Morphology and neurochemistry of two striatal neuronal subtypes expressing the GABAA receptor α3-subunit in the rat

Brain Research 876 (2000) 124–130 www.elsevier.com / locate / bres

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Morphology and neurochemistry of two striatal neuronal subtypes expressing the GABAA receptor a 3-subunit in the rat a, *, Jose´ Luis Labandeira-Garcıa ˜ a, ´ ´ a , Ana Munoz Jannette Rodrıguez-Pallares b ´ Hector J. Caruncho a

Department of Morphological Sciences, University of Santiago de Compostela, 15705 Santiago de Compostela, Galicia, Spain b Department of Fundamental Biology, University of Santiago de Compostela, 15706 Santiago de Compostela, Galicia, Spain Accepted 20 June 2000

Abstract The morphological characteristics, distribution and neurochemical phenotype of striatal neuronal subtypes expressing the GABAA receptor a 3-subunit were investigated using immunocytochemical and immunofluorescent techniques with an antibody specific for this subunit. a 3-immunopositive neurons were infrequent in the rat striatum, but two morphologically different subtypes were observed: Cholinergic neurons, and a second cellular type that may correspond to neurogliaform neurons, although it may also be a novel subtype of striatal interneuron. To identify the second cellular subtype, co-localization of the GABAA receptor a 3-subunit with markers of different classes of striatal interneurons was studied using specific antibodies. It was found that there was a lack of co-localization between all interneuronal markers used in this study and the a 3-subunit. Although the a 3-subunit immunopositive neurons represent a small percentage of the total of striatal neuronal populations, they may play an important role in the regulation of the microcircuitry of the striatum.  2000 Elsevier Science B.V. All rights reserved. Keywords: Rat; Cholinergic neurons; Striatal interneurons; GABAergic transmission

1. Introduction The neostriatum contains two types of GABAergic neurons: Medium-sized neurons with spiny dendrites and a large number of axon collaterals within the striatum [29] (this type accounts for over 90% of striatal neurons), and GABAergic interneurons that can be divided in two groups according to the presence of parvalbumin or calretinin. There are also some striatal interneurons that contain the neuropeptide somatostatin, but it is unclear whether or not they represent a third class of GABAergic interneuron [10,15,17]. In addition to the intrinsic GABAergic innervation, the striatum also receives GABAergic afferents from the globus pallidus and the substantia nigra [16,26]. The striatum also contains cholinergic interneurons and a novel subtype of interneurons characterized by their immunoreactivity for the GABAA receptor a 1-subunit that has been recently described [4,25,31]. Finally, the existence of *Corresponding author. Tel.: 134-981-563-100; fax: 134-981-547078. ´ E-mail address: [email protected] (J. Rodrıguez-Pallares).

dwarf neurogliaform interneurons in the striatum, first ´ y Cajal [24] has also been recently proposed by Ramon confirmed by Sancesario et al. [27]. The action of GABA is mediated by GABAA and GABA B receptors. GABAA receptors are pentameric complexes associated with an intrinsic chloride ion-selective channel. Multiple subunits assemble to form these receptors, whose physiological and pharmacological properties depend on their composition [9]. To date, seven classes of GABAA receptor subunit genes have been identified, encoding at least 18 different subunits: a1 – 6 , b1 – 3 , g1 – 3 , d, ´, p and r1 – 3 [21,28,30]. In situ hybridization and immunocytochemical studies [4,5,11,23,32] have shown that a 2, a 4 and b 2 / 3 are the most abundant subunits in the rat striatum, while g 2, and d are less abundant, and other subunits such as a 1 and a 3 are only weakly expressed. There is an interest in characterizing the morphology and neurochemistry of striatal neurons that are immunopositive for different GABAA receptor subunits, in order to obtain a better knowledge of GABAergic mechanisms in the striatum, and their putative roles in neurodegenerative

0006-8993 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )02636-6

´ et al. / Brain Research 876 (2000) 124 – 130 J. Rodrıguez-Pallares

diseases [6,7,20]. Although the GABAA receptor a 3subunit expression is weak in rat striatum [5,12] we have found some scattered a 3-immunopositive cells. In the present study, we used an a 3-specific antibody to characterize the morphology and localization of these cells. In addition, we used double immunolabeling approaches to investigate the possible co-localization of the a 3-subunit with other markers such as: antibodies for DARPP-32 (a phosphoprotein present in striatal projection neurons, but absent from most striatal interneurons [2]), choline acetyltransferase (ChAT), an antibody specific for GABA, GABAA receptor a 1-subunit and neuronal-specific nuclear protein (NeuN).

