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Immunocytochemical localization of GABA neurons in the rabbit and frog retina
Immunocytochemical Localization of GABA Neurons in the Rabbit and Frog Retina C. BRANDON,’
D. M. K. LAM,2 Y. Y. T. SU” AND J.-Y. WU’
Department of Cell Biology,’ and Cuilen Eye Institute2 Baylor College of Medicine, Houston, TX 77030
BRANDON, C., D. M. K. LAM, Y, Y. T. SU AND J.-Y. WU. fnfmrrr~oc,~toc.llcn?ic.~rI loc~lirtrtiorl q/‘GABA t~c/drotr.s i/l Ihe rcrhhit rrrd ,$w~ rctir~tr. BRAIN RES. BULL, 5: Suppl. 2, 21-29. 1980.-The visualization of y-aminobutyric acid (GABA) neurons in rabbit and frog retinas has been carried out, using an immunocytochemical technique for the localization of the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD). In the rabbit, immunoreactivity was restricted to a small group of amacrine cell bodies and their laminated processes in the inner plexiform layer. Electron microscopic examination showed that these processes were presynaptic to ganglion cell dendrites (infrequently), amacrine cell telodendrons, and bipolar cell terminals. Often, bipolar cell terminals were found which were densely innervated by several GAD-positive processes. No definite synapses were observed in which a GAD-positive process represented the postsynaptic element. In the frog, dense GAD immunoreactivity was observed in the inner plexiform layer, both as punctate deposits and as filled processes of stratified and diffuse amacrine cells; in the inner nuclear layer, where many cell bodies were labeled, including those of some horizontal cells; and diffusely in the outer plexiform layer. Retina
Amacrine cell
GABA
Glutamate decarboxylase
y-AMINOBUTYRIC acid has been suspected as a visual neurotransmitter since its demonstration, some twenty years ago, in the retinas of numerous vertebrate species. Since that time, this substance has also fulfilled most of the conventional criteria for the identification of a neurotransmitter; the evidence for this has been recently reviewed [3, 12, 13, 201. The specific visualization of GABA neurons in the retina was first achieved using “H-GABA autoradiography. There are at least two biochemical systems which can bind GABA, both of which contribute to the autoradiographic pattern: a high-affinity, sodium-dependent uptake system [ll, 17, 221, and the sodium-independent, high-affinity postsynaptic receptor [2, lo]. Intravitreal injection of radioisotope into intact animals led to the labeling of certain amacrine cell bodies, due to uptake sites, and to the diffuse labeling of the inner plexiform layer (IPL) due probably to the presence of both types of sites [4, 5, 81. Not all amacrine cells were labeled, and the interesting suggestion was made that GABA neurons represent at most a subgroup of amacrine cells [9]. The visualization of GABA neurons at a higher level of resolution became possible with the purification of glutamate decarboxylase (GAD) from mouse brain and the production of specific anti-GAD serum [16,24]. Using an immunohistochemical technique for the localization of GAD, these workers observed a dense labeling of the inner plexiform layer of the rat retina, in the form of several indistinct laminae [ 11. On the basis of preliminary electron microscopic observations using this same antiserum, Wood et N/. [23] and Vaughn et ul. [21] suggested that immunochemically labeled processes in the inner plexiform layer were those of a class of amacrine cells. Similar lamination of GAD-containing processes was found in the rabbit retina [4]. Also, treatment of rabbits with intravitreal colchicine in rive prior to im-
Copyright
” 1980 ANKHO
International
Immunohistochemistry
munohistochemical analysis caused dense staining of amacrine cell bodies in the inner nuclear layer, further supporting the identification of GABA neurons as a class of amacrines. Recently, Daw and co-workers [6,25] have shed some light on at least one function of these GABA neurons, by showing that the GABA anatagonist picrotoxin abolishes ganglion cell directional selectivity when infused into the blood supply of the eye. This information, suggesting a specific role for a neurochemically discrete family of neurons, prompted us to extend our immunohistochemical observations to the electron microscopic level. These results are presented here, along with some preliminary observations on GAD staining in the frog retina. METHOD Fixm tion
Eyes were removed from pentobarbital-anaesthetized albino rabbits or decapitated, pithed frogs. After removal of the cornea, the lens and vitreous were gently removed and the entire eye cup was immersed in fixative and agitated on an orbital rotator. The rabbit fixative was 2% formaldehyde (from paraformaldehyde) +O. 1% acrolein (17 PM) +0.002% CaCI, in 155 mM sodium phosphate buffer, pH 7.3; for the frog, the sodium phosphate concentration was reduced to 130 mM. After 1.5-2 hr at room temperature, the tissue was transferred to fresh fixative without acrolein and stored overnight at 4°C.
