- Email: [email protected]
Distinct cellular distribution of GABABR1 and GABAAα1 receptor immunoreactivity in the rat substantia nigra
GABAB and GABAA receptors in rat substantia nigra
Pergamon
PII: S0306-4522(00)00156-1
Neuroscience Vol. 99, No. 1, pp. 65±76, 2000 65 q 2000 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/00 $20.00+0.00
www.elsevier.com/locate/neuroscience
DISTINCT CELLULAR DISTRIBUTION OF GABABR1 AND GABAAa1 RECEPTOR IMMUNOREACTIVITY IN THE RAT SUBSTANTIA NIGRA T. K. Y. NG and K. K. L. YUNG* Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong, PR China
AbstractÐGABA is one of the most important inhibitory neurotransmitters in the substantia nigra. Functions of GABA are mediated by two major types of GABA receptors, namely the GABAA and GABAB receptors. Subunits of both the GABAA and GABAB receptors have been cloned and functional characteristics of the receptors depend on their subunit compositions. In order to characterize the cellular localization of GABABR1 and GABAAa1 subunit immunoreactivity in subpopulations of neurons in the rat substantia nigra, double and triple immuno¯uorescence was employed. Over 90% of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra pars compacta were found to display immunoreactivity for GABABR1. In contrast, immunoreactivity for GABAAa1 was found to be primarily displayed by neurons in the substantia nigra pars reticulata. Around 85% of the GABAAa1immunoreactive reticulata neurons were found to display parvalbumin immunoreactivity and some GABAAa1-positive reticulata neurons were found to be parvalbumin negative. In addition, triple-labeling experiments revealed that at the single cell level, the tyrosine hydroxylase-positive, i.e. the dopaminergic neurons in the compacta displayed intense immunoreactivity for GABABR1 but not GABAAa1 receptors. The parvalbumin-positive neurons in the reticulata displayed intense immunoreactivity for GABAAa1 but not GABABR1 receptors. The present results demonstrate in the same sections that there is a distinct pattern of localization of GABABR1 and GABAAa1 receptor immunoreactivity in different subpopulations of the rat substantia nigra and provide anatomical evidence for GABA neurotransmission in the subpopulations of nigral neurons. q 2000 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: basal ganglia, dopaminergic neurons, GABAergic neurons, parvalbumin, double and triple immuno¯uorescence, confocal microscopy.
Axonal terminals that contain the neurotransmitter GABA form inhibitory synapses to neurons of the substantia nigra (SN; for reviews see Refs 2, 11, 33 and 34). The GABAergic synaptic inputs to the SN, which mainly arise from the neostriatum and the globus pallidus, 2,11,33,34 form the two major pathways in the basal ganglia, and they also play important roles in motor regulation. The SN is divided into the substantia nigra pars compacta (SNc) which contains dopaminergic neurons and the substantia nigra pars reticulata (SNr) which mainly contains GABAergic neurons. 11 The dopaminergic neurons in the SNc provide an important feedback and regulation to neurons in the neostriatum and degeneration of the dopaminergic neurons results in Parkinson's disease. 11,44 The SNr is considered as one of the output nuclei of the basal ganglia, in which disinhibition of the neurons in the SNr form the basis for motor control in the basal ganglia (for a review see Ref. 11). The inhibitory response of GABA in the SN is mainly mediated by two classes of receptors, namely the GABAA and GABAB receptors. 9,25±27,31 The GABAB receptor is a metabotropic receptor 3,4 and is known to consist of at least two heteromeric subunits, namely, the GABABR1 and GABABR2 subunits. 18,20,21,39 Co-expression of these two subunits produces a fully functional GABAB receptor. 18,20,21,39 The GABABR1 can be further divided into its isoforms, i.e. GABABR1a and GABABR1b, that might be associated with
different neuronal elements, i.e. the presynaptic and postsynaptic elements, in the cerebellum. 1,9,19 Functionally, the GABAB receptors regulates the Ca 21 and/or K 1 channel conductance in the membranes. 3 The GABAA receptors however, are ionotropic receptors that are composed of pentameric subunits. A functional ion-channel of GABAA receptors is known to be composed of one a, three b and one g subunits. 23,24 The GABAA receptors are known to associate with membrane Cl ± channels. 23,24 Pharmacological and functional properties of the GABAA receptors are determined by the subunit compositions. 22±24 One of the most common combinations of functional GABAA receptors throughout the brain including the SN is the combination of a1, b2/3 and g2 subunits. 10 Previous physiological studies 5,6,8,13,28,29,31,36,37 suggest that there may be a functional segregation of GABAA and GABAB receptor-mediated responses in subpopulations of nigral neurons. Neurons in the SNr are tonically inhibited by GABA via the postsynaptic GABAA receptors but not the postsynaptic GABAB receptor. 6,31 Presynaptic GABAB receptor however, are suggested to have predominant roles in mediating GABA neurotransmission in the SNr. 5 In contrast, a robust GABAB response is found in the dopaminergic neurons in the SNc. 5,6,13,28,29,36,37 The GABA inhibition on the dopaminergic neurons that arises from the pallidal origin is mediated via GABAB receptors but those that arise from the reticulata neurons are suggested to mediate via GABAA receptors. 28,29,36,37 Immunoreactivity for GABABR1 receptor has been described in the rat brain with less reference to the cellular distribution in the SN. 9 A recent report has indicated that immunoreactivity for GABABR1 is intense in the SNc of monkeys. 7 This study, however, did not simultaneously provide data about the GABAA receptor localization in the
*To whom correspondence should be addressed. Tel.: 1 852-23397060; fax: 1 852-23395995. E-mail address: [email protected] (K. K. L. Yung). Abbreviations: IgG, immunoglobulin; NGS, normal goat serum; PB, phosphate buffer; PBS, phosphate-buffered saline; SN, substantia nigra; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata; TH, tyrosine hydroxylase. 65
66
T. K. Y. Ng and K. K. L. Yung
SN. There are therefore fewer anatomical data available so far to evaluate the previous physiological observations of GABAA and GABAB receptor-mediated responses in subpopulations of neurons in the SN. One important unanswered question is whether subpopulations of nigral neurons differentially express GABAA and GABAB receptor subunit immunoreactivity. In addition, previous studies in our laboratory have indicated that there is a differential localization of GABA transaminase immunoreactivity (a degradation enzyme of GABA) 43 as well as ionotropic and metabotropic glutamate receptor immunoreactivity 38,41 in distinct subpopulations of neurons in the rat SN, in particular in subpopulations of reticulata neurons that express strong immunoreactivity for parvalbumin or GABA transaminase. 38 It is therefore of interest to know whether there is a distinct pattern of localization of GABAA and GABAB receptor subunit immunoreactivity in different neuronal elements in the rat SN. The objectives of the present study were to identify and characterize the neurochemically known neuronal elements in the SN that displayed GABABR1 and GABAAa1 immunoreactivity. Subpopulations of neurons in the SN were characterized by their neurochemical markers, i.e. tyrosine hydroxylase (TH) for dopaminergic neurons in the SNc and parvalbumin for GABAergic neurons in the SNr. 38 Double and triple immuno¯uorescence experiments were employed. EXPERIMENTAL PROCEDURES
Animal and tissue preparation In the present study, six adult rats (Sprague±Dawley female, 150± 200 g, supplied by the University of Hong Kong) were used. The handling of rats and all procedures that were performed on them were approved in accordance with the Animals (Control of Experiments) Ordinance, Hong Kong, People's Republic of China, and all efforts were made by the authors to minimize the number of animals used and their suffering. The animals were deeply anesthetized with an overdose of sodium pentobarbital (60 mg/kg, i.p., Nembutal, Boehringer Mannheim). The animals were then perfused transcardially with 50± 100 ml of normal saline followed by 200 ml of ®xative. The ®xative consisted of 3% paraformaldehyde plus 0.01% glutaraldehyde in phosphate buffer (PB: 0.1 M, pH 7.4). The brains were removed and post®xed in 3% paraformaldehyde in PB at 48C for several hours. Coronal sections of the mesencephalon (60±70 mm) that contained the SN were cut under a vibrating microtome and collected in phosphate-buffered saline (PBS: 0.01 M, pH 7.4). The sections were then washed (3 £ PBS) and pre-incubated in normal goat serum (NGS; 4% in PBS) for 1 h at room temperature before proceeding to immunocytochemical procedures. Sources and preparation of antibodies Commercially available primary antibodies that were raised against GABAAa1 [rabbit polyclonal; 1: 2000 in 2% NGS and 0.1% triton X100 (PBS-Triton); Upstate Biotechnology, Lake Placid, NY, USA] and GABABR1 (guinea-pig polyclonal; 1:2000±1:5000 in PBS-Triton; Chemicon International, Temcula, CA, USA) receptors were obtained and used in the present study. The antibody against GABABR1 was raised against a common sequence to the two variants of the GABABR1 receptor, namely the GABABR1a and GABABR1b. The speci®city of the GABABR1 receptor antibody was biochemically characterized by our previous study. 40 Primary antibodies against parvalbumin (mouse monoclonal; 1:1000 in PBS-Triton; Sigma, St. Louis, MO, USA) and TH (rabbit polyclonal; 1:2000 in PBS-Triton and mouse monoclonal; 1:500 in PBS-Triton; Chemicon) were also used. Secondary antibodies were also obtained from commercial sources. Fluorochrome-conjugated secondary antibodies (Alexa 488-conjugated, Alexa 562-conjugated or cy-5-conjugated goat-anti-guinea-pig immunoglobulin (IgG), goat-anti-rabbit IgG or goat-anti-mouse IgG
in respect to the origins of the primary antibodies; Molecular Probes or Vector Labs.) were also used. Immuno¯uorescence Single and double immuno¯uorescence experiments were carried out as previously described. 38,40±42 Triple immuno¯uorescence experiments were also carried out similar to the double labeling experiments. In single labeling experiments, the nigral sections were incubated in one primary antibody. 38,40±43 In double labeling experiments, the sections were incubated in a mixture of primary antibodies, that were from different species of origins, overnight at room temperature. The GABAAa1 antibody (rabbit origin) was mixed either with parvalbumin antibody (mouse origin) or with TH antibody (mouse origin). The GABABR1 antibody (guinea-pig origin) was mixed either with parvalbumin antibody (rat origin) or with TH antibody (mouse or rabbit origin). In triple labeling experiments, GABAAa1 antibody (rabbit origin) and GABABR1 antibody (guinea-pig origin) were mixed either with parvalbumin antibody (rat origin) or with TH antibody (mouse origin). After incubation in one or in a mixture of primary antibodies, the sections were washed (3 £ PBS) and incubated in one or a mixture of secondary antibodies in respect to the origins of primary antibodies for 2 h at room temperature. The secondary antibodies were ¯uorochrome-conjugated antibodies as described above, e.g., Alexa 488-conjugated goat-anti-guinea-pig IgG, Alexa 562-conjugated goat-anti-rabbit IgG and cy5-conjugated goat-anti-mouse IgG (1:100 in PBS-Triton). Different combinations were used as described in previous studies. 38,40 After three washes (3 £ PBS), the sections were then mounted on a clean slide with mounting medium (Vectashield, Vector Labs.). The sections were then observed under a laser scanning confocal microscope (LSM 510, Zeiss with an argon±kryton laser and a helium±neon laser with three channels; excitation for Alexa 488: 488 nm; excitation for Alexa 562: 562 nm; excitation for cy-5: 688 nm). Digital images of the immunoreactive structures were captured and analysed. Controls for the single, double and triple immunocytochemistry were performed by omission of primary antibodies in turn of the mixtures in the reaction sequence. RESULTS
Confocal microscopic observations Control for immunocytochemistry. In the control sections in which the primary antibody was omitted during the single labeling procedures, no immunoreaction product was seen (data not shown). On those sections subjected to double or triple immuno¯uorescence, the two or three primary antibodies were omitted in turn during the double labeling procedures. When two primary antibodies were omitted, only one ¯uorescent labeling was observed (data not shown). Localization of GABABR1 and GABAAa 1 immunoreactivity. In the laser scanning confocal microscope, immunoreactivity for GABABR1 and GABAAa1 was identi®ed by immuno¯uorescent signal in the single-labeled sections (Fig. 1). Immunoreactivity for GABABR1 was primarily found in perikarya of the SNc. Some of the neurons in the SNr were found to display a low level of GABABR1 immunoreactivity (Fig. 1A). In contrast, immunoreactivity for GABAAa1 was primarily found in neurons in the SNr (Fig. 1B). Some of the neurons in the SNc were found to display a low level of GABAAa1 immunoreactivity (Fig. 1B). Double labeling of tyrosine hydroxylase and GABABR1. In the sections that were subjected to double labeling, immunoreactivity for TH was identi®ed by the presence of cy-5 (red) ¯uorescent signal. Immunoreactivity for TH was primarily
GABAB and GABAA receptors in rat substantia nigra
67
region, GABABR1-immunoreactive perikarya were also found (Fig. 2E). The TH-immunoreactive neurons were found to display a dense immunoreactivity for GABABR1 (Fig. 2F). Some GABABR1-immunoreactive neurons that were relatively lightly stained were found to be TH negative (Fig. 2F). Double labeling of parvalbumin and GABABR1. Similarly, immunoreactivity for parvalbumin was identi®ed in the confocal microscope by the presence of cy-5 (red) signal. Immunoreactivity for parvalbumin was primarily found in neurons in the SNr (Fig. 3A). Immunoreactive proximal dendrites were also seen (Fig. 3A). In the same sections, immunoreactivity for GABABR1 was identi®ed by the presence of Alexa 488 (green) ¯uorescence. As described as above, intense GABABR1 immunoreactivity was primarily detected in the SNc (Fig. 3B). These neurons were clearly seen to be parvalbumin negative in the superimposed image (Fig. 3C). In addition, most of the parvalbumin-immunoreactive perikarya in the SNc were found to display a lower level of GABABR1 immunoreactivity (Fig. 3C). At higher magni®cation, parvalbumin immunoreactivity was found in perikarya in the region that was at the boundary between the SNr and the SNc (Fig. 3D). Parvalbuminimmunoreactive punctuate structures were also seen (Fig. 3D). In the same sections, the parvalbumin-immunoreactive neurons were found to be GABABR1 immunoreactive (Fig. 3E±F). Moreover, some GABABR1-immunoreactive neurons were found to be parvalbumin negative (Fig. 3F).
