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The striatal gaba-ergic neurons expressing substance P receptors in the basal ganglia of mice
Neuroscience 119 (2003) 919 –925
LETTER TO NEUROSCIENCE THE STRIATAL GABA-ERGIC NEURONS EXPRESSING SUBSTANCE P RECEPTORS IN THE BASAL GANGLIA OF MICE L.-W. CHEN,a* R. CAO,a H.-L. LIU,a G. JUa AND Y. S. CHANb
nergic, serotoninergic and noradrenergic afferents from the cerebral cortex, thalamus, limbic structures, substantia nigra pars compacta, raphe nuclei, and locus coeruleus, and sends its neural projections to the key output nuclei of the basal ganglia, e.g. globus pallidus (GP) and the substantia nigra pars reticulata (SNR) (Blandini et al., 2000). Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the striatum. The majority of GABAcontaining cells are projection neurons in the striatum and characterized by medium-sized spiny neuronal profiles. These striatal GABA-ergic projection neurons constitute the direct striatonigral (substance P [SP] and dynorphin colocalized) and indirect striatopallidal (enkephalin colocalized) neural pathways of the basal ganglia (Cicchetti et al., 2000; Kawaguchi et al., 1995). Besides, GABA and calcium-binding protein (calretinin, parvalbumin or calbindin)-containing interneurons are scattered within these striatal regions (Kawaguchi et al., 1995; Koo´s and Tepper, 1999; Kubota et al., 1993; Parent et al., 1996). The GABAcontaining neurons, being a dominant inhibitory neuron population in the striatum, play a crucial role in the modulation of movement-, cognition- and emotional-related activities of the basal ganglia. Functional abnormality or structural lesions of the striatal GABA-ergic neural pathways result in various types of severe motor disturbances ranging from hypokinesia (e.g. Parkinson’s disease) to hyperkinesia (e.g. hemiballismus and Huntington’s disease) in mammals (Blandini et al., 2000; Cicchetti et al., 1996, 2000). The mammalian tachykinin family, including SP (neurokinin-1, NK-1), substance K (neurokinin-2, NK-2) and neuromedin K (NK; neurokinin-3, NK-3), acts on distinct tachykinin receptor subtypes to induce their physiological effects (Helke et al., 1990; Nakanishi, 1991; Otsuka and Yoshika, 1993). The distribution of tachykinins and tachykinin receptors has been extensively studied by ligandbinding, in situ hybridization and immunohistochemistry in the mammalian CNS (Arai and Emson, 1986; Mantyh et al., 1989; Nakaya et al., 1994). Recent studies have provided evidence that the GABA-ergic neurons are regulated by tachykinins in the brains of mammals (Maubach et al., 2001). For example, co-localization of SP receptor (SPR, or NK-1R) was found in the cerebral cortex and hippocampus (Acsady et al., 1997; Echevarria et al., 1997; Kaneko et al., 1994; Sloviter et al., 2001), and NK receptor (NKR, or NK-3R) co-localized with GABA in striatal regions (Preston et al., 2000). NKR-mediated GABA release was also
a Institute of Neurosciences, Fourth Military Medical University, Xi’an 710032, China b Department of Physiology, Faculty of Medicine, The University of Hong Kong, Hong Kong, China
Abstract—By using a double immunofluorescence, we have examined the distribution of striatal GABAergic neurons that expressed substance P receptor (SPR) in the basal ganglia of adult C57 mice. The distribution of GABA-immunoreactive neurons completely or partially overlapped with that of SPRimmunoreactive neurons in the striatum (i.e. the caudateputamen), globus pallidus, ventral pallidum, and nucleus accumbens. Neurons showing both GABA- and SPR-immunoreactivities were, however, predominantly found in the caudate-putamen, and most of them were characterized by their large-sized aspiny neuronal profile. Semi-quantification indicated that only about 13% of the total GABA-immunoreactive neurons (including large and medium-sized) displayed SPR-immunoreactivity, and these double-labeled neurons constituted about 31% of the total SPR-immunoreactive cells in the striatum. Neurons double-labeled with GABA- and SPR-immunoreactivities were hardly detected in other aforementioned regions of the basal ganglia. In addition, double immunofluorescence also showed co-localization of SPRwith glutamic acid decarboxylase-immunoreactivity, but not with parvalbumin-immunoreactivity, in the striatal neurons. Taken together with previous reports, the present study has suggested that a sub-population of striatal GABA-ergic neurons, most possibly GABA-ergic interneurons, may also receive direct physiological modulation by tachykinins through SPR in the basal ganglia of mammals. © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: tachykinin, substance P receptor, GABA, striatum, basal ganglia.
