Identification of Amacrine Neurons with a Glycinergic and Gabaergic Phenotype in the Mouse Retina

Main Article Content

Jorge Alberto Pérez-León Annie Espinal-Centeno Zelene Mendoza-Gonzalez Alejandra C. Camacho Ashley Bryan Lopez Rosa A. Perez Manuel Miranda

Abstract

The amacrine neurons in the mammalian retina comprise a large variety of cell types with distinct properties and functions that serve to integrate and modulate signals presented to output neurons. The majority of them use either glycine or GABA as inhibitory neurotransmitters and express the glycine transporter 1 (GlyT1) or glutamic acid decarboxylase (GAD67) and GABA transporters (GAT1 and GAT3), as a glycinergic or GABAergic marker respectively. We report here a novel subpopulation of amacrine neurons expressing both, GABAergic and glycinergic markers, in retinas from wild-type C57BL/6J mice and two transgenic lines. In retinal sections from the transgenic line expressing eGFP under the control of the glycine transporter 2, eGFP expression was exclusively found in cell bodies and dendrites of inhibitory amacrine neurons, identified for their immunoreactivity to syntaxin 1A. All of the glycinergic and a large portion of the GABAergic amacrine neurons contained eGFP; of these, 8-10% of GlyT1 positive neurons were also labeled either with GAD67, GAT1 or GAT3. These findings were confirmed in retinas from a wild-type and a mouse line expressing eGFP under the GAD67 promoter and two different anti-GlyT1 antibodies, showing the presence of a subpopulation with a dual phenotype. Moreover, eGFP-positive dendrites on both mouse lines were found juxtaposed to GlyR subunits and the scaffold protein gephyrin in several areas of the inner plexiform layer, demonstrating the glycinergic character of these neurons. This dual phenotype was also demonstrated in primary retina cultures, in which isolated neurons were positive for GlyT1 and GAD67 or GAT1/3. Altogether, these data provide compelling evidence of a subpopulation of dual inhibitory, glycinergic/GABAergic amacrine neurons. The co-release of both neurotransmitters may serve to strengthen the inhibition on ganglion cells under synaptic hyperexcitability.

Keywords: GABA, amacrine neuron, glycinergic, glycine receptor, Glycine Transporter 1

Article Details

How to Cite
PÉREZ-LEÓN, Jorge Alberto et al. Identification of Amacrine Neurons with a Glycinergic and Gabaergic Phenotype in the Mouse Retina. Medical Research Archives, [S.l.], v. 10, n. 1, jan. 2022. ISSN 2375-1924. Available at: <https://esmed.org/MRA/mra/article/view/2624>. Date accessed: 07 dec. 2024. doi: https://doi.org/10.18103/mra.v10i1.2624.
Section
Research Articles

References

1. Betz, H., and Laube, B. (2006) Glycine receptors: recent insights into their structural organization and functional diversity. J Neurochem 97, 1600-1610
2. Dutertre, S., Becker, C. M., and Betz, H. (2012) Inhibitory glycine receptors: an update. J Biol Chem 287, 40216-40223
3. Zhang, C., and McCall, M. A. (2012) Receptor targets of amacrine cells. Vis Neurosci 29, 11-29
4. Johnson, J. W., and Ascher, P. (1987) Glycine potentiates the NMDA response in cultured mouse brain neurons. Nature 325, 529-531
5. Pourcho, R. G., and Owczarzak, M. T. (1991) Connectivity of glycine immunoreactive amacrine cells in the cat retina. J Comp Neurol 307, 549-561
6. Ivanova, E., Muller, U., and Wassle, H. (2006) Characterization of the glycinergic input to bipolar cells of the mouse retina. Eur J Neurosci 23, 350-364