Small pieces of fixed retina and pigment epithelium (about 3 x5 mm) were encapsulated in molten low-gelling-temperature agarose (4% in phosphate-buffered saline, PBS) at 37”C,
Inc.-0361-9230/80/080021-09$01.40/O
BRANDON, LAM, SU AND WU
A
B:
“’
FIG. I. GAD imm~~or~~ti~ity in rabbit retina. OPL-outer plexiform Iayer; B&--inner nuclear layer; I%---inner plexiform layer; GCganglion cell layer. (A) Fifty micron T&on-treated, GAD-stained section shows four broad laminae of reaction product within the IPL (arrowheads). (3) Control section, incubated in preimmune serum. (Cl Fifty micron section, stained without Triton, shows the punctate nature of deposits more clearly. (l3f In a 2 grn section, some strat~~~at~o~ of stained processes near the INL is discernible. (E) Fifty micron horizontal section through the IPL shows several strings of stained varicosities (arrowheads), interspersed with individual puncta. M-MulIar cell processes. (A,ElxdOO; C,Dx 1000; Ex750).
GAD IN THE RABBIT AND FROG RETINA
FIG. 2. Montage of several GAD-stained celi bodies from a rabbit that received colchicine intravitreally two days before enucleation. (A and B) Fifty micron sections showing amacrine processes descending into the IPL (arrowheads). (C) One micron section through a similarly stained cell. (D) A probable misplaced GAD-containing amacrine cell. (E) An unidentified, stained cell body lying in the ganglion cell layer beneath a labeled amacrine ceil. (x 1000).
chilled PBS at 25 mM sodium
at 4°C for about 30 min, and sectioned at 50 /.crn into 4°C with a Vibratome. Phosphate-buffered saline was sodium phosphate/l30 mM NaCl (rabbit) or 25 mM phosphate/l~ mM NaCl (frog).
Before antibody incubation, sections were immersed in hydroxylami~e hydrochlo~de solution (25 mM in PBS) for 1 hr at 20°C. This treatment reduced non-specific staining due to unreacted aIdehyde groups. After a brief wash in PBS, sections were then incubated for 30 min in 10% dimethylsulfoxide in PBS, a step which increased the depth of antibody penetration into the sections and also somewhat intensi~cd the staining. Staining
Immu~ohistochemical staining was carried out by using a modi~cation of the peroxidase-~tiperoxidase method of Stemberger and co-workers [I41 in which the second antibody (goat antiserum to rabbit IgG) was replaced with Protein A. This method is based on the ability of the Protein A component of the cell waif of Stuphylococcus oweus to bind to the F,. portion of most mammalian IgG. Although somewhat less sensitive than the original, this method has the advantage of decreased “non-specific” background staining. In separate experiments, staining was carried out both with and without Triton X-100 in the bathing medium. In the former case, the medium was PBS containing 0.25% Triton
X- 100; in the latter, it was PBS containing 0.1% ovalbumin. All staining reagents and sera (except diaminobenzidine) were diluted with these media. In staining, sections were transferred to jndividu~ wells of a disposable multiwell titer plate containing 250 ~1 of the diluted primary antiserum (two sections per well). The anti-GAD serum used here was raised against purified mouse brain GAD (for rabbit retina) or catfish GAD (for frog retina); antigens and antisera have been extensively characterized [16, 18, 19, 241. Optimal dilutions were 1:250 and 1:500. The plate was covered and agitated for 4 hr at room temperature on an orbital rotator and then for an additional 12 hr at 4°C. Al1 subsequent operations were carried out at room temperature. At the end of this time, sections were transferred to individual 15 x 60 mm plastic petri dishes containing about 7 ml of the washing medium, agitated for 30 min on the orbital rotator, and then transferred to dishes with fresh medium and washed for an additional 30 min. Sections were then each treated with 100 ~1 of Protein A solution (50 Kugirnl)for 1.5 hr in a humid atmosphere. After two more 30 min washes, sections were treated in a spot dish with a 1: 100 dilution of peroxidase-antiperoxidase complex (45 pg/ml) for 1.5 hr, given two more 30 min washes, and then stained for peroxidase. The staining solutions contained 30 mg of diaminobenzidine.4 HCl and 25 ~1 of 30% H,O, in 50 ml of PBS; sections were generally incubated in this medium for 5-8 min. The stained sections were briefly washed in PBS, postfixed for 1 hr with 1% glutaraldehyde in PBS, treated with 1%
14
BRANDON,
LAM, SU AND WU
