Colocalization of enkephalin and glycine in amacrine cells of the chicken retina

Brain Research, 628 (1993) 349-355

349

© 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00 BRES 25899

Colocalization of enkephalin and glycine in amacrine cells of the chicken retina Carl B. Watt *, Valarie J. Florack Alice R. McPherson Laboratory of Retina Research, Centerfor Biotechnology, Baylor College of Medicine, 400 Research Forest DriL,e, The Woodlands, TX 77381, USA

(Accepted 10 August 1993)

Key words: Immunocytochemistry;Autoradiography; Neuropeptide; Classical neurotransmitter; Coexistence; Somatostatin; Neurotensin; GABA

The present double-label study combines enkephalin immunocytochemistrywith either autoradiography of glycine high-affinityuptake or glycine immunocytochemistryto investigate the coexistence of glycine in enkephalin-amacrine cells of the chicken retina. A regional analysis revealed that the percentage coexistence of glycine high-affinity uptake in enkephalin-amacrine cells did not vary appreciably throughout the retina. Overall, 54.9% of enkephalin-amacrine cells exhibited high-affinity glycine uptake. Double-label immunofluorescence cytochemistry revealed that 52.5% of enkephalin-amacrine cells expressed glycine immunoreactivity.These double-immunolabelled cells were observed throughout the center and periphery of the retina. The present study reveals a similar percentage of chicken enkephalin-amacrine cells expressing either glycine high-affinity uptake (54.9%) or glycine immunoreactivity (52.5%) and therefore, provides supportive evidence for identifying these cells as glycinergic. The present study also suggests a functional diversity in the population of enkephalin-amacrine cells in the chicken retina relative to their coexisting/non-coexisting relationship with glycine.

During the past decade, retinal enkephalinergic pathways have been the subject of both morphological and physiological analyses 19. Enkephalin has been localized primarily to amacrine cell populations in nonmammalian retinas and has been shown to be physiologically active. A number of studies have investigated enkephalin's interactive and coexisting relationships with other neuroactive peptide- and classical transmitter-specific systems 2°. One classical transmitter system that has been studied in this regard is the glycinergic system. Evidence, to date, is quite strong for establishing glycine as a major inhibitory neurotransmitter in the vertebrate retina 7'9. In the chicken retina, enkephalin and glycine have been shown to exert control over each other's activity 2'14. Chicken enkephalin-amacrine cells have also been found to exhibit a high-affinity mechanism for glycine uptake t7. A subsequent quantitative analysis demonstrated that glycine-accumulating enkephalin-amacrine cells accounted for approximately 53% of enkephalin-amacrine cells in the chicken retina 18. The present study was undertaken with the intent of providing further insight into the coexisting

* Corresponding author. Fax: (1) (713) 363-8475.

relationship between enkephalin and glycine in amacrine cells of the chicken retina. Its goals were 2-fold: first, a regional analysis of the percentage coexistence of glycine high-affinity uptake in chicken enkephalin-amacrine cells was performed; second, a double-label immunocytochemical analysis examined whether enkephalin-amacrine cells in the chicken retina express endogenous glycine-like immunoreactivity. In order to perform a regional analysis of the percentage coexistence of glycine high-affinity uptake in enkephalin immunoreactive amacrine cells, retinal sections, collected randomly from central and peripheral halves of each retinal quadrant (superior nasal, superior temporal, inferior nasal, inferior temporal), were processed for enkephalin immunocytochemistry and autoradiography of glycine high-affinity uptake 17. The two chickens (Gallus domesticus, 4 - 6 weeks old, Texas A & M University) that were used for the present study were maintained on a 12 h / 1 2 h cycled l i g h t / d a r k regimen. During the light portion of the cycle, an animal was lightly anesthetized with halothane and killed by cervical transection. Following enucleation,

350 an eyecup was isolated under room light, divided into quadrants, each of which was incubated for 10 min in 250 /xl of an oxygenated Ringer's solution containing 50 #Ci [3H]glycine (New England Nuclear Corp., 4.6 /zM, spec. act. 43.5 Ci/mmol). Following the incubation, the retinal quadrants were washed for 2 min in unlabelled oxygenated Ringer's solution. Fixation was

performed in 4% paraformaldehyde/0.l% glutaraldehyde in 0.1 M phosphate buffer for 1 h followed by overnight fixation at 4°C in 4% paraformaldehyde in 0.1 M phosphate buffer. At this point, retinal quadrants were divided into central and peripheral halves. Next, vibratome sections (50 /xm) were collected throughout each of the resulting 8 pieces of retina.