Fig. 1. Fluorescent photomicrographs of the rat substantia nigra immunostained to reveal GABABR1 receptor (A) or GABAAa1 receptor (B) immunoreactivity. The immunoreaction product is indicated by the presence of immuno¯uorescent signal. In A, intense immunoreactivity for GABABR1 (GB) is primarily found in the region of the SNc. Intense labeling is observed in perikarya of compacta neurons. In B, intense immunoreactivity for GABAAa1 (GA) is primarily observed in the region of the SNr. Intense labeling is found in perikarya of reticulata neurons. Scale bar 200 mm (B; also applies to A).
found in perikarya in the SNc (Fig. 2A). TH-immunoreactive proximal dendrites were also seen (Fig. 2A). In the same sections, immunoreactivity for GABABR1 was identi®ed by the presence of Alexa 488 (green) ¯uorescent signal. Intense immunoreactivity for GABABR1 was found in perikarya of the SNc (Fig. 2B). Most of THimmunoreactive neurons in the SNc were found to be GABABR1 immunoreactive, as seen in a superimposition of the two images [neurons that were double labeled were identi®ed by the presence of orange±yellowish signal; Fig. 2C; 94.4 ^ 0.82%; number of neurons counted (n) 338]. The TH-immunoreactive neurons that were found in the caudal region of the SNr were also found to express a robust immunoreactivity for GABABR1 (data not shown). At higher magni®cation, densely labeled TH-immunoreactive perikarya were found at the boundary between the regions of the SNc and the SNr (Fig. 2D). In the same
Double labeling of tyrosine hydroxylase and GABAAa 1. Similar to Fig. 2, immunoreactivity for TH was primarily found in perikarya of the SNc (Fig. 4A; identi®ed by cy-5 signal). In the same sections, immunoreactivity for GABAAa1 was identi®ed by the presence of Alexa 488 (green) ¯uorescent signal (Fig. 4B). An intense level of GABAAa1 immunoreactivity was found in perikarya as well as in neuropilar elements in the SNr but not in the SNc (as indicated by the superimposed image; Fig. 4C). The TH-immunoreactive perikarya were also found to display a lower level of GABAAa1 immunoreactivity (Fig. 4C). At higher magni®cation, TH-immunoreactive perikarya and their proximal dendrites were clearly seen (Fig. 4D). The TH-immunoreactive perikarya were found to be GABAAa1 immunoreactive (Fig. 4E). In addition, intensely labeled GABAAa1-immunoreactive perikarya were found to be TH negative at the boundary between the SNc and the SNr (Fig. 4F). In the SNr, the GABAAa1-immunoreactive perikarya were found to be TH negative. Double-labeled TH- and GABAAa1-immunoreactive processes were seen among the single-labeled GABAAa1-immunoreactive neurons (Fig. 4G). Double labeling of parvalbumin and GABAAa 1. Similar to Fig. 3, immunoreactivity for parvalbumin was primarily found in perikarya of the SNr (Fig. 5A). The neurons were densely labeled (Fig. 5A, D). In the same sections, immunoreactivity for GABAAa1 was also found to primarily express in neurons of the SNr and they were seen to be densely labeled (Fig. 5B, E). Superimposed images revealed that most of the parvalbumin-immunoreactive
68
T. K. Y. Ng and K. K. L. Yung
Fig. 2. Fluorescent micrographs of the rat substantia nigra immunostained to reveal TH and GABABR1 immunoreactivity. The micrographs were obtained from the same sections. (A) Immunoreactivity for TH is indicated by Alexa 562 ¯uorescent signal. Immunoreactivity for TH is mainly found in perikarya of the SNc (the white arrow indicates the same neuron in B and C). (B) In the same section, immunoreactivity for GABA BR1 (GB) is indicated by Alexa 488 ¯uorescent signal. Intense immunoreactivity for GABABR1 is also primarily found in perikarya of the SNc. They are likely to co-express TH immunoreactivity (see the neuron indicated by the white arrow). Lightly stained GABABR1-immunoreactive neurons are also observed in the SNr. (C) A superimposed image of A and B is shown. Co-localization of TH and GABABR1 immunoreactivity is indicated by a combination of the two ¯uorescent signals. Almost all of the TH-immunoreactive neurons in the SNc are found to display intense immunoreactivity for GABABR1 (the neuron shown in A and B is indicated by the white arrow). (D) At higher magni®cation, at the region of the boundary between the SNc and the SNr, THimmunoreactive perikarya are seen (the white arrow indicated the same neuron in E and F). Two perikarya of TH-negative reticulata neurons are indicated by asterisks (also in E and F). (E) In the same section, GABABR1-immunoreactive perikarya are found. The TH-immunoreactive perikarya are found to display an intense GABABR1 immunoreactivity (the same neuron indicated in D is indicated by a white arrow). The lightly stained GABA BR1immunoreactive neurons are found to be TH-negative (the two in D are indicated by asterisks). GABABR1 immunoreactivity is also observed in the neuropil. (F) The superimposed image of D and E is shown. The TH-immunoreactive perikarya are found to display strong immunoreactivity for GABABR1 (indicated by the combined signal; the same neuron in D and E is indicated by a white arrow). The lightly labeled GABABR1-immunoreactive neurons do not display TH immunoreactivity (asterisks). Scale bars 100 mm (in C, for A and B), 50 mm (in F, for D and E).
GABAB and GABAA receptors in rat substantia nigra
69
Fig. 3. Fluorescent micrographs of the rat substantia nigra immunostained to reveal parvalbumin and GABABR1 (GB) immunoreactivity. The micrographs were obtained from the same sections. (A) Immunoreactivity for parvalbumin is indicated by Alexa 562 ¯uorescent signal. Immunoreactivity for parvalbumin is only observed in neurons and in the neuropil of the SNr (one parvalbumin-positive neuron is indicated by a white arrow, the same in B and C). (B) Immunoreactivity for GABABR1 is indicated by Alexa 488 ¯uorescent signal. Intense GABABR1 immunoreactivity is detected in neurons in the SNc. Neurons in the SNr also display pale immunoreactivity for GABA BR1 (the same neuron in A is indicated by a white arrow). (C) A superimposed image is shown. Intense immunoreactivity for GABABR1 is detected in perikarya of the SNc that are parvalbumin negative. The parvalbumin-immunoreactive perikarya in the SNr thus express a low level of GABABR1 immunoreactivity. (D) At higher magni®cation, a parvalbumin-immunoreactive neuron is shown (arrow). An asterisk indicates a neuron in the SNc that is parvalbumin negative (the two neurons are shown in E and F). (E) In the same section of D, two neurons that display immunoreactivity for GABABR1 are shown. Arrow indicates the GABABR1-immunoreactive neuron in the SNr that is also parvalbumin positive (as in D). Asterisk indicates the GABABR1-immunoreactive neuron in the SNc that is parvalbumin negative. (F) A superimposed image of D and E shows the neuron in the SNr which displays both GABABR1 and parvalbumin immunoreactivity. Scale bars 100 mm (in C, for A and B), 50 mm (in F, for D and E).
70
T. K. Y. Ng and K. K. L. Yung
Fig. 4.