2. Materials and methods Six adult female Sprague–Dawley rats (all weighting about 200 g at the beginning of the experiment; Letica, Barcelona, Spain) were used to characterize the morphology and neurochemistry of GABAA receptor a 3-subunit immunopositive cells in the striatum.

2.1. Immunocytochemistry Rats were anesthetized with chloral hydrate (400 mg / kg, i.p.) and perfused transcardially with a solution of 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Brains were carefully removed, cryoprotected in the same buffer containing 20% sucrose and sectioned with a freezing microtome at 40 mm thickness for single-immunocytochemical procedures, and at 20 mm thickness for double immunolabeling. Sections including the striatum were then processed for GABAA receptor a 3-subunit, DARPP-32, ChAT, GABAA receptor a 1-subunit, GABA and NeuN immunostaining as follows: Single-immunocytochemical procedures. Samples were pre-incubated with a solution containing 5% normal serum in 0.02 M phosphate buffered saline with 1% bovine serum albumin (PBS-BSA) and 0.3% Triton X-100 (Sigma). Sections were incubated overnight at room temperature with the corresponding primary antibody diluted in PBS¨ BSA (this antibody, kindly provided by Dr. H. Mohler, was a guinea-pig polyclonal antibody specific for the GABAA receptor a 3-subunit used at 1:15000, containing 1% normal serum and 0.3% Triton X-100 [11]). Sections were then washed and incubated for 90 min with a biotinylated secondary antibody diluted 1:100 (goat antiguinea-pig (Jackson Immunoresearch, USA) and then for another 90 min with avidin–biotin-peroxidase complex (ABC, Vector; 1:100). Finally, the labeling was revealed by using 0.05% 3,39-diaminobenzidine (DAB, Sigma) and 0.04% hydrogen peroxide. Double-immunocytochemical procedures. Selected sections were processed for double immunostaining [18] of a 3-subunit and DARPP-32, ChAT, GABA, GABAA receptor a 1-subunit, or NeuN to study the possible co-

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localization of the a 3-subunit with other markers. The a 3-subunit was firstly labeled as above, using DAB-nickel sulphate to give a black staining and then, after several washes in PBS, sections were processed for the other markers using the immunoperoxidase technique (PAP). Briefly, sections were incubated overnight at 48C with the corresponding primary antibody diluted in PBS-BSA (mouse monoclonal antibodies anti-ChAT (Chemicon), or anti-NeuN (Chemicon), or anti-DARPP-32 (kindly provided by Drs. Greengard and Gustafson), and rabbit polyclonal antibodies specific for GABAA receptor a 1¨ subunit (a gift from Dr. H. Mohler) and for GABA (Sigma). The antibodies were used at the following dilutions: 1:20000 for a 1 and DARPP-32; 1:10000 for GABA; 1:1000 for NeuN; and 1:200 for ChAT. After rinsing with PBS, sections were incubated for 150 min with the corresponding secondary antibody diluted 1:50 (swine anti-rabbit for a 1, and GABA (DAKO), and goat anti-mouse for ChAT, NeuN and DARPP-32 (Sigma) and then for another 150 min with peroxidase–anti-peroxidase [PAP anti-rabbit (DAKO), and PAP anti-mouse (Sigma); 1:100]. The immunostaining was carried out with DAB without nickel sulphate as a chromogen to obtain a brown precipitate. Control sections where primary antibodies were omitted showed a lack of immunostaining. Sections were also processed for double-immunofluorescent labeling. Samples were preincubated for 60 min with a solution containing 5% normal serum and 2% polyoxyethylenesorbitan monolaurate (Tween 20, Sigma) diluted in PBS-BSA. Then, sections were incubated overnight at 48C with a mixture of the antibody for the GABAA receptor a 3-subunit and the corresponding primary antibody (i.e., anti-DARPP-32, or anti-ChAT, or anti-NeuN, or an antibody specific for GABAA receptor a 1-subunit, or anti-GABA; see above) diluted in PBS-BSA at double concentration. After several rinses, sections were incubated for 3 h in the corresponding secondary antibody diluted 1:10 (rhodamine-conjugated goat anti-guinea pig IgG (Chemicon), fluorescein-conjugated goat anti-mouse IgG (Chemicon) and fluorescein-conjugated swine anti-rabbit IgG (DAKO). After rinsing with PBS, sections were mounted onto gelatinized slides, air-drier, coverslipped with a solution of glycerol, gelatine and DABCO (25 mg / ml, Sigma) and examined under fluorescence microscopy by using appropiate filters. A drawing reconstruction of the neurons was made using a Nikon Optiphot 2 microscope equipped with a drawing tube.