The striatum (i.e. the caudate-putamen, CPu) represents the main input structure of the basal ganglia circuits. In mammals, the striatum receives glutamatergic, dopami*Corresponding author. Tel: ⫹86-29-328-5726; fax: ⫹86-29-3246270. E-mail address: [email protected] (L.-W. Chen). Abbreviations: Acb, nucleus accumbens; CPu, caudate-putamen; DTAF, dichorotriazinylamino-fluorescein; GABA, gamma-aminobutyric acid; GAD, glutamic acid decarboxylase; GP, globus pallidas; IgG, immunoglobulin G; ir, immunoreactive; NK, neuromedin K; NK-1, neurokinin-1; NK-2, neurokinin-2; NK-3, neurokinin-3; NKR, neurokinin K receptor; PB, phosphate buffer; PBS, phosphate-buffered saline; SNR, substantia nigra pars reticulata; SP, substance P; SPR, substance P receptor; TRITC, tetramethyl rhodamine isothiocyanate; VP, ventral pallidum.
0306-4522/03$30.00⫹0.00 © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S0306-4522(03)00223-9
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demonstrated in GABA-ergic interneurons of the striatum (Preston et al., 2000). Furthermore, GABA, SP and SPR are abundantly detected in striatal regions (Aubry et al., 1993; Cicchetti et al., 1996; Gerfen, 1991; Jakab and Goldman-Rakic, 1996; Jakab et al., 1996; Kaneko et al., 1993; Parent et al., 1995; Pickel et al., 2000). Administration of SP or SPR agonist also affects the activity of striatal neurons (Aosaki and Kawaguchi, 1996). It is possible that GABA-containing neurons in the striatal regions also express SPR. In the present study, therefore, co-localization of SPR in GABA-containing neurons was examined by double immunofluorescence in the striatum and other basal ganglia regions of C57 mice with the aim of elucidating the possibility of striatal GABA-ergic neurons receiving direct SP regulation. In the present study, both GABA-immunoreactive (-ir) and/or SPR-ir neurons were found in single and dually immunostained brain sections. Their distribution was consistent with that of previous observations (Kosaka et al., 1988; Kubota et al., 1993; Nakaya et al., 1994). Generally, SPR-immunoreactivity was mainly localized on the neuronal cell membrane while GABA-immunoreactivity was detected in the neuronal cytoplasm, and GABA-synthesizing enzyme glutamic acid decarboxylase (GAD)-immunoreactivity was also seen on neuronal somas (Fig. 1). SPR-ir, GABA-ir or GAD-ir neurons were predominantly distributed in the basal ganglia regions, e.g. CPu, GP, ventral pallidum (VP), nucleus accumbens (Acb), and SNR. SPR-ir neuronal somas were mainly oval in shape, large-sized with a diameter of 34⫾8 m (n⫽30), and displayed aspiny smooth or beaded dendrites, whereas only a small population of GABA-ir or GAD-ir neuronal somas were largesized with a diameter of 32⫾6 m (n⫽30). Specificity of GABA-, SPR- or GAD-immunoreactivity was confirmed by substitution or absorption control experiments (data not shown). The distribution of GABA-ir neurons completely or partially overlapped with that of SPR-ir neurons in CPu, GP, VP, and Acb except for SNR. Neurons showing both GABA- and SPR-immunoreactivities were predominantly found in the CPu (Fig. 2). The majority of double-labeled neurons were oval in shape, large-sized with a diameter of 38⫾6 m (n⫽45), and were characterized by aspiny neuronal appearance. These double-labeled neurons displayed smooth or beaded dendrites (Fig. 3). GABA/SPR-ir double cells showed low and undetectable levels of labeling in the GP, VP, Acb, and SNR (Fig. 4). Semi-quantification results indicated that about 13% of GABA-ir neurons (including large- and medium-sized) showed SPR-immunoreactivity, whereas these double-labeled neurons constituted about 31% of total SPR-ir neuronal cells in the CPu (Table 1). Besides, numerous SPR-ir dendrites or pro-
Fig. 1. Neurons showing substance P receptor (SPR)-, GABA- and GAD-immunoreactivity in coronal sections of the CPu. Representative neuronal somas are indicated by arrow heads. SPR-immunoreactivity is shown in neuronal somas and smooth or beaded dendritic processes, GABA-immunoreactivity is mainly detected in neuronal somas, and GAD-immunoreactivity is localized in both neuronal somas and axon terminals.