GABAB and GABAA receptors in rat substantia nigra
perikarya in the SNr were found to be GABAAa1 immunoreactive (Fig. 5C, F; 85.3 ^ 1.56%; n 455). However, some neurons in the SNr displayed GABAAa1 immunoreactivity alone but not parvalbumin immunoreactivity (Fig. 5C, F; 14.7 ^ 1.26%; n 455). In contrast, a lower level of GABAAa1 immunoreactivity was found in neurons in the SNc (Fig. 5B, E). At higher magni®cation, parvalbumin-immunoreactive perikarya were densely labeled (Fig. 5G). Parvalbuminimmunoreactive punctuate processes were also seen (Fig. 5G). In the same sections, the parvalbumin-immunoreactive perikarya were found to display GABAAa1 immunoreactivity (Fig. 5H, I). In the boundary between the SNc and SNr, the parvalbumin-negative neurons in the SNc were also found to display a low intensity of GABAAa1 immunoreactivity (Fig. 5H, I). In addition, the single-labeled neurons in the SNr that displayed GABAAa1 immunoreactivity alone were seen to approach by parvalbumin- and GABAAa1-immunoreactive punctuated processes (Fig. 5J). Triple labeling of GABABR1 and GABAAa 1 with tyrosine hydroxylase or parvalbumin. Triple labeling experiments revealed that the TH-immunoreactive perikarya in the SNc displayed an intense immunoreactivity for GABABR1 (Fig. 6A, B). In the same section, neurons in the SNr (TH-negative) were found to display an intense immunoreactivity for GABAAa1 (Fig. 6B, C). A very low level of GABAAa1 immunoreactivity was detected in the TH-immunoreactive perikarya (Fig. 6A, C). In contrast, the parvalbumin-immunoreactive neurons in the SNr were found to display a very low level of GABABR1 immunoreactivity (Fig. 6D, E). The parvalbumin-negative neurons in the SNc were found, however, to display an intense level of GABABR1 immunoreactivity (Fig. 6D, E). In the same sections, the parvalbumin-immunoreactive reticulata neurons were found to display a very intense level of GABAAa1 immunoreactivity (Fig. 6D, F). DISCUSSION
The present study reports the cellular localization of GABABR1 and GABAAa1 immunoreactivity in the SN of the same rats and demonstrates for the ®rst time that immunoreactivity for GABABR1 and GABAAa1 is primarily expressed by different subpopulations of neurons in the SN. In the same sections of the SN, immunoreactivity for GABABR1 is primarily found in neurons of the SNc that co-express TH immunoreactivity. In contrast, immunoreactivity for GABAAa1 is primarily found in neurons of the SNr and most of the GABAAa1-immunoreactive neurons
71
have been found to be parvalbumin immunoreactive. The present results thus provide anatomical evidence and support the previous physiological observations 5,6,13,28,29,36,37 that the compacta neurons display a robust GABAB receptor-mediated response together with GABAA receptor-mediated response and the reticulata neurons display a primarily GABAA receptor-mediated response. 6,31 Immunoreactivity for GABABR1 receptor is primarily displayed by the dopaminergic neurons The present results indicate for the ®rst time that the dopaminergic neurons in the SNc of rats are the major groups of neurons that display GABABR1 immunoreactivity. The present results are consistent with the previous results found in monkeys. 7 The two results indicate that GABABR1 immunoreactivity is expressed in neuronal elements in the SN in similar patterns among different species. The primary antibody against the GABABR1 receptor employed in the present study is raised against a common domain of GABABR1a and GABABR1b isoforms. Results of previous studies indicate that the GABABR1a isoform is likely to be the dominant isoform of GABABR1 receptors found in the regions of the basal ganglia including the SN 9 as well as in the neostriatum. 9,40 The present results thus are likely to show that immunoreactivity for GABABR1a is predominantly found in the SN. One related point is that the present results, as those found in the previous study, 7 do not provide information about the localization of the other GABAB receptor subunit, i.e. the GABABR2 subunit in the SN. The present results thus do not directly re¯ect functional information about GABAB receptors in the dopaminergic neurons as a functional GABAB receptor is known to comprise GABABR1 and GABABR2 subunits. 18,20,21,39 However, the present results provide anatomical evidence for those previous studies that observe a robust GABAB receptor-mediated response in dopaminergic neurons. 5,29,37 In addition to the GABAB receptor, the compacta neurons have been found to express strong immunoreactivity for GABAAa3, a2 but not a1, and g2 subunits. 10 It is likely that the compacta neurons contain functional GABAA channels 28,29,36,37 that have different subunit compositions from those of the reticulata neurons (discussed below). Localization of GABABR1 immunoreactivity in the substantia nigra pars reticulata The present results con®rm that immunoreactivity for GABAB is also found in neuropilar elements in the SNr. A
Fig. 4. Fluorescent micrographs of the rat substantia nigra immunostained to reveal TH and GABAAa1 (GA) immunoreactivity. The micrographs were obtained from the same sections. (A) Similar to Fig. 2, immunoreactivity for TH is found in perikarya and in the neuropil of the SNc (Alexa 562 ¯uorescent signal). One TH-immunoreactive perikaryon is indicated by an arrow (the same neuron is also indicated in B and C). (B) Intense immunoreactivity for GABAAa1 is found in the SNr. Perikarya (two are indicated by a thick arrow) as well as neuropilar elements in the SNr are intensely labeled. Perikarya in the SNc are also found to display a lower level of GABAAa1 immunoreactivity (arrow). (C) A superimposed image is shown. Intense immunoreactivity for GABAAa1 is detected in the SNr (thick arrow). TH-immunoreactive perikarya in the SNc is found to display a low level of GABAAa1 immunoreactivity. (D) At higher magni®cation, a TH-immunoreactive perikaryon is seen in the SNc (one is indicated by an arrow). A THnegative neuron is also found (one is indicated by an asterisk; also in E and F). (E) The TH-negative neuron found in D (asterisk) is seen to display intense immunoreactivity for GABAAa1. Intensely labeled proximal dendrites are also seen. The TH-positive neuron shown in D also displays a lower level of GABAAa1 immunoreactivity (arrow). (F) The superimposed image shows that the TH-immunoreactive neuron shown in D and E displays a lower level of GABAAa1 immunoreactivity (arrow). (G) In the SNr, two intensely labeled GABAAa1-immunoreactive neurons are shown (thick arrow, as indicated in B and C). The neurons are seen to be surrounded by TH-immunoreactive dendrites (arrowheads). Scale bars 100 mm (in C, for A and B), 50 mm (in G, for D±F).
72
T. K. Y. Ng and K. K. L. Yung
Fig. 5.
GABAB and GABAA receptors in rat substantia nigra
previous electron microscopic study indicated that the GABABR1 immunoreactivity was expressed by presynaptic neuronal elements in the SNr of monkeys and the GABABR1 immunoreactivity was found almost exclusively in terminals that formed asymmetrical synapses. 7 A controversial question is raised as most of the physiological studies indicated that the robust GABAB responses in the SNr were found in GABAergic axonal elements. 5,6,28,36,37 Postsynaptic GABAB responses in the SNr are known to be much weaker than presynaptic responses. 5,6 Neurons in the SNr of monkeys were found to express a low level of GABABR1 immunoreactivity. 7 The present results also indicated that the reticulata neurons of rats, albeit less, expressed a low level of GABABR1 immunoreactivity. A recent physiological study demonstrates that GABAB receptors induce a modulation of Ca 21 current but not directly on the K 1 current in neurons of the globus pallidus. 35 These results indicate that there may be a variety of GABABmediated responses in neurons including neurons in the basal ganglia, in which the previous patch clamp studies 5,6 may have only characterized a few of those responses. The cellular expressions of GABABR1, as well as other functional GABAB subunits, in the reticulata neurons therefore await further investigation. The present results thus hopefully open up an insight to further investigate the GABAB functions in the SNr. Subpopulations of nigral neurons display GABAAa 1 immunoreactivity The present ®ndings are consistent with previous ®ndings 10 that immunoreactivity for GABAAa1 is mainly found in the subgroups of neurons in the reticulata. The reticulata neurons are also found to display immunoreactivity for GABAAb2/3 and GABAAg2. 10,25 The neurons are therefore likely to display functional GABAA channels 6 with the above subunit compositions. The present observation extends the previous ®ndings that most of the GABAAa1-immunoreactive neurons in the SNr are in fact parvalbumin immunoreactive. As shown by previous reports, 12,15,16,38 parvalbumin-immunoreactive neurons are a subpopulation of the reticulata neurons and they are suggested to be the tonically active output neurons in the output pathways of the basal ganglia. 30 They are found to display a robust immunoreactivity for GABA
73
transaminase 38,43 and most of the ionotropic glutamate receptor subunits. 38 The parvalbumin-positive subpopulations of reticulata neurons are thus further proven to primarily display the GABAAa1 subunit, and likely to display functional GABAA channels with the a1, b2/3 and g6 subunit compositions. 10,25 These results, as a whole, further demonstrate that there may be a compartmentalization of glutamate and GABA interactions (and GABA metabolism) in subpopulations of reticulata neurons that display strong immunoreactivity for parvalbumin. 38 The parvalbuminimmunoreactive neurons are known to degenerate in a late stage of Parkinson's disease. 12 Recent studies in other regions of the brain indicate that blockage of GABAA receptor channels may have a neuroprotective effect on the vulnerability of the neurons towards glutamate excitotoxicity. 14,17,32 The present results thus illustrate the anatomical evidence for a possible functional interaction between the GABAergic and the glutamatergic systems in subpopulations of neurons in the SNr. The present study directly demonstrates at the cellular level that subpopulations of nigral neurons display different GABA receptor subunits. The compacta neurons primarily express GABABR1 immunoreactivity, whereas the reticulata neurons primarily express GABAAa1 immunoreactivity. The ®ndings provide anatomical evidence for a possible functional segregation of GABA functions in the rat SN as indicated by previous physiological studies. 5,6,13,28,29,36,37 Although the patterns of distribution of GABABR1 and GABAAa1 are quite distinct among the compacta as well as the reticulata neurons, the present results also indicate that the pattern is not exclusive. There are low levels of expression of GABAAa1 or GABABR1 immunoreactivity in the compacta and reticulata neurons, respectively. The exact functional implications of functional segregation of GABA receptors in different compartments of the rat SN and their roles in the circuitry in the basal ganglia await further investigation.
AcknowledgementsÐThe present work was supported by Faculty Research Grant FRG/99-00/II-29, Hong Kong Baptist University and Research Grant Council, Hong Kong. T. K. Y. Ng was supported by a postgraduate studentship, Hong Kong Baptist University. The authors would like to thank Dr W. H. Yung for critical comments. The authors would also like to thank Miss L. Y. Man for technical assistance.
Fig. 5. Fluorescent micrographs of the rat substantia nigra immunostained to reveal parvalbumin and GABAAa1 (GA) immunoreactivity. The micrographs were obtained from the same sections. (A) Similar to Fig. 3, immunoreactivity for parvalbumin is found in perikarya of the SNr (Alexa 562 signal; SNr). (B) Intense immunoreactivity for GABAAa1 is primarily detected in perikarya of the SNr (Alexa 488 signal). (C) A superimposed image of A and B is shown. Most of the parvalbumin-immunoreactive neurons are also found to display GABAAa1 immunoreactivity (combined ¯uorescent signal). However, some neurons in the SNr (one is indicated by a white arrow) are found to display GABAAa1 immunoreactivity only. (D) At the boundary between the SNr and the SNc, parvalbumin-immunoreactive perikarya are seen in the SNr (one is indicated by a white arrow). (E) Intense immunoreactivity for GABAAa1 is shown in perikarya of the SNr (the same neuron shown in D is also indicated by a white arrow). Perikarya in the SNc are found to display a lower level of GABAAa1 immunoreactivity. (F) A superimposed image is shown. Most of the parvalbumin-positive neurons in the SNr are found to display GABAAa1 immunoreactivity (combined signal; white arrow). (G) At higher magni®cation, one parvalbumin-immunoreactive neuron in the SNr is shown (arrow). One parvalbuminnegative neuron in the SNc is also shown (asterisk). (H) The neuron in G is seen to display intense immunoreactivity for GABAAa1 (arrow). The parvalbuminnegative neuron in G is also found to display a lower level of GABAAa1 immunoreactivity (asterisk). (I) A superimposed image of G and H shows the parvalbumin-immunoreactive neuron is seen to display intense immunoreactivity for GABAAa1 (combined signal; arrow). (J) In the SNr, the GABAAa1immunoreactive neuron shown in C (arrow) is shown at higher magni®cation. The neuron is seen to display GABAAa1 immunoreactivity only without PV immunoreactivity (arrow and star). The other neurons are seen to be double labeled. Scale bars 200 mm (in C, for A and B), 100 mm (in F, for D and E), 50 mm (in I and J, for G and H).