3. Results

3.1. GABAA receptor a3 -subunit immunoreactive cells Our results showed a few a 3-immunoreactive (a 3-ir) cells scattered throughout the rat striatum (Fig. 1A). Two

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Fig. 1. Immunolabeling of the GABAA receptor a 3-subunit in the rat striatum. (A) Low magnification micrograph showing scattered a 3-ir neurons (arrowheads). Note that most neurons are located close to the striatal fiber bundles. (B) Type 1 a 3-ir neuron. Both the soma and processes are immunopositive. Areas of high level of immunolabeling ‘hot spots’ are clearly evident (see arrows). (C) Type 2 a 3-ir cells (arrows). Note their close association to fiber bundles and also the presence of ‘hot spots’. (D) Large (type 1) a 3-ir neuron (asterisk) and small (type 2) a 3-ir neuron (arrow). Note that some processes of type 1 neuron are seen nearby the soma of the type 2 neuron. Calibration bar: (A) 500 mm; (B) 50 mm; (C) and (D) 20 mm.

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subtypes of a 3-subunit immunopositive cells were identified on the basis of their morphological characteristics: Type 1: These neurons were observed throughout the striatum, although they were particularly abundant in the ventral striatum. They were large neurons with oval, triangular or polygonal somata (20–35 mm in diameter) and widespread dendritic trees. The dendrites (2 to 5), originating mainly from the poles of the cell, were long and lacking in spiny projections, with few branching points (Fig. 1B). The immunoreactivity for a 3-subunit in this cell type was seen throughout the somata and dendrites, clearly associated in some cases with punctate labeling of the plasma membrane (hot spots). Type 2: The second a 3-immunopositive cell subtype was observed throughout the striatum, although generally close to the prominent myelinated fiber bundles of the internal capsule (Fig. 1C). These cells were small with a rounded (5–10 mm in diameter) or oval (5–10 mm in mean diameter) soma, bearing numerous short and thin processes. These processes (2 to 6 per cell) were smooth or slightly beaded, and profusely branched close to the cell body. Interestingly, the somata of these cells were usually located close to dendrites of type 1 a 3-ir neurons (Fig. 1D). These cells also showed punctate labeling for the a 3-subunit in the plasma membrane of the somata and processes. A drawing reconstruction of these two a 3-ir neuronal subtypes is shown in Fig. 2.