Abbreviations used in the figures aca AcbC AcbSh LS LV
anterior commissure, anterior accumbens nucleus, core accumbens nucleus, shell lateral septal nucleus lateral ventricle
MS Pir Tu VDB
medial septal nucleus piriform cortex olfactory tubercle nucleus of the diagnonal band of Broca, vertical limb
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Fig. 2. Schematic map illustrating distribution of GABA/SPR double-labeled neurons in representative coronal sections of mice brain. Each dot denotes about two to three neuronal somas exhibiting both GABA- and SPR-immunoreactivity in each side of the striatum. GABA-single, SPR-single labeled neurons are not shown in this map. Abbreviation: aca, anterior commissure, anterior; AcbC, accumbens nucleus, core; AcbSh, accumbens nucleus, shell; LS, lateral septal nucleus; LV, lateral ventricle; MS, medial septal nucleus; Pir, piriform cortex; Tu, olfactory tubercle; VDB, nucleus of diagonal band of Broca, vertical limb.
cesses of various lengths were disconnected, and widely distributed between or among neuronal somas in striatal regions. The difference in cell morphology between SPR-ir neurons with GABA- and those without GABA-immunoreactivity, however, was not clearly identified. In addition, GABA/SPR double-labeled neurons were also observed in the cerebral cortex, which was consistent with the previous observations (Echevarria et al., 1997; Kaneko et al., 1994; Sloviter et al., 2001). For further characterization of these SPR-ir neurons displaying GABA-immunoreactivity in the striatum, double immunofluorescence for SPR/GAD and SPR/parvalbumin was also performed. SPR/GAD double-labeled neurons were also scattered in the striatum. Distribution and morphology of SPR/GAD double labeled neurons were similar to that of SPR/GABA-ir neurons, but their number was only about one fifth of the latter. However, co-localization of SPR- with parvalbumin-immunoreactivity was not detected, though a number of medium-or small-sized parvalbumin-ir neuronal somas were distributed in the striatal regions (data not shown). The distribution of neurons containing GABA or SPR in mammalian brain has been examined in previous studies (Kosaka et al., 1988; Kubota et al., 1993; Nakaya et al., 1994). Our present data are consistent with these reports. We further demonstrated that about 13% of GABA-containing neurons express SPR in the striatal regions of mice, and SPR-ir neurons in these regions also contained GAD, a GABA synthesis enzyme, but not parvalbumin. Our results have provided morphological evidence for a subpopulation of striatal GABA-containing neurons that are regulated directly by tachykinins via SP receptors in the mouse basal ganglia. In previous studies, expression of SPR and NKR was observed in cholinergic or noradrenergic neurons of the
striatum, basal forebrain and locus coeruleus, indicating that these cholinergic or noradrenergic neurons receive tachykinin modulation via SPR or NKR (Aubry et al., 1993; Chen et al., 2000, 2001a,b; Gerfen, 1991; Pickel et al., 2000). Our present study has revealed SPR expression in a population of GABA-containing neurons of mouse striatum. On the basis of present observations, GABA-ergic neurons in the striatum may be categorized into two subpopulations: one major group without SPR, and one minor population with SPR. The latter population is characterized by large-sized aspiny neuronal profiles. Heterogeneous interneurons containing acetylcholine, GABA, and calcium-binding proteins (calbindin, calretinin and parvalbumin) are scattered in the CPu, and most of these interneurons are aspiny neurons (Kawaguchi et al., 1995; Kubota et al., 1993; Parent et al., 1996). It is well known that the majority of large-sized aspiny striatal neurons are cholinergic interneurons, and the other groups of neurons such as parvalbumin-, somatostatin-, calbindin- or calretinin-containing interneurons and principal spiny neurons, are mediumsized (Kawaguchi et al., 1995; Parent et al., 1996). Thus, the present result suggested that at least some of these neurons might also contain choline acetyltransferase (ChAT, or cholinergic population). Besides, co-expression of ChAT- and GABA-immunoreactivites was well demonstrated in non-pyramidal cortical neurons of the occipital lobe and spinal cord neurons (Hallanger et al., 1986; Kosaka et al., 1988). GAD mRNA was also detected in the striatal cholinergic neurons by single reverse transcriptionpolymerase chain reaction method (Richardson et al., 2000). Taken together, it can be speculated that these GABA-containing neurons with SPR are most likely interneurons, and may be modulated by SP in the striatal regions.