74
T. K. Y. Ng and K. K. L. Yung
Fig. 6. Fluorescent micrographs of the rat SN triple labeled to reveal TH or parvalbumin and GABAAa1 (GA) and GABABR1 (GB) immunoreactivity. The micrographs were obtained from the same sections. (A) At the boundary of the SNc and the SNr, TH-immunoreactive perikarya are seen (Alexa 562 signal; two are indicated by asterisks). One TH-negative perikaryon in the SNr is also shown (star). (B) Intense immunoreactivity for GABABR1 is found in THimmunoreactive neurons in the SNc (Alexa 488 signal). A TH-negative neuron is seen to display a lower level of GABABR1 immunoreactivity (star). (C) In the same section, the TH-negative neuron in the SNr is found to display intense level of GABAAa1 immunoreactivity (cy-5 combined ¯uorescent signal; star). Intensely labeled neuropilar elements are also seen. The TH-positive neurons, however, are seen to display less GABAAa1 immunoreactivity (asterisks). (D) Also at the boundary of the SNc and the SNr, parvalbumin-immunoreactive perikaryon is seen in the SNr (star). Three parvalbumin-negative perikarya in the SNc are also shown (asterisks). (E) The parvalbumin-negative neurons shown in D is seen to display intense immunoreactivity for GABABR1 (asterisks). The parvalbumin-positive neuron in the SNr is also found to display a low level of GABABR1 immunoreactivity (star). (F) In the same section, the parvalbumin-positive neuron in the SNr is seen to display intense immunoreactivity for GABAAa1 (cy-5 combined signal; star). The intensely labeled GABABR1immunoreactive neurons in the SNc are found to display a low level of GABAAa1 immunoreactivity (asterisks). Scale bar 50 mm (in F, for A±E).
REFERENCES
1. Billinton A., Upton N. and Bowery N. G. (1999) GABAB receptor isoforms GBR1a and GBR1b, appear to be associated with pre- and post-synaptic elements respectively in rat and human cerebellum. Br. J. Pharmac. 126, 1387±1392.
GABAB and GABAA receptors in rat substantia nigra
75
2. Bolam J. P. and Bennett B. D. (1995) Microcircuitry of the neostriatum. In Molecular and Cellular Mechanisms of Neostriatal Function (eds Ariano M. A. and Surmeier P. J.). R. G. Landes, Austin. 3. Bowery N. G. (1993) GABAB receptor pharmacology. A. Rev. Pharmac. Toxicol. 33, 109±147. 4. Bowery N. G., Hill D. R. and Hudson A. L. (1997) Characteristics of GABAB receptor binding sites on rat whole brain synaptic membranes. 1983. Br. J. Pharmac. 120, 452±467. 5. Chan P. K., Leung C. K. and Yung W. H. (1998) Differential expression of pre- and postsynaptic GABAB receptors in rat substantia nigra pars reticulata neurones. Eur. J. Pharmac. 349, 187±197. 6. Chan P. K. and Yung W. H. (1999) Inhibitory postsynaptic currents of rat substantia nigra pars reticulata neurons: role of GABA receptors and GABA uptake. Brain Res. 838, 18±26. 7. Charara A., Heilman C., Levey A. I. and Smith Y. (2000) Pre- and post-synaptic localization of GABAB receptors in the basal ganglia in monkeys. Neuroscience 95, 127±140. 8. Floran B., Silva I., Nava C. and Aceves J. (1988) Presynaptic modulation of the release of GABA by GABAA receptors in pars compacta and by GABAB receptors in pars reticulata of the rat substantia nigra. Eur. J. Pharmac. 150, 277±286. 9. Fritschy J. M., Meskenaite V., Weinmann O., Honer M., Benke D. and Mohler H. (1999) GABAB receptor splice variants GB1a and GB1b in rat brain: developmental regulation, cellular distribution and extrasynaptic localization. Eur. J. Neurosci. 11, 761±768. 10. Fritschy J. M. and Mohler H. (1995) GABAA-receptor heterogeneity in the adult rat brain: differential regional and cellular distribution of seven major subunits. J. comp. Neurol. 359, 154±194. 11. Gerfen C. R. and Wilson C. J. (1996) The basal ganglia. In Handbook of Chemical Neuroanatomy. Integrated Systems of the CNS Part 3 (eds Swanson L. W., BjoÈrklund A. and HoÈlfelt T.), Vol. 12, pp. 371±468. Elsevier, Amsterdam. 12. Hardman C. D., McRitchie D. A., Halliday G. M., Cartwright H. R. and Morris J. G. (1996) Substantia nigra pars reticulata neurons in Parkinson's disease. Neurodegeneration 5, 49±55. 13. Hausser M. A. and Yung W. H. (1994) Inhibitory synaptic potentials in guinea-pig substantia nigra dopamine neurones in vitro. J. Physiol., Lond. 479 (Pt 3), 401±422. 14. Hollrigel G. S., Toth K. and Soltesz I. (1996) Neuroprotection by propofol in acute mechanical injury: role of GABAergic inhibition. J. Neurophysiol. 76, 2412±2422. 15. Hontanilla B., Parent A., de las H. S. and Gimenez-Amaya J. M. (1998) Distribution of calbindin D-28k and parvalbumin neurons and ®bers in the rat basal ganglia. Brain Res. Bull. 47, 107±116. 16. Hontanilla B., Parent A. and Gimenez-Amaya J. M. (1997) Parvalbumin and calbindin D-28k in the entopeduncular nucleus, subthalamic nucleus, and substantia nigra of the rat as revealed by double-immunohistochemical methods. Synapse 25, 359±367. 17. Ingle®eld J. R., Wilson C. A. and Schwartz-Bloom R. D. (1997) Effect of transient cerebral ischemia on gamma-aminobutyric acidA receptor alpha 1subunit-immunoreactive interneurons in the gerbil CA1 hippocampus. Hippocampus 7, 511±523. 18. Jones K. A., Borowsky B., Tamm J. A., Craig D. A., Durkin M. M., Dai M., Yao W. J., Johnson M., Gunwaldsen C., Huang L. Y., Tang C., Shen Q., Salon J. A., Morse K., Laz T., Smith K. E., Nagarathnam D., Noble S. A., Branchek T. A. and Gerald C. (1998) GABAB receptors function as a heteromeric assembly of the subunits GABABR1 and GABABR2. Nature 396, 674±679. 19. 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. (1997) Expression cloning of GABAB receptors uncovers similarity to metabotropic glutamate receptors. Nature 386, 239±246. 20. Kaupmann K., Malitschek B., Schuler V., Heid J., Froestl W., Beck P., Mosbacher J., Bischoff S., Kulik A., Shigemoto R., Karschin A. and Bettler B. (1998) GABAB receptor subtypes assemble into functional heteromeric complexes. Nature 396, 683±687. 21. Kuner R., Kohr G., Grunewald S., Eisenhardt G., Bach A. and Kornau H. C. (1999) Role of heteromer formation in GABAB receptor function. Science 283, 74±77. 22. Mohler H., Benke D., Mertens S. and Fritschy J. M. (1992) GABAA-receptor subtypes differing in alpha-subunit composition display unique pharmacological properties. Adv. Biochem. Psychopharmac. 47, 41±53. 23. Mohler H., Fritschy J. M., Luscher B., Rudolph U., Benson J. and Benke D. (1996) The GABAA receptors. From subunits to diverse functions. Ion Channels 4, 89±113. 24. Mohler H., Kno¯ach F., Paysan J., Motejlek K., Benke D., Luscher B. and Fritschy J. M. (1995) Heterogeneity of GABAA-receptors: cell-speci®c expression, pharmacology, and regulation. Neurochem. Res. 20, 631±636. 25. Nicholson L. F., Faull R. L., Waldvogel H. J. and Dragunow M. (1992) The regional, cellular and subcellular localization of GABAA/benzodiazepine receptors in the substantia nigra of the rat. Neuroscience 50, 355±370. 26. Nicholson L. F., Faull R. L., Waldvogel H. J. and Dragunow M. (1995) GABA and GABAA receptor changes in the substantia nigra of the rat following quinolinic acid lesions in the striatum closely resemble Huntington's disease. Neuroscience 66, 507±521. 27. Nicholson L. F., Waldvogel H. J. and Faull R. L. (1996) GABAA receptor subtype changes in the substantia nigra of the rat following quinolinate lesions in the striatum: a correlative in situ hybridization and immunohistochemical study. Neuroscience 74, 89±98. 28. Paladini C. A., Celada P. and Tepper J. M. (1999) Striatal, pallidal, and pars reticulata evoked inhibition of nigrostriatal dopaminergic neurons is mediated by GABA(A) receptors in vivo. Neuroscience 89, 799±812. 29. Paladini C. A. and Tepper J. M. (1999) GABA(A) and GABA(B) antagonists differentially affect the ®ring pattern of substantia nigra dopaminergic neurons in vivo. Synapse 32, 165±176. 30. Rajakumar N., Elisevich K. and Flumerfelt B. A. (1994) Parvalbumin-containing GABAergic neurons in the basal ganglia output system of the rat. J. comp. Neurol. 350, 324±336. 31. Rick C. E. and Lacey M. G. (1994) Rat substantia nigra pars reticulata neurones are tonically inhibited via GABAA, but not GABAB, receptors in vitro. Brain Res. 659, 133±137. 32. Schwartz R. D., Yu X., Katzman M. R., Hayden-Hixson D. M. and Perry J. M. (1995) Diazepam, given postischemia, protects selectively vulnerable neurons in the rat hippocampus and striatum. J. Neurosci. 15, 529±539. 33. Smith A. D. and Bolam J. P. (1990) The neural network of the basal ganglia as revealed by the study of synaptic connections of identi®ed neurones. Trends Neurosci. 13, 259±265. 34. Smith Y., Bevan M. D., Shink E. and Bolam J. P. (1998) Microcircuitry of the direct and indirect pathways of the basal ganglia. Neuroscience 86, 353±387. 35. Stefani A., Spadoni F., Giacomini P., Lavaroni F. and Bernardi G. (1999) The modulation of calcium current by GABA metabotropic receptors in a subpopulation of pallidal neurons. Eur. J. Neurosci. 11, 3995±4005. 36. Tepper J. M., Martin L. P. and Anderson D. R. (1995) GABAA receptor-mediated inhibition of rat substantia nigra dopaminergic neurons by pars reticulata projection neurons. J. Neurosci. 15, 3092±3103. 37. Tepper J. M., Paladini C. A. and Celada P. (1998) GABAergic control of the ®ring pattern of substantia nigra dopaminergic neurons. Adv. Pharmac. 42, 694±699. 38. Tse Y. C. and Yung K. K. L. (2000) Cellular expression of ionotropic glutamate receptor subunits in subpopulations of neurons in the rat substantia nigra pars reticulata. Brain Res. 854, 57±69. 39. White J. H., Wise A., Main M. J., Green A., Fraser N. J., Disney G. H., Barnes A. A., Emson P., Foord S. M. and Marshall F. H. (1998) Heterodimerization is required for the formation of a functional GABAB receptor. Nature 396, 679±682.