7. Crooks, J., and Kolb, H. (1992) Localization of GABA, glycine, glutamate and tyrosine hydroxylase in the human retina. J Comp Neurol 315, 287-302
8. Davanger, S., Ottersen, O. P., and Storm-Mathisen, J. (1991) Glutamate, GABA, and glycine in the human retina: an immunocytochemical investigation. J Comp Neurol 311, 483-494
9. Menger, N., Pow, D. V., and Wassle, H. (1998) Glycinergic amacrine cells of the rat retina. The Journal of comparative neurology 401, 34-46
10. Sukhdeo, K., Koch, C. E., Miller, T. E., Zhou, H., Rivera, M., Yan, K., Cepko, C. L., Lathia, J. D., and Rich, J. N. (2014) The Lgr5 transgene is expressed specifically in glycinergic amacrine cells in the mouse retina. Experimental eye research 119, 106-110
11. Haverkamp, S., Muller, U., Zeilhofer, H. U., Harvey, R. J., and Wassle, H. (2004) Diversity of glycine receptors in the mouse retina: localization of the alpha2 subunit. J Comp Neurol 477, 399-411
12. Pourcho, R. G., and Owczarzak, M. T. (1991) Glycine receptor immunoreactivity is localized at amacrine synapses in cat retina. Visual neuroscience 7, 611-618
13. Wassle, H., Koulen, P., Brandstatter, J. H., Fletcher, E. L., and Becker, C. M. (1998) Glycine and GABA receptors in the mammalian retina. Vision Res 38, 1411-1430
14. Zafra, F., and Gimenez, C. (2008) Glycine transporters and synaptic function. IUBMB Life 60, 810-817
15. Broer, S., and Gether, U. (2012) The solute carrier 6 family of transporters. Br J Pharmacol 167, 256-278
16. Zafra, F., Aragon, C., Olivares, L., Danbolt, N. C., Gimenez, C., and Storm-Mathisen, J. (1995) Glycine transporters are differentially expressed among CNS cells. J Neurosci 15, 3952-3969
17. Eulenburg, V., Retiounskaia, M., Papadopoulos, T., Gomeza, J., and Betz, H. (2010) Glial glycine transporter 1 function is essential for early postnatal survival but dispensable in adult mice. Glia 58, 1066-1073
18. Raiteri, L., and Raiteri, M. (2010) Functional 'glial' GLYT1 glycine transporters expressed in neurons. Journal of neurochemistry 114, 647-653
19. Mohler, H., Boison, D., Singer, P., Feldon, J., Pauly-Evers, M., and Yee, B. K. (2011) Glycine transporter 1 as a potential therapeutic target for schizophrenia-related symptoms: evidence from genetically modified mouse models and pharmacological inhibition. Biochem Pharmacol 81, 1065-1077
20. Musante, V., Summa, M., Cunha, R. A., Raiteri, M., and Pittaluga, A. (2011) Pre-synaptic glycine GlyT1 transporter--NMDA receptor interaction: relevance to NMDA autoreceptor activation in the presence of Mg2+ ions. J Neurochem 117, 516-527
21. Heinze, L., Harvey, R. J., Haverkamp, S., and Wassle, H. (2007) Diversity of glycine receptors in the mouse retina: localization of the alpha4 subunit. J Comp Neurol 500, 693-707
22. Nobles, R. D., Zhang, C., Muller, U., Betz, H., and McCall, M. A. (2012) Selective glycine receptor alpha2 subunit control of crossover inhibition between the on and off retinal pathways. J Neurosci 32, 3321-3332
23. Salceda, R. (2006) Pharmacological properties of glycine uptake in the developing rat retina. Neurochem Int 49, 342-346
24. Cherry, T. J., Trimarchi, J. M., Stadler, M. B., and Cepko, C. L. (2009) Development and diversification of retinal amacrine interneurons at single cell resolution. Proc Natl Acad Sci U S A 106, 9495-9500
25. Jonas, P., Bischofberger, J., and Sandkuhler, J. (1998) Corelease of two fast neurotransmitters at a central synapse. Science 281, 419-424