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Fig. 3. Striatal neurons showing both GABA- (a– c) and SPR- (a'– c') immunoreactivity in the caudate (a, a'), dorsal (b, b') and ventral putamen (c, c') of coronal sections. Representative double-labeled neuronal somas are indicated by arrows and single-labeled somas were indicated with arrow heads. GABA/SPR double-labeled and SPR single-labeled neurons display their smooth or beaded (aspiny) dendrites. Numerous SPR-labeled dendritic processes of various lengths are disconnected and distributed between neuronal somas. The fields of a, b and c are the same as those of a', b' and c', respectively.
Modulation of GABA-containing neurons by tachykinins has been previously documented in the brain (Maubach et al., 2001; Sloviter et al., 2001). For example, co-localization of SPR with GABA was demonstrated in neurons of cortical and hippocampal regions (Acsady et al., 1997; Echevarria et al., 1997; Kaneko et al., 1994; Sloviter et al., 2001). Co-localization of calretinin and SPR was also detected in the striatal neu-
rons, which constitute about 3% of total large and medium-sized striatal neurons (Cicchetti et al., 1996). Furthermore, in situ hybridization study has demonstrated both expression of NKR in preprosomatostatin mRNA containing GABA-ergic striatal interneurons as well as NKR-mediated GABA release in the striatum slice (Preston et al., 2000). Therefore, GABA-ergic interneurons may receive tachykinin modulation via both NKR and/or
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Fig. 4. GABA- (a, b) or SPR- (a', b') immunoreactivity in the GP (a, a') and SNR (b, b'). No double-labeled neurons are detected in GP or SNR, and single-labeled neurons are representatively indicated with arrow heads. The fields of a and b are the same as those of a' and b', respectively.
SPR in the striatum. Evidence has shown that the inhibitory or GABA-ergic interneurons may exert a powerful control on the activity of projection neurons in the striatum (Koo´s and Tepper, 1999). For example, a single inhibitory interneuron may apparently inhibit the firing of over 100 spiny projection neurons. Besides, colocalized neuropeptides were found in GABA-ergic spiny projection neurons, although the function of these peptides remains unexplained. However, the release of neuropeptides from the abundant local collaterals of projection neurons may provide the interacting substrate between GABA-ergic projection neurons and interneurons in the striatum (Cicchetti et al., 2000; Koo´s and Tepper, 1999). Tachykinin receptor-mediated adaptive neural
plasticity was detected in the cortex of ischemic animals (Stumm et al., 2001). It is well known that tachykinins modulate ion channel activity, stimulate both phosphatidylinositol hydrolysis and cyclic AMP intracellular signal cascades via these tachykinin receptors (Helke et al., 1990; Nakajima et al., 1992). Our present result, together with previous observations, also suggests that the effect of SP on the basal ganglia may mostly be via activating interneurons and inducing GABA and acetylcholine release. Further studies are needed to elucidate the intracellular signaling cascades triggered by the functional operation of SP receptors on GABA-containing neurons in the striatum, as well as their pathophysiological implications in the basal ganglia of mammals.
Table 1. GABA single-, SPR single-, and GABA/SPR double-labeled neuronal somas in different regions of the mouse basal gangliaa
CPu GP VP Acb SNR
GABA
SPR
GABA/SPR (% of GABA)
GABA/SPR (% of SPR)
1433⫾124 658⫾38 605⫾77 376⫾64 893⫾118
585⫾39 235⫾43 457⫾51 112⫾34 14⫾8
184⫾27 (13%⫾6%) 0 0 0 0
184⫾27 (31%⫾5%) 0 0 0 0
a The neurons were counted on 12 sections per unilateral region from mouse brains (mean⫾S.E.M.; n⫽6). Positive neurons outside basal ganglia (e.g. cerebral cortex, basal forebrain regions) were not counted.