76
T. K. Y. Ng and K. K. L. Yung
40. Yung K. K. L., Ng T. K. Y. and Wong C. K. C. (1999) Subpopulations of neurons in the rat neostriatum display GABABR1 receptor immunoreactivity. Brain Res. 830, 345±352. 41. Yung K. K. L. (1998) Localization of ionotropic and metabotropic glutamate receptors in distinct neuronal elements of the rat substantia nigra. Neurochem. Int. 33, 313±326. 42. Yung K. K. L., Kwok K. H. H., Gao Z. G., Choi S. Y. and Kwok F. S. (1999) Expression of GABA transaminase immunoreactivity in interneurons of the rat neostriatum. Neurochem. Int. 33, 567±572. 43. Yung K. K. L., Tse Y. C., Gao Z. G., Choi S. Y. and Kwok F. S. (1998) Localization of GABA transaminase immunoreactivity in the rat substantia nigra pars reticulata. Neurosci. Lett. 253, 57±60. 44. Yurek D. M. and Sladek J. R. (1990) Dopamine cell replacement: Parkinson's disease. A. Rev. Neurosci. 13, 415±440. (Accepted 28 March 2000)
Pergamon
PII: S0306-4522(00)00156-1
Neuroscience Vol. 99, No. 1, pp. 65±76, 2000 65 q 2000 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/00 $20.00+0.00
www.elsevier.com/locate/neuroscience
DISTINCT CELLULAR DISTRIBUTION OF GABABR1 AND GABAAa1 RECEPTOR IMMUNOREACTIVITY IN THE RAT SUBSTANTIA NIGRA T. K. Y. NG and K. K. L. YUNG* Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong, PR China
AbstractÐGABA is one of the most important inhibitory neurotransmitters in the substantia nigra. Functions of GABA are mediated by two major types of GABA receptors, namely the GABAA and GABAB receptors. Subunits of both the GABAA and GABAB receptors have been cloned and functional characteristics of the receptors depend on their subunit compositions. In order to characterize the cellular localization of GABABR1 and GABAAa1 subunit immunoreactivity in subpopulations of neurons in the rat substantia nigra, double and triple immuno¯uorescence was employed. Over 90% of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra pars compacta were found to display immunoreactivity for GABABR1. In contrast, immunoreactivity for GABAAa1 was found to be primarily displayed by neurons in the substantia nigra pars reticulata. Around 85% of the GABAAa1immunoreactive reticulata neurons were found to display parvalbumin immunoreactivity and some GABAAa1-positive reticulata neurons were found to be parvalbumin negative. In addition, triple-labeling experiments revealed that at the single cell level, the tyrosine hydroxylase-positive, i.e. the dopaminergic neurons in the compacta displayed intense immunoreactivity for GABABR1 but not GABAAa1 receptors. The parvalbumin-positive neurons in the reticulata displayed intense immunoreactivity for GABAAa1 but not GABABR1 receptors. The present results demonstrate in the same sections that there is a distinct pattern of localization of GABABR1 and GABAAa1 receptor immunoreactivity in different subpopulations of the rat substantia nigra and provide anatomical evidence for GABA neurotransmission in the subpopulations of nigral neurons. q 2000 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: basal ganglia, dopaminergic neurons, GABAergic neurons, parvalbumin, double and triple immuno¯uorescence, confocal microscopy.
Axonal terminals that contain the neurotransmitter GABA form inhibitory synapses to neurons of the substantia nigra (SN; for reviews see Refs 2, 11, 33 and 34). The GABAergic synaptic inputs to the SN, which mainly arise from the neostriatum and the globus pallidus, 2,11,33,34 form the two major pathways in the basal ganglia, and they also play important roles in motor regulation. The SN is divided into the substantia nigra pars compacta (SNc) which contains dopaminergic neurons and the substantia nigra pars reticulata (SNr) which mainly contains GABAergic neurons. 11 The dopaminergic neurons in the SNc provide an important feedback and regulation to neurons in the neostriatum and degeneration of the dopaminergic neurons results in Parkinson's disease. 11,44 The SNr is considered as one of the output nuclei of the basal ganglia, in which disinhibition of the neurons in the SNr form the basis for motor control in the basal ganglia (for a review see Ref. 11). The inhibitory response of GABA in the SN is mainly mediated by two classes of receptors, namely the GABAA and GABAB receptors. 9,25±27,31 The GABAB receptor is a metabotropic receptor 3,4 and is known to consist of at least two heteromeric subunits, namely, the GABABR1 and GABABR2 subunits. 18,20,21,39 Co-expression of these two subunits produces a fully functional GABAB receptor. 18,20,21,39 The GABABR1 can be further divided into its isoforms, i.e. GABABR1a and GABABR1b, that might be associated with
different neuronal elements, i.e. the presynaptic and postsynaptic elements, in the cerebellum. 1,9,19 Functionally, the GABAB receptors regulates the Ca 21 and/or K 1 channel conductance in the membranes. 3 The GABAA receptors however, are ionotropic receptors that are composed of pentameric subunits. A functional ion-channel of GABAA receptors is known to be composed of one a, three b and one g subunits. 23,24 The GABAA receptors are known to associate with membrane Cl ± channels. 23,24 Pharmacological and functional properties of the GABAA receptors are determined by the subunit compositions. 22±24 One of the most common combinations of functional GABAA receptors throughout the brain including the SN is the combination of a1, b2/3 and g2 subunits. 10 Previous physiological studies 5,6,8,13,28,29,31,36,37 suggest that there may be a functional segregation of GABAA and GABAB receptor-mediated responses in subpopulations of nigral neurons. Neurons in the SNr are tonically inhibited by GABA via the postsynaptic GABAA receptors but not the postsynaptic GABAB receptor. 6,31 Presynaptic GABAB receptor however, are suggested to have predominant roles in mediating GABA neurotransmission in the SNr. 5 In contrast, a robust GABAB response is found in the dopaminergic neurons in the SNc. 5,6,13,28,29,36,37 The GABA inhibition on the dopaminergic neurons that arises from the pallidal origin is mediated via GABAB receptors but those that arise from the reticulata neurons are suggested to mediate via GABAA receptors. 28,29,36,37 Immunoreactivity for GABABR1 receptor has been described in the rat brain with less reference to the cellular distribution in the SN. 9 A recent report has indicated that immunoreactivity for GABABR1 is intense in the SNc of monkeys. 7 This study, however, did not simultaneously provide data about the GABAA receptor localization in the
*To whom correspondence should be addressed. Tel.: 1 852-23397060; fax: 1 852-23395995. E-mail address: [email protected] (K. K. L. Yung). Abbreviations: IgG, immunoglobulin; NGS, normal goat serum; PB, phosphate buffer; PBS, phosphate-buffered saline; SN, substantia nigra; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata; TH, tyrosine hydroxylase. 65
66
T. K. Y. Ng and K. K. L. Yung
SN. There are therefore fewer anatomical data available so far to evaluate the previous physiological observations of GABAA and GABAB receptor-mediated responses in subpopulations of neurons in the SN. One important unanswered question is whether subpopulations of nigral neurons differentially express GABAA and GABAB receptor subunit immunoreactivity. In addition, previous studies in our laboratory have indicated that there is a differential localization of GABA transaminase immunoreactivity (a degradation enzyme of GABA) 43 as well as ionotropic and metabotropic glutamate receptor immunoreactivity 38,41 in distinct subpopulations of neurons in the rat SN, in particular in subpopulations of reticulata neurons that express strong immunoreactivity for parvalbumin or GABA transaminase. 38 It is therefore of interest to know whether there is a distinct pattern of localization of GABAA and GABAB receptor subunit immunoreactivity in different neuronal elements in the rat SN. The objectives of the present study were to identify and characterize the neurochemically known neuronal elements in the SN that displayed GABABR1 and GABAAa1 immunoreactivity. Subpopulations of neurons in the SN were characterized by their neurochemical markers, i.e. tyrosine hydroxylase (TH) for dopaminergic neurons in the SNc and parvalbumin for GABAergic neurons in the SNr. 38 Double and triple immuno¯uorescence experiments were employed. EXPERIMENTAL PROCEDURES
Animal and tissue preparation In the present study, six adult rats (Sprague±Dawley female, 150± 200 g, supplied by the University of Hong Kong) were used. The handling of rats and all procedures that were performed on them were approved in accordance with the Animals (Control of Experiments) Ordinance, Hong Kong, People's Republic of China, and all efforts were made by the authors to minimize the number of animals used and their suffering. The animals were deeply anesthetized with an overdose of sodium pentobarbital (60 mg/kg, i.p., Nembutal, Boehringer Mannheim). The animals were then perfused transcardially with 50± 100 ml of normal saline followed by 200 ml of ®xative. The ®xative consisted of 3% paraformaldehyde plus 0.01% glutaraldehyde in phosphate buffer (PB: 0.1 M, pH 7.4). The brains were removed and post®xed in 3% paraformaldehyde in PB at 48C for several hours. Coronal sections of the mesencephalon (60±70 mm) that contained the SN were cut under a vibrating microtome and collected in phosphate-buffered saline (PBS: 0.01 M, pH 7.4). The sections were then washed (3 £ PBS) and pre-incubated in normal goat serum (NGS; 4% in PBS) for 1 h at room temperature before proceeding to immunocytochemical procedures. Sources and preparation of antibodies Commercially available primary antibodies that were raised against GABAAa1 [rabbit polyclonal; 1: 2000 in 2% NGS and 0.1% triton X100 (PBS-Triton); Upstate Biotechnology, Lake Placid, NY, USA] and GABABR1 (guinea-pig polyclonal; 1:2000±1:5000 in PBS-Triton; Chemicon International, Temcula, CA, USA) receptors were obtained and used in the present study. The antibody against GABABR1 was raised against a common sequence to the two variants of the GABABR1 receptor, namely the GABABR1a and GABABR1b. The speci®city of the GABABR1 receptor antibody was biochemically characterized by our previous study. 40 Primary antibodies against parvalbumin (mouse monoclonal; 1:1000 in PBS-Triton; Sigma, St. Louis, MO, USA) and TH (rabbit polyclonal; 1:2000 in PBS-Triton and mouse monoclonal; 1:500 in PBS-Triton; Chemicon) were also used. Secondary antibodies were also obtained from commercial sources. Fluorochrome-conjugated secondary antibodies (Alexa 488-conjugated, Alexa 562-conjugated or cy-5-conjugated goat-anti-guinea-pig immunoglobulin (IgG), goat-anti-rabbit IgG or goat-anti-mouse IgG
in respect to the origins of the primary antibodies; Molecular Probes or Vector Labs.) were also used. Immuno¯uorescence Single and double immuno¯uorescence experiments were carried out as previously described. 38,40±42 Triple immuno¯uorescence experiments were also carried out similar to the double labeling experiments. In single labeling experiments, the nigral sections were incubated in one primary antibody. 38,40±43 In double labeling experiments, the sections were incubated in a mixture of primary antibodies, that were from different species of origins, overnight at room temperature. The GABAAa1 antibody (rabbit origin) was mixed either with parvalbumin antibody (mouse origin) or with TH antibody (mouse origin). The GABABR1 antibody (guinea-pig origin) was mixed either with parvalbumin antibody (rat origin) or with TH antibody (mouse or rabbit origin). In triple labeling experiments, GABAAa1 antibody (rabbit origin) and GABABR1 antibody (guinea-pig origin) were mixed either with parvalbumin antibody (rat origin) or with TH antibody (mouse origin). After incubation in one or in a mixture of primary antibodies, the sections were washed (3 £ PBS) and incubated in one or a mixture of secondary antibodies in respect to the origins of primary antibodies for 2 h at room temperature. The secondary antibodies were ¯uorochrome-conjugated antibodies as described above, e.g., Alexa 488-conjugated goat-anti-guinea-pig IgG, Alexa 562-conjugated goat-anti-rabbit IgG and cy5-conjugated goat-anti-mouse IgG (1:100 in PBS-Triton). Different combinations were used as described in previous studies. 38,40 After three washes (3 £ PBS), the sections were then mounted on a clean slide with mounting medium (Vectashield, Vector Labs.). The sections were then observed under a laser scanning confocal microscope (LSM 510, Zeiss with an argon±kryton laser and a helium±neon laser with three channels; excitation for Alexa 488: 488 nm; excitation for Alexa 562: 562 nm; excitation for cy-5: 688 nm). Digital images of the immunoreactive structures were captured and analysed. Controls for the single, double and triple immunocytochemistry were performed by omission of primary antibodies in turn of the mixtures in the reaction sequence. RESULTS
Confocal microscopic observations Control for immunocytochemistry. In the control sections in which the primary antibody was omitted during the single labeling procedures, no immunoreaction product was seen (data not shown). On those sections subjected to double or triple immuno¯uorescence, the two or three primary antibodies were omitted in turn during the double labeling procedures. When two primary antibodies were omitted, only one ¯uorescent labeling was observed (data not shown). Localization of GABABR1 and GABAAa 1 immunoreactivity. In the laser scanning confocal microscope, immunoreactivity for GABABR1 and GABAAa1 was identi®ed by immuno¯uorescent signal in the single-labeled sections (Fig. 1). Immunoreactivity for GABABR1 was primarily found in perikarya of the SNc. Some of the neurons in the SNr were found to display a low level of GABABR1 immunoreactivity (Fig. 1A). In contrast, immunoreactivity for GABAAa1 was primarily found in neurons in the SNr (Fig. 1B). Some of the neurons in the SNc were found to display a low level of GABAAa1 immunoreactivity (Fig. 1B). Double labeling of tyrosine hydroxylase and GABABR1. In the sections that were subjected to double labeling, immunoreactivity for TH was identi®ed by the presence of cy-5 (red) ¯uorescent signal. Immunoreactivity for TH was primarily
GABAB and GABAA receptors in rat substantia nigra
67
region, GABABR1-immunoreactive perikarya were also found (Fig. 2E). The TH-immunoreactive neurons were found to display a dense immunoreactivity for GABABR1 (Fig. 2F). Some GABABR1-immunoreactive neurons that were relatively lightly stained were found to be TH negative (Fig. 2F). Double labeling of parvalbumin and GABABR1. Similarly, immunoreactivity for parvalbumin was identi®ed in the confocal microscope by the presence of cy-5 (red) signal. Immunoreactivity for parvalbumin was primarily found in neurons in the SNr (Fig. 3A). Immunoreactive proximal dendrites were also seen (Fig. 3A). In the same sections, immunoreactivity for GABABR1 was identi®ed by the presence of Alexa 488 (green) ¯uorescence. As described as above, intense GABABR1 immunoreactivity was primarily detected in the SNc (Fig. 3B). These neurons were clearly seen to be parvalbumin negative in the superimposed image (Fig. 3C). In addition, most of the parvalbumin-immunoreactive perikarya in the SNc were found to display a lower level of GABABR1 immunoreactivity (Fig. 3C). At higher magni®cation, parvalbumin immunoreactivity was found in perikarya in the region that was at the boundary between the SNr and the SNc (Fig. 3D). Parvalbuminimmunoreactive punctuate structures were also seen (Fig. 3D). In the same sections, the parvalbumin-immunoreactive neurons were found to be GABABR1 immunoreactive (Fig. 3E±F). Moreover, some GABABR1-immunoreactive neurons were found to be parvalbumin negative (Fig. 3F).