26. Aubrey, K. R., and Supplisson, S. (2018) Heterogeneous Signaling at GABA and Glycine Co-releasing Terminals. Frontiers in synaptic neuroscience 10, 40
27. Noh, J., Seal, R. P., Garver, J. A., Edwards, R. H., and Kandler, K. (2010) Glutamate co-release at GABA/glycinergic synapses is crucial for the refinement of an inhibitory map. Nat Neurosci 13, 232-238
28. Haverkamp, S., and Wassle, H. (2004) Characterization of an amacrine cell type of the mammalian retina immunoreactive for vesicular glutamate transporter 3. J Comp Neurol 468, 251-263
29. Lee, S., Chen, L., Chen, M., Ye, M., Seal, R. P., and Zhou, Z. J. (2014) An unconventional glutamatergic circuit in the retina formed by vGluT3 amacrine cells. Neuron 84, 708-715
30. Lee, S., Zhang, Y., Chen, M., and Zhou, Z. J. (2016) Segregated Glycine-Glutamate Co-transmission from vGluT3 Amacrine Cells to Contrast-Suppressed and Contrast-Enhanced Retinal Circuits. Neuron 90, 27-34
31. Barrera, S. P., Castrejon-Tellez, V., Trinidad, M., Robles-Escajeda, E., Vargas-Medrano, J., Varela-Ramirez, A., and Miranda, M. (2015) PKC-Dependent GlyT1 Ubiquitination Occurs Independent of Phosphorylation: Inespecificity in Lysine Selection for Ubiquitination. PLoS One 10, e0138897
32. Vargas-Medrano, J., Castrejon-Tellez, V., Plenge, F., Ramirez, I., and Miranda, M. (2011) PKCbeta-dependent phosphorylation of the glycine transporter 1. Neurochem Int 59, 1123-1132
33. Gabernet, L., Pauly-Evers, M., Schwerdel, C., Lentz, M., Bluethmann, H., Vogt, K., Alberati, D., Mohler, H., and Boison, D. (2005) Enhancement of the NMDA receptor function by reduction of glycine transporter-1 expression. Neurosci Lett 373, 79-84
34. Yee, B. K., Balic, E., Singer, P., Schwerdel, C., Grampp, T., Gabernet, L., Knuesel, I., Benke, D., Feldon, J., Mohler, H., and Boison, D. (2006) Disruption of glycine transporter 1 restricted to forebrain neurons is associated with a procognitive and antipsychotic phenotypic profile. J Neurosci 26, 3169-3181
35. Fratev, F., Miranda-Arango, M., Lopez, A. B., Padilla, E., and Sirimulla, S. (2019) Discovery of GlyT2 Inhibitors Using Structure-Based Pharmacophore Screening and Selectivity Studies by FEP+ Calculations. ACS Medicinal Chemistry Letters 10, 904-910
36. Kurolap, A., Armbruster, A., Hershkovitz, T., Hauf, K., Mory, A., Paperna, T., Hannappel, E., Tal, G., Nijem, Y., Sella, E., Mahajnah, M., Ilivitzki, A., Hershkovitz, D., Ekhilevitch, N., Mandel, H., Eulenburg, V., and Baris, H. N. (2016) Loss of Glycine Transporter 1 Causes a Subtype of Glycine Encephalopathy with Arthrogryposis and Mildly Elevated Cerebrospinal Fluid Glycine. American journal of human genetics 99, 1172-1180
37. Wojcik, S. M., Katsurabayashi, S., Guillemin, I., Friauf, E., Rosenmund, C., Brose, N., and Rhee, J. S. (2006) A shared vesicular carrier allows synaptic corelease of GABA and glycine. Neuron 50, 575-587
38. Deniz, S., Wersinger, E., Schwab, Y., Mura, C., Erdelyi, F., Szabo, G., Rendon, A., Sahel, J. A., Picaud, S., and Roux, M. J. (2011) Mammalian retinal horizontal cells are unconventional GABAergic neurons. Journal of neurochemistry 116, 350-362
39. Zhao, S., Ting, J. T., Atallah, H. E., Qiu, L., Tan, J., Gloss, B., Augustine, G. J., Deisseroth, K., Luo, M., Graybiel, A. M., and Feng, G. (2011) Cell type-specific channelrhodopsin-2 transgenic mice for optogenetic dissection of neural circuitry function. Nat Methods 8, 745-752
