- Email: [email protected]
Differential localization of GABAA receptor α and β subunits in the hamster retina and relationship with glutamic acid decarboxylase immunoreactivity
Neuroscience Letters 248 (1998) 29–32
Differential localization of GABAA receptor a and b subunits in the hamster retina and relationship with glutamic acid decarboxylase immunoreactivity Y.L. Lee a, E.Y.P. Cho b, K.K.L. Yung a ,* a
Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China b Department of Anatomy, Chinese University of Hong Kong, Shatin, Hong Kong, China Received 25 March 1998; received in revised form 6 April 1998; accepted 6 April 1998
Abstract In order to determine the cellular localization of GABAAa and b subunits in the hamster retina, single and double immunocytochemistry was performed in perfuse-fixed hamster retina using commercially-available antibodies against the two receptor subunits and glutamic acid decarboxylase. Strong GABAAb immunoreactivity was found in two strata of the inner plexiform layer and in perikarya of amacrine cells and bipolar cells in the inner nuclear layer. In contrast, no GABAAa immunoreactivity was detected. All but a few of the GABAAb-immunoreactive amacrine cells were found not to display glutamic acid decarboxylase immunoreactivity. The present results indicate that there is a differential localization of GABAAa and b subunits in different neuronal subpopulations in the hamster retina. 1998 Elsevier Science Ireland Ltd.
Keywords: GABAAa subunit; GABAAb subunit; Immunocytochemistry; Avidin-biotin-peroxidase reaction; Immunofluorescence; Light microscopy
Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system and is known to be one of the neurotransmitters found in the vertebrate retina [5,7,10,16]. The functions of GABA are mediated by GABA receptors that are divided into three major types. They are namely the GABAA receptors [9], GABAB receptors [6,9] and GABAC receptors [1,8,9]. The GABAA receptor is an ionotropic receptor that has a pentameric structure [9]. A functional ion-channel of GABAA receptor is known to be composed of five subunits that include one alpha, three beta and one gamma subunits (1a,3b,1g]. Six types of alpha subunit (al–a6), four types of beta subunit (b1–b4) and four types of gamma subunit (g1–g4) have been cloned [9,11]. The subunit compositions determine pharmacological and functional properties of the GABAA receptor [9,12]. Previous reports have indicated that injection of the
* Corresponding author. Tel.: +86 852 23397050; fax: +86 852 23397050; e-mail: [email protected]
benzodiazepine triazolum, an agonist of GABAA receptor, can induce permanent phase shifts in the circadian rhythm of locomotor activity in hamsters [15]. Although previous immunocytochemical and in situ hybridization studies have revealed that immunoreactivity for GABAA receptor is localized in different cellular elements in retinae of different mammalian species (GABAA receptor a6 subunit in rat [4]; GABAA receptor a and 13 subunits in rabbit [2,3]), less information has been reported so far about the localization of GABAAa and 13 subunit in hamster retina. It is important to determine the localization of GABAA receptors in hamster retina, as hamsters are the subjects for investigation for most of the behavioral and gene expression experiments in concern of circadian rhythms [14]. In order to determine and compare the cellular localization of the a and b subunits of GABAA in the hamster retina, immunocytochemistry was performed. In addition, another major objective of the present study was to investigate the receptor and neurotransmitter relationship in the hamster retina. In order to identify the GABAergic neurons in the hamster retina, immunoreactivity for glutamic acid decar-
0304-3940/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00326- 7
30
Y.L. Lee et al. / Neuroscience Letters 248 (1998) 29–32
boxylase (GAD), a synthetic enzyme for GABA, was detected as a neurochemical marker for GABA. In the present study, eight Syrian golden hamsters (200– 250 g, female, the Chinese University of Hong Kong) were used. The handling of hamsters and all procedures performed on them were approved in accordance to the Animals (Control of Experiments) Ordinance, Hong Kong, China. The animals were deeply anaesthetized by choral hydrate (10% in normal saline, 0.9% NaCl; intraperitoneal) and transcardially perfused by 50–100 ml of saline then followed by 200 ml of fixative (3–4% paraformaldehyde and 0.01% glutaraldehyde in 0.1 M phosphate buffer (PB), pH 7.4) as previously described [17,18]. The retinae were removed, post-fixed (3% paraformaldehyde, 1 h) and cut by a cryostat (10–20 mm). The sections were either processed to single immunocytochemistry [17,18] or immunocytochemistry combined with immunofluorescence as previously described [17]. Primary antibodies against GABAA receptor a and b subunits (mouse monoclonal;