3.2. Neurochemical characteristics In order to further characterize the neurochemistry of

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these two a 3-immunopositive neuronal subtypes, we carried out double immunolabeling of a 3 and DARPP-32, ChAT, GABA, the GABAA receptor a 1-subunit and NeuN. The co-staining of a 3-subunit and DARPP-32 revealed that none of the two a 3-immunopositive cell populations described above were positive for the phosphoprotein DARPP-32 that was shown in striatal projection neurons. Experiments using both anti-a 3 and anti-ChAT antibodies indicated that these two cytochemical markers were co-localized in almost all type 1 a 3-positive neurons. Neurons that were immunopositive for these two proteins constituted over 90% of morphological type 1, whereas the remaining 10% were not immunoreactive for ChAT (Figs. 3A–F). The second type of a 3-ir neurons was always ChAT-negative. Interestingly, all ChAT-positive neurons showed immunopositivity for a 3. Double immunolabeling using specific antibodies for a 3-subunit and GABA showed that most a 3-ir cells presented a lack of co-localization for these markers, with the exception of a small population of type 1 a 3-ir neurons (less than 5%) that showed co-localization for a 3 and GABA. To verify if a 3-ir cells type 2 were of the same neuronal type as striatal interneurons immunopositive for the GABAA receptor a 1-subunit, we used double immunolabeling for a 3 and a 1-subunits, which also showed a lack of co-localization for these two markers (Fig. 3G). In addition, NeuN and a 3-subunit immunolabeling revealed that these two markers were observed in the same cellular populations (Fig. 3H), with the exception of a few cells inside the striatum fiber bundles that were a 3-positive but negative for NeuN.

4. Discussion

Fig. 2. A schematic drawing reconstruction of rat striatal GABAA receptor a 3-subunit immunopositive neurons. Type 1 a 3-ir neuron is represented in (B), and type 2 a 3-ir neuron is shown in (A).

In this study we used immunocytochemical and immunofluorescent techniques to study the morphology, distribution and neurochemical phenotype of cells that are immunoreactive for the GABAA receptor a 3-subunit in the rat striatum. Our results confirmed previous immunocytochemical studies that have described a weak immunoreactivity for the GABAA receptor a 3-subunit in the rat striatum [5,12]. These results are also in accordance with other in situ hybridization studies [23,32], which also revealed that a 3-mRNA is weakly expressed in the striatum. In the present study, we observed some scattered a 3-ir cells in the rat striatum that could be grouped into two different morphological types: Both types were immunonegative for the phosphoprotein DARPP-32, which has been described as a marker of most striatal projection neurons [2], so these a 3-ir cells may correspond to some subtype of striatal interneurons or well represent a subclass of glial cells. Our results demonstrated that most type 1

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Fig. 3. Double immunofluorescence for GABAA receptor a 3-subunit and ChAT (A)–(F) and double immunocytochemistry for GABAA receptor a 3-subunit and different neurochemical markers (G) and (H). a 3-positive neurons (red: (A), (C), (E)) are also immunolabeled for ChAT (green: (B), (D), (F)). Different neuronal morphologies are shown: oval somata ((A), (B); see Fig. 1B) and triangular or polygonal somata ((C)–(F); see Fig. 1A and D). (G) a 3 (grey–black, long arrow) and a 1 (brown, short arrow) double labeling showing the lack of co-localization of these markers. (H) a 3 (grey–black) and NeuN (brown) double labeling showing the co-localization of both markers (arrows). Calibration bar: (A)–(F) 60 mm; (G) 100 mm; (H) 75 mm.