40. Wang, X., and Sun, Q. Q. (2012) Characterization of axo-axonic synapses in the piriform cortex of Mus musculus. The Journal of comparative neurology 520, 832-847
41. Kay, J. N., Voinescu, P. E., Chu, M. W., and Sanes, J. R. (2011) Neurod6 expression defines new retinal amacrine cell subtypes and regulates their fate. Nature neuroscience 14, 965-972
42. Xu, G. Z., Cui, L. J., Liu, A. L., Zhou, W., Gong, X., Zhong, Y. M., Yang, X. L., and Weng, S. J. (2017) Transgene is specifically and functionally expressed in retinal inhibitory interneurons in the VGAT-ChR2-EYFP mouse. Neuroscience 363, 107-119
43. Jellali, A., Stussi-Garaud, C., Gasnier, B., Rendon, A., Sahel, J. A., Dreyfus, H., and Picaud, S. (2002) Cellular localization of the vesicular inhibitory amino acid transporter in the mouse and human retina. The Journal of comparative neurology 449, 76-87
44. Dedek, K., Breuninger, T., de Sevilla Muller, L. P., Maxeiner, S., Schultz, K., Janssen-Bienhold, U., Willecke, K., Euler, T., and Weiler, R. (2009) A novel type of interplexiform amacrine cell in the mouse retina. The European journal of neuroscience 30, 217-228
45. Hu, M., Bruun, A., and Ehinger, B. (1999) Expression of GABA transporter subtypes (GAT1, GAT3) in the developing rabbit retina. Acta ophthalmologica Scandinavica 77, 261-265
46. Guo, C., Stella, S. L., Jr., Hirano, A. A., and Brecha, N. C. (2009) Plasmalemmal and vesicular gamma-aminobutyric acid transporter expression in the developing mouse retina. The Journal of comparative neurology 512, 6-26
47. Wu, Z., Guo, Z., Gearing, M., and Chen, G. (2014) Tonic inhibition in dentate gyrus impairs long-term potentiation and memory in an Alzheimer's [corrected] disease model. Nature communications 5, 4159
48. Lee, S. C., Meyer, A., Schubert, T., Huser, L., Dedek, K., and Haverkamp, S. (2015) Morphology and connectivity of the small bistratified A8 amacrine cell in the mouse retina. J Comp Neurol 523, 1529-1547
49. Haverkamp, S., and Wassle, H. (2000) Immunocytochemical analysis of the mouse retina. J Comp Neurol 424, 1-23
50. Pow, D. V., and Hendrickson, A. E. (1999) Distribution of the glycine transporter glyt-1 in mammalian and nonmammalian retinae. Visual neuroscience 16, 231-239
51. Zeilhofer, H. U., Studler, B., Arabadzisz, D., Schweizer, C., Ahmadi, S., Layh, B., Bosl, M. R., and Fritschy, J. M. (2005) Glycinergic neurons expressing enhanced green fluorescent protein in bacterial artificial chromosome transgenic mice. J Comp Neurol 482, 123-141
52. Giber, K., Diana, M. A., Plattner, V. M., Dugue, G. P., Bokor, H., Rousseau, C. V., Magloczky, Z., Havas, L., Hangya, B., Wildner, H., Zeilhofer, H. U., Dieudonne, S., and Acsady, L. (2015) A subcortical inhibitory signal for behavioral arrest in the thalamus. Nat Neurosci 18, 562-568
53. Wassle, H., Heinze, L., Ivanova, E., Majumdar, S., Weiss, J., Harvey, R. J., and Haverkamp, S. (2009) Glycinergic transmission in the Mammalian retina. Front Mol Neurosci 2, 6
54. Dumitrescu, O. N., Protti, D. A., Majumdar, S., Zeilhofer, H. U., and Wassle, H. (2006) Ionotropic glutamate receptors of amacrine cells of the mouse retina. Visual neuroscience 23, 79-90
55. Vaney, D. I., Nelson, J. C., and Pow, D. V. (1998) Neurotransmitter coupling through gap junctions in the retina. The Journal of neuroscience : the official journal of the Society for Neuroscience 18, 10594-10602