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EXPERIMENTAL PROCEDURES Animal preparation Adult male mice (C57 black strain, n⫽6) weighing 20 –22 g were used in the present study. All animal procedures conformed with the guidelines of the National Institutes of Health for the care and use of laboratory animals (NIH publications no. 80-23), and all efforts were made to minimize animal suffering and reduce the number of animals used. The animals were anesthetized with an overdose of sodium pentobarbital (100 mg/kg, i.p.) and then perfused transcardially with a volume of 20 ml of saline, followed by a volume of 100 ml of 0.1 M phosphate buffer (PB; pH 7.4) containing 4% paraformaldehyde and 0.05% glutaraldehyde for 30 min. The brains were removed immediately and placed in 0.1 M PB containing 30% sucrose overnight at 4 °C. After that, the parts of brains containing the basal ganglia were serially cut into coronal sections (30 m) on freezing microtome.
Double immunofluorescence The sections were incubated overnight at room temperature with a mixture of mouse anti-GABA immunoglobulin G (IgG; Sigma, USA; at a 1:1000 dilution) and affinity-purified rabbit anti-SPR IgG (Sigma; 1:3000 dilution) in 0.01 M phosphate-buffered saline (PBS; pH 7.4) containing 1% normal goat serum, 3% BSA and 0.1% Triton X-100. Subsequently, the sections were rinsed in 0.01 M PBS (pH 7.4), and then incubated for 4 h at room temperature with a mixture of dichorotriazinylamino-fluorescein (DTAF)conjugated donkey anti-mouse IgG and tetramethyl rhodamine isothiocyanate (TRITC)-conjugated donkey anti-rabbit IgG (Chemicon, USA, 1:200 dilution). After being washed, the sections were mounted on gelatin-coated glass slides, and coverslipped in 0.01 M PBS (pH 7.4) containing 50% glycerine and 2.5% triethylenediamine, and then examined under an Olympus fluorescence microscope (BX-60). In a corresponding protocol, double immunofluorescence of SPR with GAD65 (Sigma; 1:500 dilution), or SPR with parvalbumin (Sigma; 1:1000 dilution), and single immunoenzyme histochemistry for SPR, GABA and GAD were also performed in the sections. Blue filter or green filter was utilized to visualize DTAF-labeled neurons or TRITC-labeled neurons, respectively. The interesting images (fluorescence and bright field) were captured with a digital camera (Leica) for further semiquantification and demonstration.
Controls and data analysis For control experiments, The primary antibody was substituted with normal mouse serum (for GABA, GAD or parvalbumin immunocytochemistry) or normal rabbit serum (for SPR immunocytochemistry). Adsorption test was also performed for the specificity of GABA-immunoreactivity, in which 2 g GABA (Sigma) was added into 0.5 ml of diluted GABA antibody solution, incubated overnight at room temperature, centrifuged at 10,000⫻g for 30 min and supernatant was used for incubation of sections. No GABA-, GAD-, parvalbumin, or SPR-ir neurons were found in these control sections (data not shown). For semi-quantification, the GABA-single, SPR-single, and GABA/SPR double-labeled neuronal somas were counted on 12 sections per unilateral region from mouse brains (mean⫾S.E.M.; n⫽6). The diameters of ir neuronal somas were also evaluated under the microscope by measuring the average of their short and long diameters with the use of a morphometric micrometer and expressed as mean⫾S.E.M. The nomenclature and demarcation of brain structures were adapted with reference to the mouse brain atlas (Franklin and Paxinos, 1997). Acknowledgements—This work was supported by a Joint Research Grant from the National Natural Science Foundation of
China and the Research Grants Council of Hong Kong (NSFCRGC grant no. 30218002).