Fig. 1. Fluorescent photomicrographs of the rat substantia nigra immunostained to reveal GABABR1 receptor (A) or GABAAa1 receptor (B) immunoreactivity. The immunoreaction product is indicated by the presence of immuno¯uorescent signal. In A, intense immunoreactivity for GABABR1 (GB) is primarily found in the region of the SNc. Intense labeling is observed in perikarya of compacta neurons. In B, intense immunoreactivity for GABAAa1 (GA) is primarily observed in the region of the SNr. Intense labeling is found in perikarya of reticulata neurons. Scale bar 200 mm (B; also applies to A).
found in perikarya in the SNc (Fig. 2A). TH-immunoreactive proximal dendrites were also seen (Fig. 2A). In the same sections, immunoreactivity for GABABR1 was identi®ed by the presence of Alexa 488 (green) ¯uorescent signal. Intense immunoreactivity for GABABR1 was found in perikarya of the SNc (Fig. 2B). Most of THimmunoreactive neurons in the SNc were found to be GABABR1 immunoreactive, as seen in a superimposition of the two images [neurons that were double labeled were identi®ed by the presence of orange±yellowish signal; Fig. 2C; 94.4 ^ 0.82%; number of neurons counted (n) 338]. The TH-immunoreactive neurons that were found in the caudal region of the SNr were also found to express a robust immunoreactivity for GABABR1 (data not shown). At higher magni®cation, densely labeled TH-immunoreactive perikarya were found at the boundary between the regions of the SNc and the SNr (Fig. 2D). In the same
Double labeling of tyrosine hydroxylase and GABAAa 1. Similar to Fig. 2, immunoreactivity for TH was primarily found in perikarya of the SNc (Fig. 4A; identi®ed by cy-5 signal). In the same sections, immunoreactivity for GABAAa1 was identi®ed by the presence of Alexa 488 (green) ¯uorescent signal (Fig. 4B). An intense level of GABAAa1 immunoreactivity was found in perikarya as well as in neuropilar elements in the SNr but not in the SNc (as indicated by the superimposed image; Fig. 4C). The TH-immunoreactive perikarya were also found to display a lower level of GABAAa1 immunoreactivity (Fig. 4C). At higher magni®cation, TH-immunoreactive perikarya and their proximal dendrites were clearly seen (Fig. 4D). The TH-immunoreactive perikarya were found to be GABAAa1 immunoreactive (Fig. 4E). In addition, intensely labeled GABAAa1-immunoreactive perikarya were found to be TH negative at the boundary between the SNc and the SNr (Fig. 4F). In the SNr, the GABAAa1-immunoreactive perikarya were found to be TH negative. Double-labeled TH- and GABAAa1-immunoreactive processes were seen among the single-labeled GABAAa1-immunoreactive neurons (Fig. 4G). Double labeling of parvalbumin and GABAAa 1. Similar to Fig. 3, immunoreactivity for parvalbumin was primarily found in perikarya of the SNr (Fig. 5A). The neurons were densely labeled (Fig. 5A, D). In the same sections, immunoreactivity for GABAAa1 was also found to primarily express in neurons of the SNr and they were seen to be densely labeled (Fig. 5B, E). Superimposed images revealed that most of the parvalbumin-immunoreactive
68
T. K. Y. Ng and K. K. L. Yung
Fig. 2. Fluorescent micrographs of the rat substantia nigra immunostained to reveal TH and GABABR1 immunoreactivity. The micrographs were obtained from the same sections. (A) Immunoreactivity for TH is indicated by Alexa 562 ¯uorescent signal. Immunoreactivity for TH is mainly found in perikarya of the SNc (the white arrow indicates the same neuron in B and C). (B) In the same section, immunoreactivity for GABA BR1 (GB) is indicated by Alexa 488 ¯uorescent signal. Intense immunoreactivity for GABABR1 is also primarily found in perikarya of the SNc. They are likely to co-express TH immunoreactivity (see the neuron indicated by the white arrow). Lightly stained GABABR1-immunoreactive neurons are also observed in the SNr. (C) A superimposed image of A and B is shown. Co-localization of TH and GABABR1 immunoreactivity is indicated by a combination of the two ¯uorescent signals. Almost all of the TH-immunoreactive neurons in the SNc are found to display intense immunoreactivity for GABABR1 (the neuron shown in A and B is indicated by the white arrow). (D) At higher magni®cation, at the region of the boundary between the SNc and the SNr, THimmunoreactive perikarya are seen (the white arrow indicated the same neuron in E and F). Two perikarya of TH-negative reticulata neurons are indicated by asterisks (also in E and F). (E) In the same section, GABABR1-immunoreactive perikarya are found. The TH-immunoreactive perikarya are found to display an intense GABABR1 immunoreactivity (the same neuron indicated in D is indicated by a white arrow). The lightly stained GABA BR1immunoreactive neurons are found to be TH-negative (the two in D are indicated by asterisks). GABABR1 immunoreactivity is also observed in the neuropil. (F) The superimposed image of D and E is shown. The TH-immunoreactive perikarya are found to display strong immunoreactivity for GABABR1 (indicated by the combined signal; the same neuron in D and E is indicated by a white arrow). The lightly labeled GABABR1-immunoreactive neurons do not display TH immunoreactivity (asterisks). Scale bars 100 mm (in C, for A and B), 50 mm (in F, for D and E).
GABAB and GABAA receptors in rat substantia nigra
69
Fig. 3. Fluorescent micrographs of the rat substantia nigra immunostained to reveal parvalbumin and GABABR1 (GB) immunoreactivity. The micrographs were obtained from the same sections. (A) Immunoreactivity for parvalbumin is indicated by Alexa 562 ¯uorescent signal. Immunoreactivity for parvalbumin is only observed in neurons and in the neuropil of the SNr (one parvalbumin-positive neuron is indicated by a white arrow, the same in B and C). (B) Immunoreactivity for GABABR1 is indicated by Alexa 488 ¯uorescent signal. Intense GABABR1 immunoreactivity is detected in neurons in the SNc. Neurons in the SNr also display pale immunoreactivity for GABA BR1 (the same neuron in A is indicated by a white arrow). (C) A superimposed image is shown. Intense immunoreactivity for GABABR1 is detected in perikarya of the SNc that are parvalbumin negative. The parvalbumin-immunoreactive perikarya in the SNr thus express a low level of GABABR1 immunoreactivity. (D) At higher magni®cation, a parvalbumin-immunoreactive neuron is shown (arrow). An asterisk indicates a neuron in the SNc that is parvalbumin negative (the two neurons are shown in E and F). (E) In the same section of D, two neurons that display immunoreactivity for GABABR1 are shown. Arrow indicates the GABABR1-immunoreactive neuron in the SNr that is also parvalbumin positive (as in D). Asterisk indicates the GABABR1-immunoreactive neuron in the SNc that is parvalbumin negative. (F) A superimposed image of D and E shows the neuron in the SNr which displays both GABABR1 and parvalbumin immunoreactivity. Scale bars 100 mm (in C, for A and B), 50 mm (in F, for D and E).
70
T. K. Y. Ng and K. K. L. Yung
Fig. 4.