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Fig. 1. Autoradiograms of 1-/xm-thick plastic embedded sections double-labelled for enkephalin immunocytochemistry and autoradiography of high-affinity [3H]glycine uptake. The photomicrographs are focused at the level of the emulsion layer and show sections collected from central (A,B) and peripheral (C) regions of the chicken retina. The solid block arrows in A, B and C point to cells in the inner nuclear layer (INL) that are double-labelled for enkephalin and glycine. The hollow block arrow in B points to a cell that is labelled only for enkephalin. The inserts in A, B and C are higher magnification views of the single- (hollow block arrow) and double-(solid block arrows) labelled enkephalin-amacrine cells. These photomicrographs are focused at the level of the immunoperoxidase staining so to reveal more clearly immunolabelled enkephalin cells. The arrowheads point to a few of numerous cells that are labelled only for glycine. The dotted line in A demarcates the border between the INL and the inner plexiform layer (IPL). Bar for A - C = 20/xm.

351 Unless stated otherwise, all subsequent steps were performed at room temperature in phosphate-buffered saline (PBS, pH 7.3) containing 0.3% Triton X-100. Vibratome sections that were collected from central and peripheral regions of each retinal quadrant, were subjected to primary incubation in the mouse anti[LeuS]enkephalin monoclonal antibody (MAS 083c, Sera Lab, 1 : 100,000) for overnight at 4°C. Immunoperoxidase staining was performed using the avidin-biotin technique (Vector Laboratories) and diaminobenzidine tetrahydrochloride as the chromogen (10 mg/30 ml PBS, 10 /zl 3% H 2 0 2, Polyscience). At this point, appropriately immunostained sections were embedded in plastic and autoradiograms of 1 /xm-thick sections were prepared according to routine procedures 16. Double-label immunofluorescence histochemistry was used to determine whether chicken enkephalinamacrine cells express endogenous glycine-like immunoreactivity. The eyecups from two chickens were isolated and fixed in the manner described above for uptake studies. A complete set of cryo-prepared retinal sections (8 /zm) were collected throughout each eyecup. Sections selected from throughout this series were processed for double-label immunocytochemistry using antibodies raised in different species. Unless stated otherwise, all ensuing steps were performed at room temperature in PBS containing 0.3% Triton X-100. Primary incubation was performed overnight at 4°C in an antibody mixture containing the monoclonal mouse anti-[LeuS]enkephalin antibody (MAS 083c, Sera Labs, 1:300,000) and the polyclonal rabbit anti-glycine antibody 28 (1 : 100). Following the primary incubation, retinal sections were rinsed for 30 min in PBS and incubated in the secondary antibody mixture containing fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (1:300) and Texas red (TXR)-conjugated goat anti-rabbit IgG (1:300) and for 1 h. The sections were rinsed in PBS for 30 min, coverslipped (9:1 glycerol:PBS, 1 mg/ml p-phenylenediamine dihydrochloride, pH 8.5) and viewed with a Zeiss Axioplan light microscope equipped with filter sets (Carl Zeiss Inc.) that differentiate fluorescein (filter no. 487410, wavelength range 450-490 nm) and Texas red (filter no. 487400, wavelength range 530-585 nm) fluorescence. The control experiment testing the specificity of the mouse anti-[LeuS]enkephalin antibody involved its adsorption with an excess amount of synthetic [Leu 5] or [Met55]enkephalin (100/xg/ml of diluted antiserum or 10 -4 M, Peninsula). The rabbit anti-glycine antibody was replaced with normal rabbit serum. In each case, these procedures resulted in an absence of specific labelling. The specificities of the secondary fluorescing

TABLE I

Quantitative analysis of the expression of high-affinity GL Y uptake by ENK imrnunoreactive amacrine cells in the chicken retina ENK, enkephalin; GLY, glycine.