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a 3-ir cells are characterized by ChAT staining. Waldvogel et al. [30] have just reported that some a 3-ir neurons were immunopositive for both ChAT and a 3-subunit in the human striatum. Interestingly, we have provided evidence showing that different types of ChAT-positive neurons in the rat striatum [19] express the GABAA receptor a 3subunit, indicating that these interneurons may exhibit characteristic responses to GABAergic innervation. Gao et al. [13] have also observed that cholinergic neurons in the medial septum-diagonal band complex in the basal forebrain of rat are characterized by the presence of a 3subunit, although they have also observed many a 3-ir neurons that were not immunoreactive for ChAT. Different studies [1,8,14,22] have demonstrated that striatal acetylcholine (ACh) output in the striatum is directly modulated by GABA via GABAA receptors located on cholinergic neurons, and indirectly modulated via GABA B receptors located presynaptically in other neurons that form synapses on cholinergic neurons. Interestingly, these studies showed that the change of the output of striatal ACh by different substances that are agonistic and antagonistic to GABA receptors were ineffective at low concentrations. Our findings showed that cholinergic neurons in the rat striatum contain the GABAA receptor a 3-subunit, whose pharmacological characteristics [9] could explain the previous results. However, a small percentage of neurons with type 1 morphology were immunonegative for ChAT. A possibility is that some cholinergic neurons may contain a small amount of ChAT that was not detected by the immunolabeling techniques used here. In addition, some of these neurons appeared to be positive for GABA, and could correspond to a subtype of human GABAergic striatal neurons expressing the a 3-subunit recently described by Waldvogel et al. [30]. Whereas the vast majority of type 1 a 3-ir neurons were positive for ChAT, the type 2 a 3-ir cells were negative for this marker. Therefore, we used different cytomarkers to find if these cells corresponded to another neuronal subtype: Type 2 a 3-ir cells showed a similar morphology to those previously described as type 1 a 1-ir neurons [4,31]. Both neuronal types have a rounded or oval somata and a widely branching dendritic tree, although they have a distinct topographic distribution; type 1 a 1-ir neurons are mainly located in the dorsolateral regions of the striatum, while type 2 a 3-ir cells are a homogeneously distributed throughout the striatum. Finally, our double-immunocytochemical experiments indicated that most type 2 neurons showed a lack of co-localization for these markers. However, Waldvogel et al. [30] have just reported that in the human striatum some a 3-ir neurons are strongly immunopositive for the GABAA receptor a 1-subunit. These data suggest that it could be different subtypes of striatal cells, since our type 2 a 3-ir neurons are generally located close to or sometimes inside the myelinated fiber bundles. Moreover, our study has also shown that GABA was not present, at least in detectable amounts with the

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techniques employed, in these a 3-ir neurons. In addition, most type 2 a 3-ir cells were positive for NeuN, suggesting that type 2 a 3-ir cells could represent an additional neuronal subtype. There is a class of dwarf or neurogliaform neurons that was first described in the human ´ y Cajal [24], and recently demonstrated striatum by Ramon in detail in the rat striatum [27], and type 2 a 3-ir cells closely resemble these dwarf neurons. It has been suggested that neurogliaform cells in the rat striatum may integrate information over a short distance and may have a satellite function, since they exhibit dye-coupling with other striatal neurons [27]. Our findings appear to agree with these suggestions, as type 2 a 3-ir neurons are cells with short processes that extend close to the cell body, and whose somata are usually situated beside dendrites of other neurons, mainly type 1 a 3-ir neurons. This might indicate an interaction between these neurons due to release of trophic factors by neighboring cells or that type 2 a 3-ir cells send processes that might establish synapse-like contacts with dendrites of other (i.e., type 1 a 3-ir) neurons. At the cellular level, both types of a 3-immunopositive neurons have a 3-ir in the somata and dendrites. Staining for this GABAA receptor subunit is observed in ‘hot spots’ at the cell surface [12]. These aggregates may indicate postsynaptic complexes [3]. Moreover, type 1 a 3-immunopositive neurons also show an intracellular immunoreactivity for this subunit. GABAA receptors containing the a 3-subunit, assembled with b and g2 -subunits, show low affinity and maximal efficacy for GABA [9]. The fact that some interneurons in the rat striatum, mainly cholinergic neurons, contain the GABAA receptor a 3-subunit suggests that these neurons may receive a strong GABAergic input, since the GABAA receptor complexes contain an a -subunit that shows lowaffinity to GABA, or even that the strength of this innervation could be regulated by endogenous GABAA receptor allosteric modulators. In conclusion, this study has demonstrated that in the rat striatum cholinergic neurons contain the GABAA receptor a 3-subunit, and that exists a second cellular type immunoreactive for this subunit that may correspond to neurogliaform neurons, although it may also be a novel subtype of striatal interneuron. These neurons represent a small percentage of the total of striatal neuronal populations, however they may play an important role in the regulation of the microcircuitry of the striatum.

Acknowledgements ¨ We are grateful to Dr. H. Mohler for the generous gift of the GABAA receptor subunit specific antibodies, and Drs. P. Greengard and E.L. Gustafson who supplied the DARPP-32 antibody. This work was supported by XUGA and the Spanish DGESIC (PGC).

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