56. Sorkina, T., Doolen, S., Galperin, E., Zahniser, N. R., and Sorkin, A. (2003) Oligomerization of Dopamine Transporters Visualized in Living Cells by Fluorescence Resonance Energy Transfer Microscopy. J. Biol. Chem. 278, 28274-28283
57. Torres, G. E., Carneiro, A., Seamans, K., Fiorentini, C., Sweeney, A., Yao, W.-D., and Caron, M. G. (2003) Oligomerization and Trafficking of the Human Dopamine Transporter. Mutational analysis identifies critical domains important for the functional expression of the transporter. J. Biol. Chem. 278, 2731-2739
58. Schmid, J. A., Scholze, P., Kudlacek, O., Freissmuth, M., Singer, E. A., and Sitte, H. H. (2001) Oligomerization of the Human Serotonin Transporter and of the Rat GABA Transporter 1 Visualized by Fluorescence Resonance Energy Transfer Microscopy in Living Cells. J. Biol. Chem. 276, 3805-3810
59. Bartholomaus, I., Milan-Lobo, L., Nicke, A., Dutertre, S., Hastrup, H., Jha, A., Gether, U., Sitte, H. H., Betz, H., and Eulenburg, V. (2008) Glycine transporter dimers: evidence for occurrence in the plasma membrane. The Journal of biological chemistry 283, 10978-10991
60. Eulenburg, V., Knop, G., Sedmak, T., Schuster, S., Hauf, K., Schneider, J., Feigenspan, A., Joachimsthaler, A., and Brandstatter, J. H. (2018) GlyT1 determines the glycinergic phenotype of amacrine cells in the mouse retina. Brain structure & function 223, 3251-3266
61. Mosinger, J. L., Yazulla, S., and Studholme, K. M. (1986) GABA-like immunoreactivity in the vertebrate retina: a species comparison. Experimental eye research 42, 631-644
62. Grunert, U., and Wassle, H. (1990) GABA-like immunoreactivity in the macaque monkey retina: a light and electron microscopic study. The Journal of comparative neurology 297, 509-524
63. Marquardt, T., Ashery-Padan, R., Andrejewski, N., Scardigli, R., Guillemot, F., and Gruss, P. (2001) Pax6 is required for the multipotent state of retinal progenitor cells. Cell 105, 43-55
64. Johnson, J., Chen, T. K., Rickman, D. W., Evans, C., and Brecha, N. C. (1996) Multiple gamma-Aminobutyric acid plasma membrane transporters (GAT-1, GAT-2, GAT-3) in the rat retina. J Comp Neurol 375, 212-224
65. Brecha, N. C., and Weigmann, C. (1994) Expression of GAT-1, a high-affinity gamma-aminobutyric acid plasma membrane transporter in the rat retina. J Comp Neurol 345, 602-611
66. Weltzien, F., Percival, K. A., Martin, P. R., and Grunert, U. (2015) Analysis of bipolar and amacrine populations in marmoset retina. The Journal of comparative neurology 523, 313-334
67. Kalloniatis, M., Marc, R. E., and Murry, R. F. (1996) Amino acid signatures in the primate retina. The Journal of neuroscience : the official journal of the Society for Neuroscience 16, 6807-6829
68. Koontz, M. A., Hendrickson, L. E., Brace, S. T., and Hendrickson, A. E. (1993) Immunocytochemical localization of GABA and glycine in amacrine and displaced amacrine cells of macaque monkey retina. Vision research 33, 2617-2628
69. Rousseau, F., Aubrey, K. R., and Supplisson, S. (2008) The glycine transporter GlyT2 controls the dynamics of synaptic vesicle refilling in inhibitory spinal cord neurons. J Neurosci 28, 9755-9768
70. Aubrey, K. R., Rossi, F. M., Ruivo, R., Alboni, S., Bellenchi, G. C., Le Goff, A., Gasnier, B., and Supplisson, S. (2007) The transporters GlyT2 and VIAAT cooperate to determine the vesicular glycinergic phenotype. J Neurosci 27, 6273-6281
71. Roux, M. J., and Supplisson, S. (2000) Neuronal and glial glycine transporters have different stoichiometries. Neuron 25, 373-383