0~0, in PBS for 2-3 hr, stained en bloc with 1% uranyl acetate in 0.1 M sodium acetate, dehydrated with graded ethanols, and infiltrated with Epon. For embedding, sections were placed in smah drops of resin at intervals on a smooth sheet of ~uminum foil, then covered with a weighted microscope slide that had been treated with d~chlor~imethy~silane to prevent epoxy from adhering to it. After polymerization, the foil was easily peeled off to yield a 50 Frn sheet of epoxy containing the sections mounted with the stained face upward. Sections could be coverslipped with oil or glycerol for examination and photography. For microtomy, small pieces of the stained sections were cut out with a razor blade, glued to smooth epoxy blocks with cyano-acryiate glue and sectioned en fuce. RESULTS
Rabbit Retina Light microscopy. GAD staining, carried out in the presence of 0.25% Triton X-100 to enhance antibody penetration, yielded the pattern shown in Fig. 1A. In these 50 pm sections, GAD-positive deposits formed four broad laminae within the inner plexiform layer. The Iaminae were of approximateIy equal intensity and were evenly spaced throughout the layer. Control sections, incubated with pre-immune serum, were free of reaction product (Fig. 1B). When Triton was omitted, GAD reaction product was observed in much more discrete deposits, while lamination was less distinct (Fig. 1C). When sections were cut to a thickness of 2 pm, stained processes sometimes appeared as long strings of varicosities, or as dense, punctate structures within the IPL (Fig. ID). Varicosities were seen to better effect in horizontat sections through the IPL, where they were again interspersed with individual punctate deposits (Fig. 1E). Administration of colchicine l-2 days before staining, a technique first described by Ribak et al. [IS], caused a blockage of axon transport and an accumulation of GAD product in cell bodies. Under these conditions amacrine cells were the only cell type stained (Fig. 2). Partially filled processes were seen to descend into the IPL (Fig. 2A and 2B), never in the opposite direction. On rare occasions, labeled cells with the characteristic morphology of amacrine cells were observed in the ganglion ceil layer (Fig. 2D and 2E), and were most probably displaced amacrines. Electrott microscopy. GAD-stained processes; when sectioned longitudinally, often appeared as dense varicosities (Fig. 3A). These swellings were characterized by accretions of synaptic vesicles and microtubules, both of which were associated with reaction product. Regions between varicosities were generally not stained. Stained processes were involved in several types of synaptic relationships, some more frequent than others. An amacrine cell may be presynaptic to any of three cell types (ganglion, bipolar, amacrine), or postsynaptic to two (bipolar, amacrine). Among the first group, GABA-amacrine to ganglion cell synapses were observed the least often .(Fig. 3B); GABA-amacrine:bipolar and GABA-amacrine:amacrine synapses were far more numerous. Postsynaptic amacrine processes (Fig. 4A-D) were identified by their small size and relatively low vesicle density, by the presence of membranous tubular structures, and sometimes by the presence of varicosities containing synaptic vesicles (e.g., Fig. 4C). GABA-amacrine:bipolar synapses were identified most
FIG. 3. Low-power electron micrographs of GAD-positive processes in the IPL of the rabbit. GAM-GABA-amacrine cell process: GCD-ganglion ceil dendrite; mt-microtubules; sv-synaptic vesicles. (A) Amacrine cell varicosity (arrowheads) containing numerous stained synaptic vesicles and micr~tubules. (B) A GADpositive process presynaptic to a large ganglion cell dendrite; reaction product is again clustered around synaptic vesicles. (Ax28,200; B x 39,250).
easily when the postsynaptic process contained a synaptic ribbon; several of this type are shown in Figs. 5 and 6. Often, bipolar terminals were strikingly surrounded by a dense plexus of GAD-positive amacrine boutons (Fig. 5). Frog Retina Light microscopy.
As might be expected,
the pattern of
GAD IN THE RABrJlT AND FROG RETINA
25
26
BRANDON,
LAM, SU AND WU
FIG. 5. Bipolar cell terminals in the rabbit retina. BT-bipolar terminal. GAD-stained terminals were often seen surrounding bipolar terminals, which were usually identified by their characteristic ribbons (arrowheads). (A,B~39,250; Cx48,750; D~45,800).
GAD IN THE RABBIT
AND FROG
27
RETINA
FIG, 6, Bipolar cell terminals in the rabbit retina. Bipolar terminals were not always profusely innervated by GADcontaining processes. (A,B) The postsynaptic element is invofved in the dyadic relationship characteristic of bipolar terminals [arrowheads). In (B), the larger labeled process appears to be an oblique section through a large varicosity, like those seen in Fig. 3. (Ax52,300; Bx39,250).
FIG. 7_ GAD in frog retina. {A) Dense reaction product fills the 1PL of
a five micron section and gives
are labeled (asterisks). (B) A one micron section shows the punctate natures of the staining, as well as several stained amacrine and horizontal cell bodies. (x750) some indication
of lamination
(arrowheads).