used at 1:2000 dilution in PBS supplemented with 0.1% Triton X–100 and 2% NGS [PBS–Triton]; Chemicon International, Temcula, CA) and antibody against GAD (rat monoclonal; used at 1:50 dilution in PBS-Triton, Chemicon) was used. In addition, secondary antibodies (biotinylated goat–anti-rat or goat–anti-mouse IgG, 1:100–200 dilutions in PBS–Triton) were used. Immunoreactivity for receptor and GAD was revealed by the avidin–biotin peroxidase (ABC) method (1:100; ABC kit, Vector Labs.). In the case of double immunolabeling, after revealing immunoreactivity for the receptors, GAD immunoreactivity was then revealed in the same sections by incubating in fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (1:100–200; PBS–Triton). Immunoreactivity for GABAA receptor b subunit was found in two strata of the inner plexiform layer (IPL; Fig. 1A). At a higher magnification, the perikarya of neurons were densely labeled and GABAAb-immunoreactive cell processes were seen running along the vertical axis of the inner nuclear layer (INL; Fig. 1B–C). Most of these cells
Fig. 1. Light micrographs of the hamster retina immunostained to reveal immunoreactivity for GABAAb (A–C) and a (D) subunits. (A) At low magnification, immunoreactivity for GABAAb subunit is found in two strata of the inner plexiform layer (IPL; indicated by white arrowheads). In addition. GABAAb-immunoreactive perikarya are also observed in the inner nuclear layer (INL). (B,C) At higher magnification, many of the GABAAb-immunoreactive cells in the INL are found to have a single process extending from the cell bodies (arrows). Occasionally, some immunoreactive bipolar cells are also seen (open arrow). (D) No immunoreactivity for the GABAAa subunit is observed in all layers of the hamster retina. ONL, Outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bars (A,D) 500 mm; (B,C) 100 mm.
Y.L. Lee et al. / Neuroscience Letters 248 (1998) 29–32
were seen to be ‘axonless’ or possessed only one process (Fig. 1B–C). In addition, GABAAb immunoreactivity was found in the perikarya of bipolar cells (Fig. 1C). In contrast, no immunoreactivity for the GABAAa subunit was detected in all the layers of the hamster retina (Fig. 1D). Immunoreactivity for GAD was observed in the IPL and amacrine cells of INL as revealed by immunofluorescence (Fig. 2B,D). In the IPL, GAD immunoreactivity was detected in three strata (data not shown) and also in perikarya of amacrine cells in the INL (Fig. 2B,D). In those sections, double-labeled to reveal GABAAb and GAD immunoreactivity, in most of the cases GABAAb-immunoreactive perikarya did not display GAD immunoreactivity (Fig. 2A–B). However, occasionally a few GABAAb-immunoreactive perikarya (about 7.5%, n = 282) were found to be also GAD-immunoreactive (Fig. 2C–D). The results of the present study indicate that there is a differential localization of GABAA receptor a and b subunits in the hamster retina. No GABAAa immunoreactivity was detected. This result indicates the a subunit is not expressed by neuronal elements in the hamster retina. In contrast to the present results, immunoreactivity for a subunits has been found in retinae of other mammalian species [2–4]. This discrepancy is likely due to the species variation of the animal studied as the a subunit may not be expressed in the hamster retina. However, the present results do not prelude the possibility that the primary antibody does not recognize the a subunit expressed in hamster retina. The present results indicate that some of the distinct retinal neuronal populations in hamster express the GABAAb subunit. Immunoreactivity for GABAAb subunit is mainly observed in perikarya in the INL and in two strata of neuropilar elements in the IPL. Morphology of most of the labeled cells resembles that of the amacrine cells [13]. The amacrine cells are therefore the major types of neurons that display immunoreactivity for GABAAb subunit. In addition to amacrine cells, occasionally some bipolar cells in the INL have been found to contain the GABAA receptor immunoreactivity. It is however, extremely difficult to identify the subpopulations of bipolar cells that -display GABAA b immunoreactivity in the INL by light microscopy. As described by previous study [7], immunoreactivity for GAD was observed in the neuropil of the IPL and in amacrine cells of the INL. Since GAD is the synthetic enzyme of GABA (see refs. in [7]), GAD immunoreactivity can indicate the presence of GABA. In the present results, most of the GABAAb-immunoreactive cells do not display GAD immunoreactivity, which implies that they are not GABAergic neurons. In contrast, there is a small population of GAD immunoreactive amacrine cells found to display GABAAb immunoreactivity. This indicates a small subpopulation of GABAergic amacrine cells may also express subunits of the GABAA receptor. The functional role of the b subunit remains to be established (see below). Although the present report do not provide functional
31
Fig. 2. Light micrographs of the hamster retina double immunostained to reveal immunoreactivity for GABAAb subunit and glutamic acid decarboxylase (GAD). (A) A GABAAb-immunoreactive amacrine cell is shown in the inner plexiform layer (arrowhead). (B) The neuron shown in (A) is shown (white arrowhead). It is GAD-negative. Another amacrine cell nearby is found to be GAD-immunoreactive (white arrow). (C) In other section, a GABAAb-immunoreactive amacrine cell is shown (arrowhead). (D) The neuron shown in (C) is shown (white arrowhead). It is found to express GAD immunoreactivity as indicates by immunofluorescence. Another nearby cell is shown to be only GAD-immunoreactive (white arrow). ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer. Scale bar (A-D) 100 mm.