GABAB and GABAA receptors in rat substantia nigra
perikarya in the SNr were found to be GABAAa1 immunoreactive (Fig. 5C, F; 85.3 ^ 1.56%; n 455). However, some neurons in the SNr displayed GABAAa1 immunoreactivity alone but not parvalbumin immunoreactivity (Fig. 5C, F; 14.7 ^ 1.26%; n 455). In contrast, a lower level of GABAAa1 immunoreactivity was found in neurons in the SNc (Fig. 5B, E). At higher magni®cation, parvalbumin-immunoreactive perikarya were densely labeled (Fig. 5G). Parvalbuminimmunoreactive punctuate processes were also seen (Fig. 5G). In the same sections, the parvalbumin-immunoreactive perikarya were found to display GABAAa1 immunoreactivity (Fig. 5H, I). In the boundary between the SNc and SNr, the parvalbumin-negative neurons in the SNc were also found to display a low intensity of GABAAa1 immunoreactivity (Fig. 5H, I). In addition, the single-labeled neurons in the SNr that displayed GABAAa1 immunoreactivity alone were seen to approach by parvalbumin- and GABAAa1-immunoreactive punctuated processes (Fig. 5J). Triple labeling of GABABR1 and GABAAa 1 with tyrosine hydroxylase or parvalbumin. Triple labeling experiments revealed that the TH-immunoreactive perikarya in the SNc displayed an intense immunoreactivity for GABABR1 (Fig. 6A, B). In the same section, neurons in the SNr (TH-negative) were found to display an intense immunoreactivity for GABAAa1 (Fig. 6B, C). A very low level of GABAAa1 immunoreactivity was detected in the TH-immunoreactive perikarya (Fig. 6A, C). In contrast, the parvalbumin-immunoreactive neurons in the SNr were found to display a very low level of GABABR1 immunoreactivity (Fig. 6D, E). The parvalbumin-negative neurons in the SNc were found, however, to display an intense level of GABABR1 immunoreactivity (Fig. 6D, E). In the same sections, the parvalbumin-immunoreactive reticulata neurons were found to display a very intense level of GABAAa1 immunoreactivity (Fig. 6D, F). DISCUSSION
The present study reports the cellular localization of GABABR1 and GABAAa1 immunoreactivity in the SN of the same rats and demonstrates for the ®rst time that immunoreactivity for GABABR1 and GABAAa1 is primarily expressed by different subpopulations of neurons in the SN. In the same sections of the SN, immunoreactivity for GABABR1 is primarily found in neurons of the SNc that co-express TH immunoreactivity. In contrast, immunoreactivity for GABAAa1 is primarily found in neurons of the SNr and most of the GABAAa1-immunoreactive neurons
71
have been found to be parvalbumin immunoreactive. The present results thus provide anatomical evidence and support the previous physiological observations 5,6,13,28,29,36,37 that the compacta neurons display a robust GABAB receptor-mediated response together with GABAA receptor-mediated response and the reticulata neurons display a primarily GABAA receptor-mediated response. 6,31 Immunoreactivity for GABABR1 receptor is primarily displayed by the dopaminergic neurons The present results indicate for the ®rst time that the dopaminergic neurons in the SNc of rats are the major groups of neurons that display GABABR1 immunoreactivity. The present results are consistent with the previous results found in monkeys. 7 The two results indicate that GABABR1 immunoreactivity is expressed in neuronal elements in the SN in similar patterns among different species. The primary antibody against the GABABR1 receptor employed in the present study is raised against a common domain of GABABR1a and GABABR1b isoforms. Results of previous studies indicate that the GABABR1a isoform is likely to be the dominant isoform of GABABR1 receptors found in the regions of the basal ganglia including the SN 9 as well as in the neostriatum. 9,40 The present results thus are likely to show that immunoreactivity for GABABR1a is predominantly found in the SN. One related point is that the present results, as those found in the previous study, 7 do not provide information about the localization of the other GABAB receptor subunit, i.e. the GABABR2 subunit in the SN. The present results thus do not directly re¯ect functional information about GABAB receptors in the dopaminergic neurons as a functional GABAB receptor is known to comprise GABABR1 and GABABR2 subunits. 18,20,21,39 However, the present results provide anatomical evidence for those previous studies that observe a robust GABAB receptor-mediated response in dopaminergic neurons. 5,29,37 In addition to the GABAB receptor, the compacta neurons have been found to express strong immunoreactivity for GABAAa3, a2 but not a1, and g2 subunits. 10 It is likely that the compacta neurons contain functional GABAA channels 28,29,36,37 that have different subunit compositions from those of the reticulata neurons (discussed below). Localization of GABABR1 immunoreactivity in the substantia nigra pars reticulata The present results con®rm that immunoreactivity for GABAB is also found in neuropilar elements in the SNr. A
Fig. 4. Fluorescent micrographs of the rat substantia nigra immunostained to reveal TH and GABAAa1 (GA) immunoreactivity. The micrographs were obtained from the same sections. (A) Similar to Fig. 2, immunoreactivity for TH is found in perikarya and in the neuropil of the SNc (Alexa 562 ¯uorescent signal). One TH-immunoreactive perikaryon is indicated by an arrow (the same neuron is also indicated in B and C). (B) Intense immunoreactivity for GABAAa1 is found in the SNr. Perikarya (two are indicated by a thick arrow) as well as neuropilar elements in the SNr are intensely labeled. Perikarya in the SNc are also found to display a lower level of GABAAa1 immunoreactivity (arrow). (C) A superimposed image is shown. Intense immunoreactivity for GABAAa1 is detected in the SNr (thick arrow). TH-immunoreactive perikarya in the SNc is found to display a low level of GABAAa1 immunoreactivity. (D) At higher magni®cation, a TH-immunoreactive perikaryon is seen in the SNc (one is indicated by an arrow). A THnegative neuron is also found (one is indicated by an asterisk; also in E and F). (E) The TH-negative neuron found in D (asterisk) is seen to display intense immunoreactivity for GABAAa1. Intensely labeled proximal dendrites are also seen. The TH-positive neuron shown in D also displays a lower level of GABAAa1 immunoreactivity (arrow). (F) The superimposed image shows that the TH-immunoreactive neuron shown in D and E displays a lower level of GABAAa1 immunoreactivity (arrow). (G) In the SNr, two intensely labeled GABAAa1-immunoreactive neurons are shown (thick arrow, as indicated in B and C). The neurons are seen to be surrounded by TH-immunoreactive dendrites (arrowheads). Scale bars 100 mm (in C, for A and B), 50 mm (in G, for D±F).
72
T. K. Y. Ng and K. K. L. Yung
Fig. 5.
GABAB and GABAA receptors in rat substantia nigra
previous electron microscopic study indicated that the GABABR1 immunoreactivity was expressed by presynaptic neuronal elements in the SNr of monkeys and the GABABR1 immunoreactivity was found almost exclusively in terminals that formed asymmetrical synapses. 7 A controversial question is raised as most of the physiological studies indicated that the robust GABAB responses in the SNr were found in GABAergic axonal elements. 5,6,28,36,37 Postsynaptic GABAB responses in the SNr are known to be much weaker than presynaptic responses. 5,6 Neurons in the SNr of monkeys were found to express a low level of GABABR1 immunoreactivity. 7 The present results also indicated that the reticulata neurons of rats, albeit less, expressed a low level of GABABR1 immunoreactivity. A recent physiological study demonstrates that GABAB receptors induce a modulation of Ca 21 current but not directly on the K 1 current in neurons of the globus pallidus. 35 These results indicate that there may be a variety of GABABmediated responses in neurons including neurons in the basal ganglia, in which the previous patch clamp studies 5,6 may have only characterized a few of those responses. The cellular expressions of GABABR1, as well as other functional GABAB subunits, in the reticulata neurons therefore await further investigation. The present results thus hopefully open up an insight to further investigate the GABAB functions in the SNr. Subpopulations of nigral neurons display GABAAa 1 immunoreactivity The present ®ndings are consistent with previous ®ndings 10 that immunoreactivity for GABAAa1 is mainly found in the subgroups of neurons in the reticulata. The reticulata neurons are also found to display immunoreactivity for GABAAb2/3 and GABAAg2. 10,25 The neurons are therefore likely to display functional GABAA channels 6 with the above subunit compositions. The present observation extends the previous ®ndings that most of the GABAAa1-immunoreactive neurons in the SNr are in fact parvalbumin immunoreactive. As shown by previous reports, 12,15,16,38 parvalbumin-immunoreactive neurons are a subpopulation of the reticulata neurons and they are suggested to be the tonically active output neurons in the output pathways of the basal ganglia. 30 They are found to display a robust immunoreactivity for GABA
73
transaminase 38,43 and most of the ionotropic glutamate receptor subunits. 38 The parvalbumin-positive subpopulations of reticulata neurons are thus further proven to primarily display the GABAAa1 subunit, and likely to display functional GABAA channels with the a1, b2/3 and g6 subunit compositions. 10,25 These results, as a whole, further demonstrate that there may be a compartmentalization of glutamate and GABA interactions (and GABA metabolism) in subpopulations of reticulata neurons that display strong immunoreactivity for parvalbumin. 38 The parvalbuminimmunoreactive neurons are known to degenerate in a late stage of Parkinson's disease. 12 Recent studies in other regions of the brain indicate that blockage of GABAA receptor channels may have a neuroprotective effect on the vulnerability of the neurons towards glutamate excitotoxicity. 14,17,32 The present results thus illustrate the anatomical evidence for a possible functional interaction between the GABAergic and the glutamatergic systems in subpopulations of neurons in the SNr. The present study directly demonstrates at the cellular level that subpopulations of nigral neurons display different GABA receptor subunits. The compacta neurons primarily express GABABR1 immunoreactivity, whereas the reticulata neurons primarily express GABAAa1 immunoreactivity. The ®ndings provide anatomical evidence for a possible functional segregation of GABA functions in the rat SN as indicated by previous physiological studies. 5,6,13,28,29,36,37 Although the patterns of distribution of GABABR1 and GABAAa1 are quite distinct among the compacta as well as the reticulata neurons, the present results also indicate that the pattern is not exclusive. There are low levels of expression of GABAAa1 or GABABR1 immunoreactivity in the compacta and reticulata neurons, respectively. The exact functional implications of functional segregation of GABA receptors in different compartments of the rat SN and their roles in the circuitry in the basal ganglia await further investigation.
AcknowledgementsÐThe present work was supported by Faculty Research Grant FRG/99-00/II-29, Hong Kong Baptist University and Research Grant Council, Hong Kong. T. K. Y. Ng was supported by a postgraduate studentship, Hong Kong Baptist University. The authors would like to thank Dr W. H. Yung for critical comments. The authors would also like to thank Miss L. Y. Man for technical assistance.
Fig. 5. Fluorescent micrographs of the rat substantia nigra immunostained to reveal parvalbumin and GABAAa1 (GA) immunoreactivity. The micrographs were obtained from the same sections. (A) Similar to Fig. 3, immunoreactivity for parvalbumin is found in perikarya of the SNr (Alexa 562 signal; SNr). (B) Intense immunoreactivity for GABAAa1 is primarily detected in perikarya of the SNr (Alexa 488 signal). (C) A superimposed image of A and B is shown. Most of the parvalbumin-immunoreactive neurons are also found to display GABAAa1 immunoreactivity (combined ¯uorescent signal). However, some neurons in the SNr (one is indicated by a white arrow) are found to display GABAAa1 immunoreactivity only. (D) At the boundary between the SNr and the SNc, parvalbumin-immunoreactive perikarya are seen in the SNr (one is indicated by a white arrow). (E) Intense immunoreactivity for GABAAa1 is shown in perikarya of the SNr (the same neuron shown in D is also indicated by a white arrow). Perikarya in the SNc are found to display a lower level of GABAAa1 immunoreactivity. (F) A superimposed image is shown. Most of the parvalbumin-positive neurons in the SNr are found to display GABAAa1 immunoreactivity (combined signal; white arrow). (G) At higher magni®cation, one parvalbumin-immunoreactive neuron in the SNr is shown (arrow). One parvalbuminnegative neuron in the SNc is also shown (asterisk). (H) The neuron in G is seen to display intense immunoreactivity for GABAAa1 (arrow). The parvalbuminnegative neuron in G is also found to display a lower level of GABAAa1 immunoreactivity (asterisk). (I) A superimposed image of G and H shows the parvalbumin-immunoreactive neuron is seen to display intense immunoreactivity for GABAAa1 (combined signal; arrow). (J) In the SNr, the GABAAa1immunoreactive neuron shown in C (arrow) is shown at higher magni®cation. The neuron is seen to display GABAAa1 immunoreactivity only without PV immunoreactivity (arrow and star). The other neurons are seen to be double labeled. Scale bars 200 mm (in C, for A and B), 100 mm (in F, for D and E), 50 mm (in I and J, for G and H).