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(Accepted 4 March 2003)
LETTER TO NEUROSCIENCE THE STRIATAL GABA-ERGIC NEURONS EXPRESSING SUBSTANCE P RECEPTORS IN THE BASAL GANGLIA OF MICE L.-W. CHEN,a* R. CAO,a H.-L. LIU,a G. JUa AND Y. S. CHANb
nergic, serotoninergic and noradrenergic afferents from the cerebral cortex, thalamus, limbic structures, substantia nigra pars compacta, raphe nuclei, and locus coeruleus, and sends its neural projections to the key output nuclei of the basal ganglia, e.g. globus pallidus (GP) and the substantia nigra pars reticulata (SNR) (Blandini et al., 2000). Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the striatum. The majority of GABAcontaining cells are projection neurons in the striatum and characterized by medium-sized spiny neuronal profiles. These striatal GABA-ergic projection neurons constitute the direct striatonigral (substance P [SP] and dynorphin colocalized) and indirect striatopallidal (enkephalin colocalized) neural pathways of the basal ganglia (Cicchetti et al., 2000; Kawaguchi et al., 1995). Besides, GABA and calcium-binding protein (calretinin, parvalbumin or calbindin)-containing interneurons are scattered within these striatal regions (Kawaguchi et al., 1995; Koo´s and Tepper, 1999; Kubota et al., 1993; Parent et al., 1996). The GABAcontaining neurons, being a dominant inhibitory neuron population in the striatum, play a crucial role in the modulation of movement-, cognition- and emotional-related activities of the basal ganglia. Functional abnormality or structural lesions of the striatal GABA-ergic neural pathways result in various types of severe motor disturbances ranging from hypokinesia (e.g. Parkinson’s disease) to hyperkinesia (e.g. hemiballismus and Huntington’s disease) in mammals (Blandini et al., 2000; Cicchetti et al., 1996, 2000). The mammalian tachykinin family, including SP (neurokinin-1, NK-1), substance K (neurokinin-2, NK-2) and neuromedin K (NK; neurokinin-3, NK-3), acts on distinct tachykinin receptor subtypes to induce their physiological effects (Helke et al., 1990; Nakanishi, 1991; Otsuka and Yoshika, 1993). The distribution of tachykinins and tachykinin receptors has been extensively studied by ligandbinding, in situ hybridization and immunohistochemistry in the mammalian CNS (Arai and Emson, 1986; Mantyh et al., 1989; Nakaya et al., 1994). Recent studies have provided evidence that the GABA-ergic neurons are regulated by tachykinins in the brains of mammals (Maubach et al., 2001). For example, co-localization of SP receptor (SPR, or NK-1R) was found in the cerebral cortex and hippocampus (Acsady et al., 1997; Echevarria et al., 1997; Kaneko et al., 1994; Sloviter et al., 2001), and NK receptor (NKR, or NK-3R) co-localized with GABA in striatal regions (Preston et al., 2000). NKR-mediated GABA release was also
a Institute of Neurosciences, Fourth Military Medical University, Xi’an 710032, China b Department of Physiology, Faculty of Medicine, The University of Hong Kong, Hong Kong, China
Abstract—By using a double immunofluorescence, we have examined the distribution of striatal GABAergic neurons that expressed substance P receptor (SPR) in the basal ganglia of adult C57 mice. The distribution of GABA-immunoreactive neurons completely or partially overlapped with that of SPRimmunoreactive neurons in the striatum (i.e. the caudateputamen), globus pallidus, ventral pallidum, and nucleus accumbens. Neurons showing both GABA- and SPR-immunoreactivities were, however, predominantly found in the caudate-putamen, and most of them were characterized by their large-sized aspiny neuronal profile. Semi-quantification indicated that only about 13% of the total GABA-immunoreactive neurons (including large and medium-sized) displayed SPR-immunoreactivity, and these double-labeled neurons constituted about 31% of the total SPR-immunoreactive cells in the striatum. Neurons double-labeled with GABA- and SPR-immunoreactivities were hardly detected in other aforementioned regions of the basal ganglia. In addition, double immunofluorescence also showed co-localization of SPRwith glutamic acid decarboxylase-immunoreactivity, but not with parvalbumin-immunoreactivity, in the striatal neurons. Taken together with previous reports, the present study has suggested that a sub-population of striatal GABA-ergic neurons, most possibly GABA-ergic interneurons, may also receive direct physiological modulation by tachykinins through SPR in the basal ganglia of mammals. © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: tachykinin, substance P receptor, GABA, striatum, basal ganglia.