32
Y.L. Lee et al. / Neuroscience Letters 248 (1998) 29–32
information, it is unlikely that the GABAA channels present in the hamster retina are functional, as no GABAAa subunit immunoreactivity is detected. A previous report indicates that the phase shifts in the circadian rhythm in the hamster induced by benzodiazepine may not be mediated by the eye [15], there may be no functional GABAA channels in the hamster retina. The present report provides anatomical evidence for the above functional observation. This may indicate that the distribution of functional GABAA channels in hamster is very different from those in other mammalian species, in which functional GABAA channels are found [10,12]. Further investigation is necessary in order to establish the role of GABAAb subunit expressed by neurons in hamster retina. In summary, there is a differential localization of GABAAa and b subunits in different neuronal elements of the hamster retina and the distribution of GABAA subunit immunoreactivity in hamster retina is different from other mammalian species and may be related to the GABA neurotransmission in the hamster retina.
[6]
[7]
[8] [9]
[10]
[11]
[12]
The authors would like to thank Miss Lousie Ng for technical assistance in the present project. The present work was supported by Faculty Research Grants FRG/9596/I-17, FRG/95-96/II-73 and FRG/96-97/II-19, Hong Kong Baptist University (to KKLY). [1] Enz, R., Brandsta¨tter, J.H., Wa¨ssle, H. and Bormann, J., Immunocytochemical localization of the GABAC receptor r subunits in the mammalian retina, J. Neurosci., 16 (1995) 4479–4490. [2] Greferath, U., Grunert, U., Muller, F. and Wassle, H., Localization of GABAA receptors in the rabbit retina, Cell Tissue Res., 276 (1994) 295–307. [3] Greferath, U., Grunert, U., Fritschy, J.M., Stephenson, A., Mohler, H. and Wassle, H., GABAA receptor subunits have differential distributions in the rat retina: in situ hybridization and immunohistochemistry, J. Comp. Neurol., 353 (1995) 55357l. [4] Gutie´rrez, A., Khan, Z.U. and De Blas, A.L., Immunocytochemical localization of the a6 subunit of the g-Aminobutyric acidA receptor in the rat nervous system, J. Comp. Neurol., 365 (1996) 504–510. [5] Hamassaki-Britto, D.E., Brzozowska-Prechtl, A., Karten, H.J., Lindstrom, J.M. and Keyser, K.T., GABA-like immunoreactive
[13] [14]
[15]
[16]
[17] [18]
cells containing nicotinic acetylcholine receptors in the chick retina, J. Comp. Neurol., 313 (1991) 394–408. Kaupmann, K., Huggel, K., Heid, J., Flor, P.J., Bischoff, S., Mickel, S.J., McMaster, G., Angst, C., Bittiger, H., Froestl, W. and Bettler, B., Expression cloning of GABAB receptors uncovers similarity to metabotropic glutamate receptors, Nature, 386 (1997) 239–246. Lugo-Garcia, N. and Blanco, R.E., Localization of GAD- and GABA-like immunoreactivity in ground squirrel retina: retrograde labeling demonstrates GAD-positive ganglion cells, Brain Res., 564 (1991) 19–26. Lukasiewicz, P.D., GABAC receptors in the vertebrate