Some amacrine
cell bodies
28
BRANDON,
LAM, SU AND WU
FIG. 8. GAD in frog retina. Deeper areas of stained, 50 wrn sections showed golgi-like patterns of amacrine cell staining. (A) Montage showing several GAD-stained processes of apparent stratified amacrine cells. One of them forms a varicosity just above the ganglion cell layer (arrowhead). which may represent a synaptic contact. (3) Montage showing several bushy, somewhat diffuse GABA amacrines; in the first two, processes can be seen emanating from the INL (arrowheads). (X 1000)
GAD immunoreactiv~ty in this species was complex, for at least two reasons. First, the synaptic organization of the frog retina is complicated (see e.g., [7]). Second, GAD immunoreactivity was apparently so great that cells were not just stained at synaptic sites, but were often completely filled with reaction product, even in the soma. In a 5 pm section of Triton-treated tissue (Fig. 7A), both stratified and diffuse labeling of the IPL could be seen. In addition, numerous cell bodies throughout the IPL were GAD-positive, and a diffuse labeling of the outer plexiform layer was noted. In 1 pm plastic sections, the staining in the IPL was found to consist of the usual punctate deposits, and the perikaryal cytoplasm of some amacrine and horizontal cells was labeled (Fig. 7B). Deeper into the stained sections, the arborizations of both stratified and diffuse amacrine cells were sometimes filled with reaction product, yielding a Golgi-like picture (Fig. 8A and 8B).
other amacrine cell processes, or bipolar cell terminals, although the first type of interaction is rarely observed. Labeled processes were not seen that were postsynaptic. This might be expected if substantial distances separated pre- and postsynaptic sites of a given amacrine cell, and if the sites where it was postsynaptic contained few vesicles and little GAD, In the rat retina, on the other hand, GADcontaining amacrines were found that were postsynaptic to both amacrines and bipolars 121,231. Second, a type of synaptic relationship was observed in which numerous GAD-containing terminals synapsed on the same bipolar cell terminal. This a~~gement provides a morphological substrate for a potent GABA-mediated inhibition of a certain type of bipolar cell. #ether or not such an arrangement is involved in the generation of directionally selective receptive fields remains a subject for further study. ACKNOWLEDGEMENTS
DISCUSSION
Two basic conclusions may be drawn from the observations reported here on the rabbit retina. First, GAD-positive terminals may be presynaptic to ganglion cell dendrites,
This work was supported National
Institutes
of Health
by research (NS
grants from the U. S.
13224 to JYW and EY 02423 to
DMKL). DMKL is a recipieni of a Research Career Development Award from the U. S. National Eye institute.
GAD IN THE RABBIT AND FROG RETINA
29
REFERENCES 1. Barber, R. P. and K. Saito. In: GABA in Nrrvous System Fumdon. edited by E. Roberts, T. N. Chase and D. B. Tower. New York: Raven Press, 1976, pp. 113-132. 2. Beaumont, K., W. S. Chilton, H. E. Yamamura and S. J. Enna. Muscimol binding in rat brain: association with synaptic GABA receptors. Bruin Rc.s. 148: 153-163, 1978. 3. Bonting, S. L., ed., Transmitfcrs in the Visuul Process. New York: Pergamon Press, 1976. 4. Brandon, C., D. M. K. Lam and J.-Y. Wu. The y-aminobutyric acid system in rabbit retina: localization by immunocytochemistry and autoradiography. Proc. nntn. Aurd. Sc,i. U.S.A. 16: 3557-3561, 1979. 5. Bruun, A. and B. Ehinger.
Uptake of certain possible neurotransmitters into retinal neurones of some mammals. E.wpl E.ve Res. 19: 435-447, 1914. 6. Caldwell, J. H., N. W. Daw and H. J. Wyatt. Effects of picrotoxin and strychnine on rabbit retinal ganglion cells: lateral interactions for cells with more complex receptive fields. J. Physiol. 276: 277-298, 1978. 7. Dubin, M. The inner plexiform layer of the vertebrate
retina: a quantitative and comparative electron microscopic analysis. J. camp. Nrurol. 140: 479-506, 1970. 8. Ehinger, B. Autoradiographic identification of rabbit retinal neurons that take up GABA. Experienria. 26: 1063-1064, 1970. 9. Ehinger, B. and B. Falck. Autoradiography of some suspected neurotransmitter substances: GABA, glycine, glutamic acid, histamine, dopamine, and L-dopa. Bruin Res. 33: 157-172, 1971. 10. Enna, S. J. and S. H. Snyder. Gamma-aminobutyric acid (GABA) receptor binding in mammalian retina. Bruin Res. 115: 174179, 1976. 11. Goodchild, M. and M. J. Neal. The uptake of RH-gammaaminobutyric acid by the retina. Br. ./. Phormcrc,. 47: 52%542. 1973. 12. Graham, Jr., L. T. Comparative aspects of neurotransmitters in the retina. In: The Eye, edited by H. Davson and L. T. Graham, Jr. New York: Academic Press, 1974, pp. 283-342. 13. Neal, M. J. Amino acid transmitter substances in the vertebrate retina. Gcn. Phnrmcrc,. 7: 321-332. 1976.