72. Bagnall, M. W., Zingg, B., Sakatos, A., Moghadam, S. H., Zeilhofer, H. U., and du Lac, S. (2009) Glycinergic projection neurons of the cerebellum. J Neurosci 29, 10104-10110
73. Korbolina, E. E., Ershov, N. I., Bryzgalov, L. O., and Kolosova, N. G. (2014) Application of quantitative trait locus mapping and transcriptomics to studies of the senescence-accelerated phenotype in rats. BMC Genomics 15 Suppl 12, S3
74. Provencio, I., Rollag, M. D., and Castrucci, A. M. (2002) Photoreceptive net in the mammalian retina. This mesh of cells may explain how some blind mice can still tell day from night. Nature 415, 493
75. Ecker, J. L., Dumitrescu, O. N., Wong, K. Y., Alam, N. M., Chen, S. K., LeGates, T., Renna, J. M., Prusky, G. T., Berson, D. M., and Hattar, S. (2010) Melanopsin-expressing retinal ganglion-cell photoreceptors: cellular diversity and role in pattern vision. Neuron 67, 49-60
76. Schmidt, T. M., Chen, S. K., and Hattar, S. (2011) Intrinsically photosensitive retinal ganglion cells: many subtypes, diverse functions. Trends Neurosci 34, 572-580
77. Schmidt, T. M., and Kofuji, P. (2011) Structure and function of bistratified intrinsically photosensitive retinal ganglion cells in the mouse. J Comp Neurol 519, 1492-1504
78. Pena-Rangel, M. T., Riesgo-Escovar, J. R., Sanchez-Chavez, G., and Salceda, R. (2008) Glycine transporters (glycine transporter 1 and glycine transporter 2) are expressed in retina. Neuroreport 19, 1295-1299
79. Gabriel, R., Erdelyi, F., Szabo, G., Lawrence, J. J., and Wilhelm, M. (2016) Ectopic transgene expression in the retina of four transgenic mouse lines. Brain structure & function 221, 3729-3741
80. Apostolides, P. F., and Trussell, L. O. (2013) Rapid, activity-independent turnover of vesicular transmitter content at a mixed glycine/GABA synapse. J Neurosci 33, 4768-4781
81. Dufour, A., Tell, F., Kessler, J. P., and Baude, A. (2010) Mixed GABA-glycine synapses delineate a specific topography in the nucleus tractus solitarii of adult rat. J Physiol 588, 1097-1115
82. Wang, Y., Kakizaki, T., Sakagami, H., Saito, K., Ebihara, S., Kato, M., Hirabayashi, M., Saito, Y., Furuya, N., and Yanagawa, Y. (2009) Fluorescent labeling of both GABAergic and glycinergic neurons in vesicular GABA transporter (VGAT)-venus transgenic mouse. Neuroscience 164, 1031-1043
83. Gillespie, D. C., Kim, G., and Kandler, K. (2005) Inhibitory synapses in the developing auditory system are glutamatergic. Nat Neurosci 8, 332-338
84. Hirasawa, H., Puopolo, M., and Raviola, E. (2009) Extrasynaptic release of GABA by retinal dopaminergic neurons. Journal of neurophysiology 102, 146-158
85. Hirasawa, H., Betensky, R. A., and Raviola, E. (2012) Corelease of dopamine and GABA by a retinal dopaminergic neuron. The Journal of neuroscience : the official journal of the Society for Neuroscience 32, 13281-13291
86. Protti, D. A., Gerschenfeld, H. M., and Llano, I. (1997) GABAergic and glycinergic IPSCs in ganglion cells of rat retinal slices. J Neurosci 17, 6075-6085
87. Tian, N., Hwang, T. N., and Copenhagen, D. R. (1998) Analysis of excitatory and inhibitory spontaneous synaptic activity in mouse retinal ganglion cells. J Neurophysiol 80, 1327-1340
88. Frech, M. J., Perez-Leon, J., Wassle, H., and Backus, K. H. (2001) Characterization of the spontaneous synaptic activity of amacrine cells in the mouse retina. J Neurophysiol 86, 1632-1643