retina, Mol. Neurobiol., 12 (1996) 181–194. Morrow, A.L., Regulation of GABAA receptor function and gene expression in the central nervous system, Int. Rev. Neurobiol., 38 (1995) 1–41. Osborne, N.N., Wood, J. and Mouller, A., The influence of experimental ischaemia on protein kinase C and the GABAergic system in the rabbit retina, Neuropharmacology, 34 (1995) 1279–1288. Persohn, E., Malherbe, P. and Richards, J.G., Comparative molecular neuroanatomy of cloned GABAA receptor subunits in the rat CNS, J. Comp. Neurol., 326 (1992) 193–216. Reichelt, W., Herandez, M., Danlian, R.T., Kisaalita, W.S. and Jordan, B.L., GABAA receptor currents recorded form Mu¨ller glial cells of the baboon (Papio cynocephalus) retina, Neurosci. Lett., 203 (1996) 159–162. Rodieck, R.W. The Vertebrate Retina, W.H. Freeman, San Francisco, 1973. Stamp, J.A., Piggins, H.D., Rusak, B. and Semba, K., Distribution of ionotropic glutamate receptor subunit immunoreactivity in the suprachiasmatic nucleus and intergeniculate leaflet of the hamster, Brain Res., 756 (1997) 215–224. van Reeth, O., Losee-Olson, S. and Turek, F.W.N., Phase shifts in the circadian activity rhythm induced by triazolam are not mediated by the eyes or the pineal gland in the hamster, Neurosci. Lett., 80 (1987) 185–190. Yeh, H.H., Grigorenko, E.V. and Veruki, M.L., Correlation between a biculline-resistant response to GABA and GABAA receptor r1 subunit expression in single rat retinal bipolar cells, Vis. Neurosci., 13 (1996) 283–292. Yung, K.K.L., Localization of glutamate receptors in dorsal horn of rat spinal cord, NeuroReport, 9 (1998) 1261–1265. Yung, K.K.L. and Lo, Y.L., Immunocytochemical localization of muscarinic m2 receptor in the rat spinal cord, Neurosci. Lett., 229 (1997) 81–84.
Differential localization of GABAA receptor a and b subunits in the hamster retina and relationship with glutamic acid decarboxylase immunoreactivity Y.L. Lee a, E.Y.P. Cho b, K.K.L. Yung a ,* a
Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China b Department of Anatomy, Chinese University of Hong Kong, Shatin, Hong Kong, China Received 25 March 1998; received in revised form 6 April 1998; accepted 6 April 1998
Abstract In order to determine the cellular localization of GABAAa and b subunits in the hamster retina, single and double immunocytochemistry was performed in perfuse-fixed hamster retina using commercially-available antibodies against the two receptor subunits and glutamic acid decarboxylase. Strong GABAAb immunoreactivity was found in two strata of the inner plexiform layer and in perikarya of amacrine cells and bipolar cells in the inner nuclear layer. In contrast, no GABAAa immunoreactivity was detected. All but a few of the GABAAb-immunoreactive amacrine cells were found not to display glutamic acid decarboxylase immunoreactivity. The present results indicate that there is a differential localization of GABAAa and b subunits in different neuronal subpopulations in the hamster retina. 1998 Elsevier Science Ireland Ltd.