14 Petrali, J. P., D. M. Hinton, G. C. Moriarty and L. A. Sternberger. The unlabeled antibody enzyme method of immunocytochemistry. Quantitative comparison of sensitivities with and without peroxidase-anti-peroxidase complex (PAP). J. Histochem. C~rochem. 22: 782-801, 1974. 15 Ribak, C. E., J. E. Vaughn and K. Saito. Immunocytochemical localization of glutamic acid decarboxylase in neuronal somata following colchicine inhibition of axonal transport. Bruin Res. 140: 315-332, 1978.
16. Saito, K., J.-Y. Wu and E. Roberts. lmmunochemical comparisons of vertebrate glutamic acid decarboxylase. Bruin Rcs. 6.5: 277-285, 1974.
17. Starr, M. S. and M. J. Voaden. The uptake of “C-yaminobutyric acid by the isolated retina of the rat. Vision Rc.s. 12: 549-557. 1972. 18. Su, Y. Y. T., J.-Y. Wu and D. M. K. Lam. Purification
of L-glutamic acid decarboxylase from catfish brain. .I. Neurochcm. 33: 169 179, 1979. 19. Su, Y. Y. T., J.-Y. Wu and D. M. K. Lam. Immunochemical studies of anti-catfish GAD IgG. Ah.srrrrct qf fhc, 8th Annrrrrl MeefinP. Socirrv for Nc,urosciettce. 1979. n. 599. 20. Van Harreveld,.A’. Pharmacology of the vertebrate retina. Prog. ‘Vcrtrohiol. 8: l-43, 1976. 21. Vaughn, J. E., R. P. Barber, K. Saito, E. Roberts and E. V. Famiglietti, Jr. Immunocytochemical identification of GABAergic neurons in rat retina. Anat. Rrc. 190: 571-572, 1978. 22. Voaden. M. J.. J. Marshall and N. Murani. The uptake of (“H)y-aminobutyric acid and (“H)glycine by the isolated retina of the frog. Bruin Res. 67: 115-134, 1974. 23. Wood, J. G.. B. J. McLaughlin and J. E. Vaughn. In: GABA in Nen~ts S,vstom Furzction. edited by E. Roberts. T. N. Chase and D. B. Tower. New York: Raven Press, 1976, pp. 133-148. 24. Wu. J.-Y., T. Matsuda and E. Roberts. Purification and characterization of glutamate decarboxylase from mouse brain. .I. hid Chcm. 248: 302%3034, 1973. 25. Wyatt, H. J. and N. W. Daw. Specific effects of neurotransmitter antagonists on ganglion cells in rabbit retina. Scic,nc,cT191: 204-205. 1976.
D. M. K. LAM,2 Y. Y. T. SU” AND J.-Y. WU’
Department of Cell Biology,’ and Cuilen Eye Institute2 Baylor College of Medicine, Houston, TX 77030
BRANDON, C., D. M. K. LAM, Y, Y. T. SU AND J.-Y. WU. fnfmrrr~oc,~toc.llcn?ic.~rI loc~lirtrtiorl q/‘GABA t~c/drotr.s i/l Ihe rcrhhit rrrd ,$w~ rctir~tr. BRAIN RES. BULL, 5: Suppl. 2, 21-29. 1980.-The visualization of y-aminobutyric acid (GABA) neurons in rabbit and frog retinas has been carried out, using an immunocytochemical technique for the localization of the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD). In the rabbit, immunoreactivity was restricted to a small group of amacrine cell bodies and their laminated processes in the inner plexiform layer. Electron microscopic examination showed that these processes were presynaptic to ganglion cell dendrites (infrequently), amacrine cell telodendrons, and bipolar cell terminals. Often, bipolar cell terminals were found which were densely innervated by several GAD-positive processes. No definite synapses were observed in which a GAD-positive process represented the postsynaptic element. In the frog, dense GAD immunoreactivity was observed in the inner plexiform layer, both as punctate deposits and as filled processes of stratified and diffuse amacrine cells; in the inner nuclear layer, where many cell bodies were labeled, including those of some horizontal cells; and diffusely in the outer plexiform layer. Retina
Amacrine cell
GABA
Glutamate decarboxylase
y-AMINOBUTYRIC acid has been suspected as a visual neurotransmitter since its demonstration, some twenty years ago, in the retinas of numerous vertebrate species. Since that time, this substance has also fulfilled most of the conventional criteria for the identification of a neurotransmitter; the evidence for this has been recently reviewed [3, 12, 13, 201. The specific visualization of GABA neurons in the retina was first achieved using “H-GABA autoradiography. There are at least two biochemical systems which can bind GABA, both of which contribute to the autoradiographic pattern: a high-affinity, sodium-dependent uptake system [ll, 17, 221, and the sodium-independent, high-affinity postsynaptic receptor [2, lo]. Intravitreal injection of radioisotope