentle shaking, in a saline solution (for amphibians: 100 mM NaCI, 2.5 mM KCI, 10 mM glucose, 10 mM Na-HEPES [pH 6.21; for mammals: 140 mM NaCI, 3.6 mM KCI, 10 mM glucose, 3 mM Na-HEPES [pH 6.21) supplemented with 10 U/ml papain (Worthington), 1.2 mM EDTA, and 5.5 mM cysteine. It was then washed with cold saline (pH 7.4) containing bovine serum albumin (0.1 mglml). Dissociation of the retina into individual cells was effected by gentle trituration of the treated retina in cold saline (pH 7.4) with a wide-bore transfer pipette. Aliquots of freshly dissociated cells were placed in a test tube and fixed with 4% paraformaldehyde in phosphate buffer overnight at 4OC. The fixed cells were pipetted onto polyo-lysine-coated slides and left to settle for 2 hr. The subsequent immunostaining procedures were identical to those previously described for retinal sections.

Acknowledgments We thank Dr. Samuel M. Wu for comments and suggestions on themanuscript.This workwassupported in part by USPHSgrant EY06837 to K.-W. Y., MH18531 and Research Scientist Award DA00074 to S. H. S., fellowship MH-09953 to A. H. S., and a gift from Bristol-Meyers Squibb (S. H. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

December

11, 1990; revised

February

1, 1991.

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,