The striatum (i.e. the caudate-putamen, CPu) represents the main input structure of the basal ganglia circuits. In mammals, the striatum receives glutamatergic, dopami*Corresponding author. Tel: ⫹86-29-328-5726; fax: ⫹86-29-3246270. E-mail address: [email protected] (L.-W. Chen). Abbreviations: Acb, nucleus accumbens; CPu, caudate-putamen; DTAF, dichorotriazinylamino-fluorescein; GABA, gamma-aminobutyric acid; GAD, glutamic acid decarboxylase; GP, globus pallidas; IgG, immunoglobulin G; ir, immunoreactive; NK, neuromedin K; NK-1, neurokinin-1; NK-2, neurokinin-2; NK-3, neurokinin-3; NKR, neurokinin K receptor; PB, phosphate buffer; PBS, phosphate-buffered saline; SNR, substantia nigra pars reticulata; SP, substance P; SPR, substance P receptor; TRITC, tetramethyl rhodamine isothiocyanate; VP, ventral pallidum.
0306-4522/03$30.00⫹0.00 © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S0306-4522(03)00223-9
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demonstrated in GABA-ergic interneurons of the striatum (Preston et al., 2000). Furthermore, GABA, SP and SPR are abundantly detected in striatal regions (Aubry et al., 1993; Cicchetti et al., 1996; Gerfen, 1991; Jakab and Goldman-Rakic, 1996; Jakab et al., 1996; Kaneko et al., 1993; Parent et al., 1995; Pickel et al., 2000). Administration of SP or SPR agonist also affects the activity of striatal neurons (Aosaki and Kawaguchi, 1996). It is possible that GABA-containing neurons in the striatal regions also express SPR. In the present study, therefore, co-localization of SPR in GABA-containing neurons was examined by double immunofluorescence in the striatum and other basal ganglia regions of C57 mice with the aim of elucidating the possibility of striatal GABA-ergic neurons receiving direct SP regulation. In the present study, both GABA-immunoreactive (-ir) and/or SPR-ir neurons were found in single and dually immunostained brain sections. Their distribution was consistent with that of previous observations (Kosaka et al., 1988; Kubota et al., 1993; Nakaya et al., 1994). Generally, SPR-immunoreactivity was mainly localized on the neuronal cell membrane while GABA-immunoreactivity was detected in the neuronal cytoplasm, and GABA-synthesizing enzyme glutamic acid decarboxylase (GAD)-immunoreactivity was also seen on neuronal somas (Fig. 1). SPR-ir, GABA-ir or GAD-ir neurons were predominantly distributed in the basal ganglia regions, e.g. CPu, GP, ventral pallidum (VP), nucleus accumbens (Acb), and SNR. SPR-ir neuronal somas were mainly oval in shape, large-sized with a diameter of 34⫾8 m (n⫽30), and displayed aspiny smooth or beaded dendrites, whereas only a small population of GABA-ir or GAD-ir neuronal somas were largesized with a diameter of 32⫾6 m (n⫽30). Specificity of GABA-, SPR- or GAD-immunoreactivity was confirmed by substitution or absorption control experiments (data not shown). The distribution of GABA-ir neurons completely or partially overlapped with that of SPR-ir neurons in CPu, GP, VP, and Acb except for SNR. Neurons showing both GABA- and SPR-immunoreactivities were predominantly found in the CPu (Fig. 2). The majority of double-labeled neurons were oval in shape, large-sized with a diameter of 38⫾6 m (n⫽45), and were characterized by aspiny neuronal appearance. These double-labeled neurons displayed smooth or beaded dendrites (Fig. 3). GABA/SPR-ir double cells showed low and undetectable levels of labeling in the GP, VP, Acb, and SNR (Fig. 4). Semi-quantification results indicated that about 13% of GABA-ir neurons (including large- and medium-sized) showed SPR-immunoreactivity, whereas these double-labeled neurons constituted about 31% of total SPR-ir neuronal cells in the CPu (Table 1). Besides, numerous SPR-ir dendrites or pro-
Fig. 1. Neurons showing substance P receptor (SPR)-, GABA- and GAD-immunoreactivity in coronal sections of the CPu. Representative neuronal somas are indicated by arrow heads. SPR-immunoreactivity is shown in neuronal somas and smooth or beaded dendritic processes, GABA-immunoreactivity is mainly detected in neuronal somas, and GAD-immunoreactivity is localized in both neuronal somas and axon terminals.