into intact animals led to the labeling of certain amacrine cell bodies, due to uptake sites, and to the diffuse labeling of the inner plexiform layer (IPL) due probably to the presence of both types of sites [4, 5, 81. Not all amacrine cells were labeled, and the interesting suggestion was made that GABA neurons represent at most a subgroup of amacrine cells [9]. The visualization of GABA neurons at a higher level of resolution became possible with the purification of glutamate decarboxylase (GAD) from mouse brain and the production of specific anti-GAD serum [16,24]. Using an immunohistochemical technique for the localization of GAD, these workers observed a dense labeling of the inner plexiform layer of the rat retina, in the form of several indistinct laminae [ 11. On the basis of preliminary electron microscopic observations using this same antiserum, Wood et N/. [23] and Vaughn et ul. [21] suggested that immunochemically labeled processes in the inner plexiform layer were those of a class of amacrine cells. Similar lamination of GAD-containing processes was found in the rabbit retina [4]. Also, treatment of rabbits with intravitreal colchicine in rive prior to im-
Copyright
” 1980 ANKHO
International
Immunohistochemistry
munohistochemical analysis caused dense staining of amacrine cell bodies in the inner nuclear layer, further supporting the identification of GABA neurons as a class of amacrines. Recently, Daw and co-workers [6,25] have shed some light on at least one function of these GABA neurons, by showing that the GABA anatagonist picrotoxin abolishes ganglion cell directional selectivity when infused into the blood supply of the eye. This information, suggesting a specific role for a neurochemically discrete family of neurons, prompted us to extend our immunohistochemical observations to the electron microscopic level. These results are presented here, along with some preliminary observations on GAD staining in the frog retina. METHOD Fixm tion
Eyes were removed from pentobarbital-anaesthetized albino rabbits or decapitated, pithed frogs. After removal of the cornea, the lens and vitreous were gently removed and the entire eye cup was immersed in fixative and agitated on an orbital rotator. The rabbit fixative was 2% formaldehyde (from paraformaldehyde) +O. 1% acrolein (17 PM) +0.002% CaCI, in 155 mM sodium phosphate buffer, pH 7.3; for the frog, the sodium phosphate concentration was reduced to 130 mM. After 1.5-2 hr at room temperature, the tissue was transferred to fresh fixative without acrolein and stored overnight at 4°C.
Small pieces of fixed retina and pigment epithelium (about 3 x5 mm) were encapsulated in molten low-gelling-temperature agarose (4% in phosphate-buffered saline, PBS) at 37”C,
Inc.-0361-9230/80/080021-09$01.40/O
BRANDON, LAM, SU AND WU
A
B:
“’
FIG. I. GAD imm~~or~~ti~ity in rabbit retina. OPL-outer plexiform Iayer; B&--inner nuclear layer; I%---inner plexiform layer; GCganglion cell layer. (A) Fifty micron T&on-treated, GAD-stained section shows four broad laminae of reaction product within the IPL (arrowheads). (3) Control section, incubated in preimmune serum. (Cl Fifty micron section, stained without Triton, shows the punctate nature of deposits more clearly. (l3f In a 2 grn section, some strat~~~at~o~ of stained processes near the INL is discernible. (E) Fifty micron horizontal section through the IPL shows several strings of stained varicosities (arrowheads), interspersed with individual puncta. M-MulIar cell processes. (A,ElxdOO; C,Dx 1000; Ex750).
GAD IN THE RABBIT AND FROG RETINA
FIG. 2. Montage of several GAD-stained celi bodies from a rabbit that received colchicine intravitreally two days before enucleation. (A and B) Fifty micron sections showing amacrine processes descending into the IPL (arrowheads). (C) One micron section through a similarly stained cell. (D) A probable misplaced GAD-containing amacrine cell. (E) An unidentified, stained cell body lying in the ganglion cell layer beneath a labeled amacrine ceil. (x 1000).
chilled PBS at 25 mM sodium
at 4°C for about 30 min, and sectioned at 50 /.crn into 4°C with a Vibratome. Phosphate-buffered saline was sodium phosphate/l30 mM NaCl (rabbit) or 25 mM phosphate/l~ mM NaCl (frog).