Quadrant

Superior nasal Central Peripheral Superior temporal Central Peripheral Inferior temporal Central Peripheral Inferior nasal Central Peripheral Total

Total Total ENK-cells ENK-cells expressing observed G L Y uptake

% Colocalization

134 112

77 61

57.5 54.5

158 138

90 76

57.0 55.1

112 118

63 57

56.3 48.3

176 118

91 70

51.7 59.3

1,066

585

54.9

antibodies were tested by omitting one or the other primary antibodies in separate experiments. These control experiments revealed that the secondary antibodies were specific for their designated antigens. Double-label paradigms utilized in the present study (Figs. 1 and 2) provided patterns of localization of enkephalinergic 19 and glycinergic7 neurons comparable to those observed when each of these techniques are employed separately. In the present study, enkephalin was localized to a population of amacrine ceils whose cell bodies were situated in the second or third tier of cells from the border of the adjacent inner plexiform layer. Enkephalin processes ramified in sublayers 1, 3, 4 and 5 of the inner plexiform layer. Glycine was localized to a large population of cells in the inner nuclear layer. The vast majority of these ceils were situated in the amacrine cell layers. A smaller number of glycine-labelled cells were located at a more distal level of the inner nuclear layer and were identified as bipolar cells. Glycine process labelling was observed throughout the inner plexiform layer and sporadically in the outer plexiform layer. As revealed in Table I, more than one-thousand enkephalin-amacrine cells were examined in autoradiograms prepared for the regional survey of the percentage colocalization of glycine high-affinity uptake in enkephalin-amacrine cells of the chicken retina. The data were obtained primarily from an examination of retinal regions collected from a single retina. Percentages of coexistence observed in retinal regions collected from a second chicken did not vary appreciably from the first and therefore, the data from both animals were combined for the purpose of preparing Table I. Double-labelled cells were observed through-

352 out central and peripheral regions of each retinal q u a d r a n t (Fig. 1). For the most part, only the m o r e heavily immunostained somas of enkephalin-amacrine cells were observed to co-label for glycine uptake (see below). T h e percentage coexistence of glycine high-affinity uptake in chicken enkephalin-amacrine cells was observed to be fairly consistent throughout each region of the retina that was examined (Table I). Overall, 54.9% of enkephalin-amacrine ceils in the chicken retina were found to express a high-affinity uptake mechanism for glycine. As would be expected on the basis of their relative numbers and locations, n u m e r o u s glycine-accumulating cells were observed not to co-label for enkephalin immunoreactivity (Fig. 1). A total of 484 enkephalin-amacrine cells were observed in retinal sections double-labelled for both enkephalin and glycine immunocytochemistry (Table II). Sections were selected from t h r o u g h o u t series of cryo-prepared sections that were collected from the retinas of two chickens. No appreciable difference was observed between chickens with respect to the percentage coexistence of glycine in enkephalin amacrine ceils. Therefore, data from both animals were combined for the purpose of preparing Table II. Fifty-two point five percent of enkephalin-immunoreactive amacrine ceils were found to co-label for glycine immunoreactivity. Double-labelled ceils were observed t h r o u g h o u t central and peripheral regions of the retina (Fig. 2). For the most part, only enkephalin-amacrine ceils that were heavily immunolabelled co-expressed glycine immunoreactivity. Also, as noted above for glycine-accumulating cells, n u m e r o u s glycine i m m u n o r e a c t i v e ceils were found not to co-express enkephalin immunoreactivity (Fig. 2). In the p r e s e n t study, e n d o g e n o u s enkephalin- and glycine-like immunoreactivities were found to co-occur in amacrine ceils of the chicken retina. To our knowledge, this is the first report of the colocalization of these two markers in neurons of the central nervous system. In the retina, this coexisting relationship appears to be species specific, as evidenced by the observation that enkephalin-amacrine cells in the tiger sala-