Keywords: GABAAa subunit; GABAAb subunit; Immunocytochemistry; Avidin-biotin-peroxidase reaction; Immunofluorescence; Light microscopy
Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system and is known to be one of the neurotransmitters found in the vertebrate retina [5,7,10,16]. The functions of GABA are mediated by GABA receptors that are divided into three major types. They are namely the GABAA receptors [9], GABAB receptors [6,9] and GABAC receptors [1,8,9]. The GABAA receptor is an ionotropic receptor that has a pentameric structure [9]. A functional ion-channel of GABAA receptor is known to be composed of five subunits that include one alpha, three beta and one gamma subunits (1a,3b,1g]. Six types of alpha subunit (al–a6), four types of beta subunit (b1–b4) and four types of gamma subunit (g1–g4) have been cloned [9,11]. The subunit compositions determine pharmacological and functional properties of the GABAA receptor [9,12]. Previous reports have indicated that injection of the
* Corresponding author. Tel.: +86 852 23397050; fax: +86 852 23397050; e-mail: [email protected]
benzodiazepine triazolum, an agonist of GABAA receptor, can induce permanent phase shifts in the circadian rhythm of locomotor activity in hamsters [15]. Although previous immunocytochemical and in situ hybridization studies have revealed that immunoreactivity for GABAA receptor is localized in different cellular elements in retinae of different mammalian species (GABAA receptor a6 subunit in rat [4]; GABAA receptor a and 13 subunits in rabbit [2,3]), less information has been reported so far about the localization of GABAAa and 13 subunit in hamster retina. It is important to determine the localization of GABAA receptors in hamster retina, as hamsters are the subjects for investigation for most of the behavioral and gene expression experiments in concern of circadian rhythms [14]. In order to determine and compare the cellular localization of the a and b subunits of GABAA in the hamster retina, immunocytochemistry was performed. In addition, another major objective of the present study was to investigate the receptor and neurotransmitter relationship in the hamster retina. In order to identify the GABAergic neurons in the hamster retina, immunoreactivity for glutamic acid decar-
0304-3940/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00326- 7
30
Y.L. Lee et al. / Neuroscience Letters 248 (1998) 29–32
boxylase (GAD), a synthetic enzyme for GABA, was detected as a neurochemical marker for GABA. In the present study, eight Syrian golden hamsters (200– 250 g, female, the Chinese University of Hong Kong) were used. The handling of hamsters and all procedures performed on them were approved in accordance to the Animals (Control of Experiments) Ordinance, Hong Kong, China. The animals were deeply anaesthetized by choral hydrate (10% in normal saline, 0.9% NaCl; intraperitoneal) and transcardially perfused by 50–100 ml of saline then followed by 200 ml of fixative (3–4% paraformaldehyde and 0.01% glutaraldehyde in 0.1 M phosphate buffer (PB), pH 7.4) as previously described [17,18]. The retinae were removed, post-fixed (3% paraformaldehyde, 1 h) and cut by a cryostat (10–20 mm). The sections were either processed to single immunocytochemistry [17,18] or immunocytochemistry combined with immunofluorescence as previously described [17]. Primary antibodies against GABAA receptor a and b subunits (mouse monoclonal;
used at 1:2000 dilution in PBS supplemented with 0.1% Triton X–100 and 2% NGS [PBS–Triton]; Chemicon International, Temcula, CA) and antibody against GAD (rat monoclonal; used at 1:50 dilution in PBS-Triton, Chemicon) was used. In addition, secondary antibodies (biotinylated goat–anti-rat or goat–anti-mouse IgG, 1:100–200 dilutions in PBS–Triton) were used. Immunoreactivity for receptor and GAD was revealed by the avidin–biotin peroxidase (ABC) method (1:100; ABC kit, Vector Labs.). In the case of double immunolabeling, after revealing immunoreactivity for the receptors, GAD immunoreactivity was then revealed in the same sections by incubating in fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (1:100–200; PBS–Triton). Immunoreactivity for GABAA receptor b subunit was found in two strata of the inner plexiform layer (IPL; Fig. 1A). At a higher magnification, the perikarya of neurons were densely labeled and GABAAb-immunoreactive cell processes were seen running along the vertical axis of the inner nuclear layer (INL; Fig. 1B–C). Most of these cells
Fig. 1. Light micrographs of the hamster retina immunostained to reveal immunoreactivity for GABAAb (A–C) and a (D) subunits. (A) At low magnification, immunoreactivity for GABAAb subunit is found in two strata of the inner plexiform layer (IPL; indicated by white arrowheads). In addition. GABAAb-immunoreactive perikarya are also observed in the inner nuclear layer (INL). (B,C) At higher magnification, many of the GABAAb-immunoreactive cells in the INL are found to have a single process extending from the cell bodies (arrows). Occasionally, some immunoreactive bipolar cells are also seen (open arrow). (D) No immunoreactivity for the GABAAa subunit is observed in all layers of the hamster retina. ONL, Outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bars (A,D) 500 mm; (B,C) 100 mm.