Before antibody incubation, sections were immersed in hydroxylami~e hydrochlo~de solution (25 mM in PBS) for 1 hr at 20°C. This treatment reduced non-specific staining due to unreacted aIdehyde groups. After a brief wash in PBS, sections were then incubated for 30 min in 10% dimethylsulfoxide in PBS, a step which increased the depth of antibody penetration into the sections and also somewhat intensi~cd the staining. Staining
Immu~ohistochemical staining was carried out by using a modi~cation of the peroxidase-~tiperoxidase method of Stemberger and co-workers [I41 in which the second antibody (goat antiserum to rabbit IgG) was replaced with Protein A. This method is based on the ability of the Protein A component of the cell waif of Stuphylococcus oweus to bind to the F,. portion of most mammalian IgG. Although somewhat less sensitive than the original, this method has the advantage of decreased “non-specific” background staining. In separate experiments, staining was carried out both with and without Triton X-100 in the bathing medium. In the former case, the medium was PBS containing 0.25% Triton
X- 100; in the latter, it was PBS containing 0.1% ovalbumin. All staining reagents and sera (except diaminobenzidine) were diluted with these media. In staining, sections were transferred to jndividu~ wells of a disposable multiwell titer plate containing 250 ~1 of the diluted primary antiserum (two sections per well). The anti-GAD serum used here was raised against purified mouse brain GAD (for rabbit retina) or catfish GAD (for frog retina); antigens and antisera have been extensively characterized [16, 18, 19, 241. Optimal dilutions were 1:250 and 1:500. The plate was covered and agitated for 4 hr at room temperature on an orbital rotator and then for an additional 12 hr at 4°C. Al1 subsequent operations were carried out at room temperature. At the end of this time, sections were transferred to individual 15 x 60 mm plastic petri dishes containing about 7 ml of the washing medium, agitated for 30 min on the orbital rotator, and then transferred to dishes with fresh medium and washed for an additional 30 min. Sections were then each treated with 100 ~1 of Protein A solution (50 Kugirnl)for 1.5 hr in a humid atmosphere. After two more 30 min washes, sections were treated in a spot dish with a 1: 100 dilution of peroxidase-antiperoxidase complex (45 pg/ml) for 1.5 hr, given two more 30 min washes, and then stained for peroxidase. The staining solutions contained 30 mg of diaminobenzidine.4 HCl and 25 ~1 of 30% H,O, in 50 ml of PBS; sections were generally incubated in this medium for 5-8 min. The stained sections were briefly washed in PBS, postfixed for 1 hr with 1% glutaraldehyde in PBS, treated with 1%
14
BRANDON,
LAM, SU AND WU
0~0, in PBS for 2-3 hr, stained en bloc with 1% uranyl acetate in 0.1 M sodium acetate, dehydrated with graded ethanols, and infiltrated with Epon. For embedding, sections were placed in smah drops of resin at intervals on a smooth sheet of ~uminum foil, then covered with a weighted microscope slide that had been treated with d~chlor~imethy~silane to prevent epoxy from adhering to it. After polymerization, the foil was easily peeled off to yield a 50 Frn sheet of epoxy containing the sections mounted with the stained face upward. Sections could be coverslipped with oil or glycerol for examination and photography. For microtomy, small pieces of the stained sections were cut out with a razor blade, glued to smooth epoxy blocks with cyano-acryiate glue and sectioned en fuce. RESULTS
Rabbit Retina Light microscopy. GAD staining, carried out in the presence of 0.25% Triton X-100 to enhance antibody penetration, yielded the pattern shown in Fig. 1A. In these 50 pm sections, GAD-positive deposits formed four broad laminae within the inner plexiform layer. The Iaminae were of approximateIy equal intensity and were evenly spaced throughout the layer. Control sections, incubated with pre-immune serum, were free of reaction product (Fig. 1B). When Triton was omitted, GAD reaction product was observed in much more discrete deposits, while lamination was less distinct (Fig. 1C). When sections were cut to a thickness of 2 pm, stained processes sometimes appeared as long strings of varicosities, or as dense, punctate structures within the IPL (Fig. ID). Varicosities were seen to better effect in horizontat sections through the IPL, where they were again interspersed with individual punctate deposits (Fig. 1E). Administration of colchicine l-2 days before staining, a technique first described by Ribak et al. [IS], caused a blockage of axon transport and an accumulation of GAD product in cell bodies. Under these conditions amacrine cells were the only cell type stained (Fig. 2). Partially filled processes were seen to descend into the IPL (Fig. 2A and 2B), never in the opposite direction. On rare occasions, labeled cells with the characteristic morphology of amacrine cells were observed in the ganglion ceil layer (Fig. 2D and 2E), and were most probably displaced amacrines. Electrott microscopy. GAD-stained processes; when sectioned longitudinally, often appeared as dense varicosities (Fig. 3A). These swellings were characterized by accretions of synaptic vesicles and microtubules, both of which were associated with reaction product. Regions between varicosities were generally not stained. Stained processes were involved in several types of synaptic relationships, some more frequent than others. An amacrine cell may be presynaptic to any of three cell types (ganglion, bipolar, amacrine), or postsynaptic to two (bipolar, amacrine). Among the first group, GABA-amacrine to ganglion cell synapses were observed the least often .(Fig. 3B); GABA-amacrine:bipolar and GABA-amacrine:amacrine synapses were far more numerous. Postsynaptic amacrine processes (Fig. 4A-D) were identified by their small size and relatively low vesicle density, by the presence of membranous tubular structures, and sometimes by the presence of varicosities containing synaptic vesicles (e.g., Fig. 4C). GABA-amacrine:bipolar synapses were identified most
FIG. 3. Low-power electron micrographs of GAD-positive processes in the IPL of the rabbit. GAM-GABA-amacrine cell process: GCD-ganglion ceil dendrite; mt-microtubules; sv-synaptic vesicles. (A) Amacrine cell varicosity (arrowheads) containing numerous stained synaptic vesicles and micr~tubules. (B) A GADpositive process presynaptic to a large ganglion cell dendrite; reaction product is again clustered around synaptic vesicles. (Ax28,200; B x 39,250).