m a n d e r retina do not co-label for either glycine imm u n o r e a c t i v i t y or glycine high-affinity u p t a k e 22, Enkephalin is also found to colocalize with the neuropeptides, n e u r o t e n s i n 24 and s o m a t o s t a t i n 5,6 in amacrine cells of the chicken retina and with the classical inhibitory transmitter, G A B A , in amacrine cell populations of a variety of n o n - m a m m a l i a n retinas 2°, Glycine colocalizes with neurotensin in turtle 27 and chicken ~7 retinal amacrine ceils, with substance P in larval tiger salamander retinal amacrine cells 25 and with somatostatin in interplexiform cells of Xenopus laevis 12 and Rana pipiens 13 retinas. Glycine also coexists with G A B A in amacrine cell populations of the goldfish 1, tiger salamander 3°, cat H and h u m a n 3 retinas. A n u m b e r of criteria must be established before a substance can be designated as a transmitter for a neuronal population 29. It must be d e m o n s t r a t e d that the substance is synthesized, stored and released by m e m b e r s of this population and that u p o n release, it exerts postsynaptic actions. Morphological analyses form an important part of these studies. I m m u n o c y t o chemical studies determine w h e t h e r e n d o g e n o u s transmitter-like substances are present in a cell, while autoradiography uptake studies determine w h e t h e r a cell possesses a mechanism for neurotransmitter reuptake. In a previous study, 53% of enkephalin-amacrine cells in the chicken retina were found to exhibit high-affinity uptake of glycine ~8. T h e present study both corroborates and extends this earlier report. In the present study, a similar percentage (54.9%) of enkephalinamacrine cells were observed to exhibit glycine high-affinity uptake. The present study demonstrates further that these cells are fairly evenly distributed t h r o u g h o u t the retina. The present double-label i m m u n o c y t o c h e m ical analysis, which reveals a similar percentage (52.5%) of enkephalin-amacrine cells expressing glycine immunoreactivity, provides evidence that glycine-accumulating enkephalin-amacrine cells synthesize glycine and are therefore, true glycinergic neurons. This type of corroborative evidence is essential for establishing the transmitter specificity of a cell population. For

Fig. 2. Paired fluorescent micrographs (A-B, C-D and E-F) showing the same retinal sections double-labelled for glycine (TXR; A,C,E) and enkephalin (FITC; B,D,F) immunofluorescence histochemistry. The thin arrows in A-B and C-D point to cells in the central retina that are double-labelled for both glycine (A,C) and enkephalin (B,D). The thin arrows in E-F point to a cell in the periphery of the retina that is double-labelled for glycine (E) and enkephalin (F). Note that these cells, which are located in the inner nuclear layer (INL), exhibit relatively intense enkephalin immunofluorescence. For example, the cell in F is somewhat larger than it appears in E because of its brightly glowing appearance. The hollow block arrows in C-D point to a less intensely labelled enkephalin-cell (D) that also labels for glycine (C). The solid block arrow in D points to an enkephalin-cell that is not labelled for glycine. The small asterisk in C indicates the location of this cell and reveals its non-expression of glycine immunoreactivity. Note the numerous cells in A,C and E that are labelled only for glycine immunoreactivity. The large asterisk in A denotes glycine labelling throughout the inner plexiform layer (IPL). The open arrow in A indicates glycine labelling in the outer plexiform layer. The small and large stars in D denote enkephalin-labelled processes ramifying in the distal and proximal IPL, respectively. The dotted line in C demarcates the border between INL and the IPL. Bar for A-F = 20 ~zm.

353

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354 TABLE II Percentage co-expression of GL Y immunoreactivity by ENK immunoreactive amacrine cells of the chicken retina

ENK, enkephalin; GLY, glycine. Cell type

No. of cells observed

% of total ENK-cells

ENK only ENK and GLY Total ENK cells

230 254 484

47.5 52.5

example, serotonin-accumulating amacrine cells in the goldfish retina are divisible into two populations relative to their expression/non-expression of serotoninlike immunoreactivity 8. In the present study, the vast majority of chicken enkephalin-amacrine cells, which express markers of glycine activity, were heavily immunolabelled. As reported previously, it is not uncommon to observe heavily and lightly immunolabelled chicken enkephalinamacrine cells in studies employing either immunoperoxidase or immunofluorescence labelling techniques 25. These ceils, which make up 80% and 20% of chicken enkephalin-amacrine cells, respectively, are distributed evenly throughout the retina. Recently, G A B A was found to be localized only to enkephalin-amacrine cells that are less intensely immunostained 2s. It remains for future studies to determine possible morphological and functional distinctions between ,lightly and heavily immunolabelled enkephalin-amacrine cells in the chicken retina. In conclusion, the present study provides evidence suggesting that a population of chicken enkephalinamacrine cells are glycinergic. At present, the functional implications of multiple signalling through cells that are both enkephalinergic and glycinergic is uncertain. Recent studies reveal that enkephalin and glycine modulate each other's activity in the chicken retina. Enkephalin inhibits the potassium-induced release of glycine from the chicken retina 14, while glycine exerts an inhibitory control over the level of leucineS-en kephalin-like immunoreactivity in chicken amacrine cells during the light, an action apparently responsive to changes in ambient lighting 2. It remains to be determined which mechanism(s) mediate these actions. Self-regulation has been postulated as a mechanism mediating interactions between transmitter substances which are involved in coexisting relationships 2°, However, even if an autoregulatory mechanism is operable in the chicken retina, it is also possible that other mechanisms (mono-, poly- a n d / o r parasynaptic) mediate the inhibitory actions exerted between enkephalinand glycine-cells. Insight into the nature of the synaptic