74
T. K. Y. Ng and K. K. L. Yung
Fig. 6. Fluorescent micrographs of the rat SN triple labeled to reveal TH or parvalbumin and GABAAa1 (GA) and GABABR1 (GB) immunoreactivity. The micrographs were obtained from the same sections. (A) At the boundary of the SNc and the SNr, TH-immunoreactive perikarya are seen (Alexa 562 signal; two are indicated by asterisks). One TH-negative perikaryon in the SNr is also shown (star). (B) Intense immunoreactivity for GABABR1 is found in THimmunoreactive neurons in the SNc (Alexa 488 signal). A TH-negative neuron is seen to display a lower level of GABABR1 immunoreactivity (star). (C) In the same section, the TH-negative neuron in the SNr is found to display intense level of GABAAa1 immunoreactivity (cy-5 combined ¯uorescent signal; star). Intensely labeled neuropilar elements are also seen. The TH-positive neurons, however, are seen to display less GABAAa1 immunoreactivity (asterisks). (D) Also at the boundary of the SNc and the SNr, parvalbumin-immunoreactive perikaryon is seen in the SNr (star). Three parvalbumin-negative perikarya in the SNc are also shown (asterisks). (E) The parvalbumin-negative neurons shown in D is seen to display intense immunoreactivity for GABABR1 (asterisks). The parvalbumin-positive neuron in the SNr is also found to display a low level of GABABR1 immunoreactivity (star). (F) In the same section, the parvalbumin-positive neuron in the SNr is seen to display intense immunoreactivity for GABAAa1 (cy-5 combined signal; star). The intensely labeled GABABR1immunoreactive neurons in the SNc are found to display a low level of GABAAa1 immunoreactivity (asterisks). Scale bar 50 mm (in F, for A±E).
REFERENCES
1. Billinton A., Upton N. and Bowery N. G. (1999) GABAB receptor isoforms GBR1a and GBR1b, appear to be associated with pre- and post-synaptic elements respectively in rat and human cerebellum. Br. J. Pharmac. 126, 1387±1392.
GABAB and GABAA receptors in rat substantia nigra
75
2. Bolam J. P. and Bennett B. D. (1995) Microcircuitry of the neostriatum. In Molecular and Cellular Mechanisms of Neostriatal Function (eds Ariano M. A. and Surmeier P. J.). R. G. Landes, Austin. 3. Bowery N. G. (1993) GABAB receptor pharmacology. A. Rev. Pharmac. Toxicol. 33, 109±147. 4. Bowery N. G., Hill D. R. and Hudson A. L. (1997) Characteristics of GABAB receptor binding sites on rat whole brain synaptic membranes. 1983. Br. J. Pharmac. 120, 452±467. 5. Chan P. K., Leung C. K. and Yung W. H. (1998) Differential expression of pre- and postsynaptic GABAB receptors in rat substantia nigra pars reticulata neurones. Eur. J. Pharmac. 349, 187±197. 6. Chan P. K. and Yung W. H. (1999) Inhibitory postsynaptic currents of rat substantia nigra pars reticulata neurons: role of GABA receptors and GABA uptake. Brain Res. 838, 18±26. 7. Charara A., Heilman C., Levey A. I. and Smith Y. (2000) Pre- and post-synaptic localization of GABAB receptors in the basal ganglia in monkeys. Neuroscience 95, 127±140. 8. Floran B., Silva I., Nava C. and Aceves J. (1988) Presynaptic modulation of the release of GABA by GABAA receptors in pars compacta and by GABAB receptors in pars reticulata of the rat substantia nigra. Eur. J. Pharmac. 150, 277±286. 9. Fritschy J. M., Meskenaite V., Weinmann O., Honer M., Benke D. and Mohler H. (1999) GABAB receptor splice variants GB1a and GB1b in rat brain: developmental regulation, cellular distribution and extrasynaptic localization. Eur. J. Neurosci. 11, 761±768. 10. Fritschy J. M. and Mohler H. (1995) GABAA-receptor heterogeneity in the adult rat brain: differential regional and cellular distribution of seven major subunits. J. comp. Neurol. 359, 154±194. 11. Gerfen C. R. and Wilson C. J. (1996) The basal ganglia. In Handbook of Chemical Neuroanatomy. Integrated Systems of the CNS Part 3 (eds Swanson L. W., BjoÈrklund A. and HoÈlfelt T.), Vol. 12, pp. 371±468. Elsevier, Amsterdam. 12. Hardman C. D., McRitchie D. A., Halliday G. M., Cartwright H. R. and Morris J. G. (1996) Substantia nigra pars reticulata neurons in Parkinson's disease. Neurodegeneration 5, 49±55. 13. Hausser M. A. and Yung W. H. (1994) Inhibitory synaptic potentials in guinea-pig substantia nigra dopamine neurones in vitro. J. Physiol., Lond. 479 (Pt 3), 401±422. 14. Hollrigel G. S., Toth K. and Soltesz I. (1996) Neuroprotection by propofol in acute mechanical injury: role of GABAergic inhibition. J. Neurophysiol. 76, 2412±2422. 15. Hontanilla B., Parent A., de las H. S. and Gimenez-Amaya J. M. (1998) Distribution of calbindin D-28k and parvalbumin neurons and ®bers in the rat basal ganglia. Brain Res. Bull. 47, 107±116. 16. Hontanilla B., Parent A. and Gimenez-Amaya J. M. (1997) Parvalbumin and calbindin D-28k in the entopeduncular nucleus, subthalamic nucleus, and substantia nigra of the rat as revealed by double-immunohistochemical methods. Synapse 25, 359±367. 17. Ingle®eld J. R., Wilson C. A. and Schwartz-Bloom R. D. (1997) Effect of transient cerebral ischemia on gamma-aminobutyric acidA receptor alpha 1subunit-immunoreactive interneurons in the gerbil CA1 hippocampus. Hippocampus 7, 511±523. 18. Jones K. A., Borowsky B., Tamm J. A., Craig D. A., Durkin M. M., Dai M., Yao W. J., Johnson M., Gunwaldsen C., Huang L. Y., Tang C., Shen Q., Salon J. A., Morse K., Laz T., Smith K. E., Nagarathnam D., Noble S. A., Branchek T. A. and Gerald C. (1998) GABAB receptors function as a heteromeric assembly of the subunits GABABR1 and GABABR2. Nature 396, 674±679. 19. 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. (1997) Expression cloning of GABAB receptors uncovers similarity to metabotropic glutamate receptors. Nature 386, 239±246. 20. Kaupmann K., Malitschek B., Schuler V., Heid J., Froestl W., Beck P., Mosbacher J., Bischoff S., Kulik A., Shigemoto R., Karschin A. and Bettler B. (1998) GABAB receptor subtypes assemble into functional heteromeric complexes. Nature 396, 683±687. 21. Kuner R., Kohr G., Grunewald S., Eisenhardt G., Bach A. and Kornau H. C. (1999) Role of heteromer formation in GABAB receptor function. Science 283, 74±77. 22. Mohler H., Benke D., Mertens S. and Fritschy J. M. (1992) GABAA-receptor subtypes differing in alpha-subunit composition display unique pharmacological properties. Adv. Biochem. Psychopharmac. 47, 41±53. 23. Mohler H., Fritschy J. M., Luscher B., Rudolph U., Benson J. and Benke D. (1996) The GABAA receptors. From subunits to diverse functions. Ion Channels 4, 89±113. 24. Mohler H., Kno¯ach F., Paysan J., Motejlek K., Benke D., Luscher B. and Fritschy J. M. (1995) Heterogeneity of GABAA-receptors: cell-speci®c expression, pharmacology, and regulation. Neurochem. Res. 20, 631±636. 25. Nicholson L. F., Faull R. L., Waldvogel H. J. and Dragunow M. (1992) The regional, cellular and subcellular localization of GABAA/benzodiazepine receptors in the substantia nigra of the rat. Neuroscience 50, 355±370. 26. Nicholson L. F., Faull R. L., Waldvogel H. J. and Dragunow M. (1995) GABA and GABAA receptor changes in the substantia nigra of the rat following quinolinic acid lesions in the striatum closely resemble Huntington's disease. Neuroscience 66, 507±521. 27. Nicholson L. F., Waldvogel H. J. and Faull R. L. (1996) GABAA receptor subtype changes in the substantia nigra of the rat following quinolinate lesions in the striatum: a correlative in situ hybridization and immunohistochemical study. Neuroscience 74, 89±98. 28. Paladini C. A., Celada P. and Tepper J. M. (1999) Striatal, pallidal, and pars reticulata evoked inhibition of nigrostriatal dopaminergic neurons is mediated by GABA(A) receptors in vivo. Neuroscience 89, 799±812. 29. Paladini C. A. and Tepper J. M. (1999) GABA(A) and GABA(B) antagonists differentially affect the ®ring pattern of substantia nigra dopaminergic neurons in vivo. Synapse 32, 165±176. 30. Rajakumar N., Elisevich K. and Flumerfelt B. A. (1994) Parvalbumin-containing GABAergic neurons in the basal ganglia output system of the rat. J. comp. Neurol. 350, 324±336. 31. Rick C. E. and Lacey M. G. (1994) Rat substantia nigra pars reticulata neurones are tonically inhibited via GABAA, but not GABAB, receptors in vitro. Brain Res. 659, 133±137. 32. Schwartz R. D., Yu X., Katzman M. R., Hayden-Hixson D. M. and Perry J. M. (1995) Diazepam, given postischemia, protects selectively vulnerable neurons in the rat hippocampus and striatum. J. Neurosci. 15, 529±539. 33. Smith A. D. and Bolam J. P. (1990) The neural network of the basal ganglia as revealed by the study of synaptic connections of identi®ed neurones. Trends Neurosci. 13, 259±265. 34. Smith Y., Bevan M. D., Shink E. and Bolam J. P. (1998) Microcircuitry of the direct and indirect pathways of the basal ganglia. Neuroscience 86, 353±387. 35. Stefani A., Spadoni F., Giacomini P., Lavaroni F. and Bernardi G. (1999) The modulation of calcium current by GABA metabotropic receptors in a subpopulation of pallidal neurons. Eur. J. Neurosci. 11, 3995±4005. 36. Tepper J. M., Martin L. P. and Anderson D. R. (1995) GABAA receptor-mediated inhibition of rat substantia nigra dopaminergic neurons by pars reticulata projection neurons. J. Neurosci. 15, 3092±3103. 37. Tepper J. M., Paladini C. A. and Celada P. (1998) GABAergic control of the ®ring pattern of substantia nigra dopaminergic neurons. Adv. Pharmac. 42, 694±699. 38. Tse Y. C. and Yung K. K. L. (2000) Cellular expression of ionotropic glutamate receptor subunits in subpopulations of neurons in the rat substantia nigra pars reticulata. Brain Res. 854, 57±69. 39. White J. H., Wise A., Main M. J., Green A., Fraser N. J., Disney G. H., Barnes A. A., Emson P., Foord S. M. and Marshall F. H. (1998) Heterodimerization is required for the formation of a functional GABAB receptor. Nature 396, 679±682.
76
T. K. Y. Ng and K. K. L. Yung
40. Yung K. K. L., Ng T. K. Y. and Wong C. K. C. (1999) Subpopulations of neurons in the rat neostriatum display GABABR1 receptor immunoreactivity. Brain Res. 830, 345±352. 41. Yung K. K. L. (1998) Localization of ionotropic and metabotropic glutamate receptors in distinct neuronal elements of the rat substantia nigra. Neurochem. Int. 33, 313±326. 42. Yung K. K. L., Kwok K. H. H., Gao Z. G., Choi S. Y. and Kwok F. S. (1999) Expression of GABA transaminase immunoreactivity in interneurons of the rat neostriatum. Neurochem. Int. 33, 567±572. 43. Yung K. K. L., Tse Y. C., Gao Z. G., Choi S. Y. and Kwok F. S. (1998) Localization of GABA transaminase immunoreactivity in the rat substantia nigra pars reticulata. Neurosci. Lett. 253, 57±60. 44. Yurek D. M. and Sladek J. R. (1990) Dopamine cell replacement: Parkinson's disease. A. Rev. Neurosci. 13, 415±440. (Accepted 28 March 2000)