Y.L. Lee et al. / Neuroscience Letters 248 (1998) 29–32
were seen to be ‘axonless’ or possessed only one process (Fig. 1B–C). In addition, GABAAb immunoreactivity was found in the perikarya of bipolar cells (Fig. 1C). In contrast, no immunoreactivity for the GABAAa subunit was detected in all the layers of the hamster retina (Fig. 1D). Immunoreactivity for GAD was observed in the IPL and amacrine cells of INL as revealed by immunofluorescence (Fig. 2B,D). In the IPL, GAD immunoreactivity was detected in three strata (data not shown) and also in perikarya of amacrine cells in the INL (Fig. 2B,D). In those sections, double-labeled to reveal GABAAb and GAD immunoreactivity, in most of the cases GABAAb-immunoreactive perikarya did not display GAD immunoreactivity (Fig. 2A–B). However, occasionally a few GABAAb-immunoreactive perikarya (about 7.5%, n = 282) were found to be also GAD-immunoreactive (Fig. 2C–D). The results of the present study indicate that there is a differential localization of GABAA receptor a and b subunits in the hamster retina. No GABAAa immunoreactivity was detected. This result indicates the a subunit is not expressed by neuronal elements in the hamster retina. In contrast to the present results, immunoreactivity for a subunits has been found in retinae of other mammalian species [2–4]. This discrepancy is likely due to the species variation of the animal studied as the a subunit may not be expressed in the hamster retina. However, the present results do not prelude the possibility that the primary antibody does not recognize the a subunit expressed in hamster retina. The present results indicate that some of the distinct retinal neuronal populations in hamster express the GABAAb subunit. Immunoreactivity for GABAAb subunit is mainly observed in perikarya in the INL and in two strata of neuropilar elements in the IPL. Morphology of most of the labeled cells resembles that of the amacrine cells [13]. The amacrine cells are therefore the major types of neurons that display immunoreactivity for GABAAb subunit. In addition to amacrine cells, occasionally some bipolar cells in the INL have been found to contain the GABAA receptor immunoreactivity. It is however, extremely difficult to identify the subpopulations of bipolar cells that -display GABAA b immunoreactivity in the INL by light microscopy. As described by previous study [7], immunoreactivity for GAD was observed in the neuropil of the IPL and in amacrine cells of the INL. Since GAD is the synthetic enzyme of GABA (see refs. in [7]), GAD immunoreactivity can indicate the presence of GABA. In the present results, most of the GABAAb-immunoreactive cells do not display GAD immunoreactivity, which implies that they are not GABAergic neurons. In contrast, there is a small population of GAD immunoreactive amacrine cells found to display GABAAb immunoreactivity. This indicates a small subpopulation of GABAergic amacrine cells may also express subunits of the GABAA receptor. The functional role of the b subunit remains to be established (see below). Although the present report do not provide functional
31
Fig. 2. Light micrographs of the hamster retina double immunostained to reveal immunoreactivity for GABAAb subunit and glutamic acid decarboxylase (GAD). (A) A GABAAb-immunoreactive amacrine cell is shown in the inner plexiform layer (arrowhead). (B) The neuron shown in (A) is shown (white arrowhead). It is GAD-negative. Another amacrine cell nearby is found to be GAD-immunoreactive (white arrow). (C) In other section, a GABAAb-immunoreactive amacrine cell is shown (arrowhead). (D) The neuron shown in (C) is shown (white arrowhead). It is found to express GAD immunoreactivity as indicates by immunofluorescence. Another nearby cell is shown to be only GAD-immunoreactive (white arrow). ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer. Scale bar (A-D) 100 mm.
32
Y.L. Lee et al. / Neuroscience Letters 248 (1998) 29–32
information, it is unlikely that the GABAA channels present in the hamster retina are functional, as no GABAAa subunit immunoreactivity is detected. A previous report indicates that the phase shifts in the circadian rhythm in the hamster induced by benzodiazepine may not be mediated by the eye [15], there may be no functional GABAA channels in the hamster retina. The present report provides anatomical evidence for the above functional observation. This may indicate that the distribution of functional GABAA channels in hamster is very different from those in other mammalian species, in which functional GABAA channels are found [10,12]. Further investigation is necessary in order to establish the role of GABAAb subunit expressed by neurons in hamster retina. In summary, there is a differential localization of GABAAa and b subunits in different neuronal elements of the hamster retina and the distribution of GABAA subunit immunoreactivity in hamster retina is different from other mammalian species and may be related to the GABA neurotransmission in the hamster retina.