mechanisms mediating enkephalin's and glycine's inhibitory effects on one another can be obtained from double-label immunoelectron microscopic analyses. Such analyses have recently revealed substantial synaptic interaction between enkephalinergic and G A B A e r gic amacrine cells in the chicken retina 4. Moreover, similar to glycine, G A B A was found to be present in approximately 15% of chicken enkephalin-amacrine cells and to inhibit the potassium-induced release of G A B A from the chicken retina 16'1s'22. It should be noted that the functional nature of enkephalinglycine-amacrine cells may be even more complex, as evidenced by recent studies predicting that these cells contain two additional neuropeptides, somatostatin and neurotensin 5'z1'23. It has been proposed that the coordinated co-release of fast-acting excitatory (acetylcholine) and inhibitory (GABA) transmitters from rabbit starburst amacrine cells brings about directional selectivity in a population of ganglion cells 1°'1s. To date, studies have not explored the interactive relationships between enkephalin-glycine-amacrine cells and ganglion cells in the chicken retina. Therefore, it remains to be determined how enkephalin, glycine and possibly other neuroactive peptides, either coreleased from the same amacrine cells or released from other noncoexisting cells, interact to affect the activity of ganglion cells as well a s other cell types during the processing of visual information in the chicken retina. This study was supported by grants from the National Institutes of Health (EYO5622) and the Retina Research Foundation (Houston). We would like to thank Dr. Robert Wenthold for the generous gift of the glycine antiserum. We would also like to thank Ms. Rene Meyer for expert technical assistance with the word processing. 1 Ball, A.K. and Brandon, C., Localization of 3H-GABA, -muscimol and -glycine in goldfish retinas stained for glutamate decarboxylase, J. Neurosci., 6 (1986) 1621-1627. 2 Boelen, M.K., Wellard, J., Dowton, M., Chubb, I.W. and Morgan, I.G., Glycinergic control of [leuS]enkephalin levels in chicken retina, Brain Res., 557 (1991) 221-226. 3 Davanger, S., Ottersen, O.P. and Storm-Mathisen, J., Glutamate, GABA and in the human retina: an immunocytochemicalinvestigation, J. Comp. Neurol., 311 (1991) 483-494. 4 Glazebrook, P.A., Fry, K.R. and Watt, C.B., Interaction between enkephalin and GABA in the chicken retina: a double-label immunoelectron microscopic study, Soc. Neurosci. Abstr., 19 (1993) in press. 5 Hamano, K., Katayama-Kumoi, Y., Kiyama, H., Ishimoto, I., Manabe, R. and Tohyama, H., Coexistence of enkephalin and somatostatin in the chicken retina, Brain Res., 489 (1989) 254-260. 6 Li, H.B., Watt, C.B. and Lam, D.M.K., Double-label analyses of somatostatin's coexistence with enkephalin and gamma-aminobutyric acid in amacrine cells of the chicken retina, Brain Res., 525 (1990) 304-309. 7 Marc, R.E., The role of glycine in retinal circuitry. In Morgan, W.W., Retinal Transmitters and Modulators: Models"for the Brain, CRC Press, Boca Raton, 1985, pp. 119-158. 8 Marc, R.E., Liu, W.L., Scholz, K. and Muller, J.F., Serotoninergic and serotonin-accumulating neurons in the goldfish retina, Z Neurosci., 8 (1988) 3427-3450.

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