easily when the postsynaptic process contained a synaptic ribbon; several of this type are shown in Figs. 5 and 6. Often, bipolar terminals were strikingly surrounded by a dense plexus of GAD-positive amacrine boutons (Fig. 5). Frog Retina Light microscopy.
As might be expected,
the pattern of
GAD IN THE RABrJlT AND FROG RETINA
25
26
BRANDON,
LAM, SU AND WU
FIG. 5. Bipolar cell terminals in the rabbit retina. BT-bipolar terminal. GAD-stained terminals were often seen surrounding bipolar terminals, which were usually identified by their characteristic ribbons (arrowheads). (A,B~39,250; Cx48,750; D~45,800).
GAD IN THE RABBIT
AND FROG
27
RETINA
FIG, 6, Bipolar cell terminals in the rabbit retina. Bipolar terminals were not always profusely innervated by GADcontaining processes. (A,B) The postsynaptic element is invofved in the dyadic relationship characteristic of bipolar terminals [arrowheads). In (B), the larger labeled process appears to be an oblique section through a large varicosity, like those seen in Fig. 3. (Ax52,300; Bx39,250).
FIG. 7_ GAD in frog retina. {A) Dense reaction product fills the 1PL of
a five micron section and gives
are labeled (asterisks). (B) A one micron section shows the punctate natures of the staining, as well as several stained amacrine and horizontal cell bodies. (x750) some indication
of lamination
(arrowheads).
Some amacrine
cell bodies
28
BRANDON,
LAM, SU AND WU
FIG. 8. GAD in frog retina. Deeper areas of stained, 50 wrn sections showed golgi-like patterns of amacrine cell staining. (A) Montage showing several GAD-stained processes of apparent stratified amacrine cells. One of them forms a varicosity just above the ganglion cell layer (arrowhead). which may represent a synaptic contact. (3) Montage showing several bushy, somewhat diffuse GABA amacrines; in the first two, processes can be seen emanating from the INL (arrowheads). (X 1000)
GAD immunoreactiv~ty in this species was complex, for at least two reasons. First, the synaptic organization of the frog retina is complicated (see e.g., [7]). Second, GAD immunoreactivity was apparently so great that cells were not just stained at synaptic sites, but were often completely filled with reaction product, even in the soma. In a 5 pm section of Triton-treated tissue (Fig. 7A), both stratified and diffuse labeling of the IPL could be seen. In addition, numerous cell bodies throughout the IPL were GAD-positive, and a diffuse labeling of the outer plexiform layer was noted. In 1 pm plastic sections, the staining in the IPL was found to consist of the usual punctate deposits, and the perikaryal cytoplasm of some amacrine and horizontal cells was labeled (Fig. 7B). Deeper into the stained sections, the arborizations of both stratified and diffuse amacrine cells were sometimes filled with reaction product, yielding a Golgi-like picture (Fig. 8A and 8B).
other amacrine cell processes, or bipolar cell terminals, although the first type of interaction is rarely observed. Labeled processes were not seen that were postsynaptic. This might be expected if substantial distances separated pre- and postsynaptic sites of a given amacrine cell, and if the sites where it was postsynaptic contained few vesicles and little GAD, In the rat retina, on the other hand, GADcontaining amacrines were found that were postsynaptic to both amacrines and bipolars 121,231. Second, a type of synaptic relationship was observed in which numerous GAD-containing terminals synapsed on the same bipolar cell terminal. This a~~gement provides a morphological substrate for a potent GABA-mediated inhibition of a certain type of bipolar cell. #ether or not such an arrangement is involved in the generation of directionally selective receptive fields remains a subject for further study. ACKNOWLEDGEMENTS
DISCUSSION
Two basic conclusions may be drawn from the observations reported here on the rabbit retina. First, GAD-positive terminals may be presynaptic to ganglion cell dendrites,
This work was supported National
Institutes
of Health
by research (NS
grants from the U. S.
13224 to JYW and EY 02423 to
DMKL). DMKL is a recipieni of a Research Career Development Award from the U. S. National Eye institute.
GAD IN THE RABBIT AND FROG RETINA
29
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