[6]
[7]
[8] [9]
[10]
[11]
[12]
The authors would like to thank Miss Lousie Ng for technical assistance in the present project. The present work was supported by Faculty Research Grants FRG/9596/I-17, FRG/95-96/II-73 and FRG/96-97/II-19, Hong Kong Baptist University (to KKLY). [1] Enz, R., Brandsta¨tter, J.H., Wa¨ssle, H. and Bormann, J., Immunocytochemical localization of the GABAC receptor r subunits in the mammalian retina, J. Neurosci., 16 (1995) 4479–4490. [2] Greferath, U., Grunert, U., Muller, F. and Wassle, H., Localization of GABAA receptors in the rabbit retina, Cell Tissue Res., 276 (1994) 295–307. [3] Greferath, U., Grunert, U., Fritschy, J.M., Stephenson, A., Mohler, H. and Wassle, H., GABAA receptor subunits have differential distributions in the rat retina: in situ hybridization and immunohistochemistry, J. Comp. Neurol., 353 (1995) 55357l. [4] Gutie´rrez, A., Khan, Z.U. and De Blas, A.L., Immunocytochemical localization of the a6 subunit of the g-Aminobutyric acidA receptor in the rat nervous system, J. Comp. Neurol., 365 (1996) 504–510. [5] Hamassaki-Britto, D.E., Brzozowska-Prechtl, A., Karten, H.J., Lindstrom, J.M. and Keyser, K.T., GABA-like immunoreactive
[13] [14]
[15]
[16]
[17] [18]
cells containing nicotinic acetylcholine receptors in the chick retina, J. Comp. Neurol., 313 (1991) 394–408. Kaupmann, K., Huggel, K., Heid, J., Flor, P.J., Bischoff, S., Mickel, S.J., McMaster, G., Angst, C., Bittiger, H., Froestl, W. and Bettler, B., Expression cloning of GABAB receptors uncovers similarity to metabotropic glutamate receptors, Nature, 386 (1997) 239–246. Lugo-Garcia, N. and Blanco, R.E., Localization of GAD- and GABA-like immunoreactivity in ground squirrel retina: retrograde labeling demonstrates GAD-positive ganglion cells, Brain Res., 564 (1991) 19–26. Lukasiewicz, P.D., GABAC receptors in the vertebrate retina, Mol. Neurobiol., 12 (1996) 181–194. Morrow, A.L., Regulation of GABAA receptor function and gene expression in the central nervous system, Int. Rev. Neurobiol., 38 (1995) 1–41. Osborne, N.N., Wood, J. and Mouller, A., The influence of experimental ischaemia on protein kinase C and the GABAergic system in the rabbit retina, Neuropharmacology, 34 (1995) 1279–1288. Persohn, E., Malherbe, P. and Richards, J.G., Comparative molecular neuroanatomy of cloned GABAA receptor subunits in the rat CNS, J. Comp. Neurol., 326 (1992) 193–216. Reichelt, W., Herandez, M., Danlian, R.T., Kisaalita, W.S. and Jordan, B.L., GABAA receptor currents recorded form Mu¨ller glial cells of the baboon (Papio cynocephalus) retina, Neurosci. Lett., 203 (1996) 159–162. Rodieck, R.W. The Vertebrate Retina, W.H. Freeman, San Francisco, 1973. Stamp, J.A., Piggins, H.D., Rusak, B. and Semba, K., Distribution of ionotropic glutamate receptor subunit immunoreactivity in the suprachiasmatic nucleus and intergeniculate leaflet of the hamster, Brain Res., 756 (1997) 215–224. van Reeth, O., Losee-Olson, S. and Turek, F.W.N., Phase shifts in the circadian activity rhythm induced by triazolam are not mediated by the eyes or the pineal gland in the hamster, Neurosci. Lett., 80 (1987) 185–190. Yeh, H.H., Grigorenko, E.V. and Veruki, M.L., Correlation between a biculline-resistant response to GABA and GABAA receptor r1 subunit expression in single rat retinal bipolar cells, Vis. Neurosci., 13 (1996) 283–292. Yung, K.K.L., Localization of glutamate receptors in dorsal horn of rat spinal cord, NeuroReport, 9 (1998) 1261–1265. Yung, K.K.L. and Lo, Y.L., Immunocytochemical localization of muscarinic m2 receptor in the rat spinal cord, Neurosci. Lett., 229 (1997) 81–84.