Abbreviations used in the figures aca AcbC AcbSh LS LV
anterior commissure, anterior accumbens nucleus, core accumbens nucleus, shell lateral septal nucleus lateral ventricle
MS Pir Tu VDB
medial septal nucleus piriform cortex olfactory tubercle nucleus of the diagnonal band of Broca, vertical limb
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Fig. 2. Schematic map illustrating distribution of GABA/SPR double-labeled neurons in representative coronal sections of mice brain. Each dot denotes about two to three neuronal somas exhibiting both GABA- and SPR-immunoreactivity in each side of the striatum. GABA-single, SPR-single labeled neurons are not shown in this map. Abbreviation: aca, anterior commissure, anterior; AcbC, accumbens nucleus, core; AcbSh, accumbens nucleus, shell; LS, lateral septal nucleus; LV, lateral ventricle; MS, medial septal nucleus; Pir, piriform cortex; Tu, olfactory tubercle; VDB, nucleus of diagonal band of Broca, vertical limb.
cesses of various lengths were disconnected, and widely distributed between or among neuronal somas in striatal regions. The difference in cell morphology between SPR-ir neurons with GABA- and those without GABA-immunoreactivity, however, was not clearly identified. In addition, GABA/SPR double-labeled neurons were also observed in the cerebral cortex, which was consistent with the previous observations (Echevarria et al., 1997; Kaneko et al., 1994; Sloviter et al., 2001). For further characterization of these SPR-ir neurons displaying GABA-immunoreactivity in the striatum, double immunofluorescence for SPR/GAD and SPR/parvalbumin was also performed. SPR/GAD double-labeled neurons were also scattered in the striatum. Distribution and morphology of SPR/GAD double labeled neurons were similar to that of SPR/GABA-ir neurons, but their number was only about one fifth of the latter. However, co-localization of SPR- with parvalbumin-immunoreactivity was not detected, though a number of medium-or small-sized parvalbumin-ir neuronal somas were distributed in the striatal regions (data not shown). The distribution of neurons containing GABA or SPR in mammalian brain has been examined in previous studies (Kosaka et al., 1988; Kubota et al., 1993; Nakaya et al., 1994). Our present data are consistent with these reports. We further demonstrated that about 13% of GABA-containing neurons express SPR in the striatal regions of mice, and SPR-ir neurons in these regions also contained GAD, a GABA synthesis enzyme, but not parvalbumin. Our results have provided morphological evidence for a subpopulation of striatal GABA-containing neurons that are regulated directly by tachykinins via SP receptors in the mouse basal ganglia. In previous studies, expression of SPR and NKR was observed in cholinergic or noradrenergic neurons of the
striatum, basal forebrain and locus coeruleus, indicating that these cholinergic or noradrenergic neurons receive tachykinin modulation via SPR or NKR (Aubry et al., 1993; Chen et al., 2000, 2001a,b; Gerfen, 1991; Pickel et al., 2000). Our present study has revealed SPR expression in a population of GABA-containing neurons of mouse striatum. On the basis of present observations, GABA-ergic neurons in the striatum may be categorized into two subpopulations: one major group without SPR, and one minor population with SPR. The latter population is characterized by large-sized aspiny neuronal profiles. Heterogeneous interneurons containing acetylcholine, GABA, and calcium-binding proteins (calbindin, calretinin and parvalbumin) are scattered in the CPu, and most of these interneurons are aspiny neurons (Kawaguchi et al., 1995; Kubota et al., 1993; Parent et al., 1996). It is well known that the majority of large-sized aspiny striatal neurons are cholinergic interneurons, and the other groups of neurons such as parvalbumin-, somatostatin-, calbindin- or calretinin-containing interneurons and principal spiny neurons, are mediumsized (Kawaguchi et al., 1995; Parent et al., 1996). Thus, the present result suggested that at least some of these neurons might also contain choline acetyltransferase (ChAT, or cholinergic population). Besides, co-expression of ChAT- and GABA-immunoreactivites was well demonstrated in non-pyramidal cortical neurons of the occipital lobe and spinal cord neurons (Hallanger et al., 1986; Kosaka et al., 1988). GAD mRNA was also detected in the striatal cholinergic neurons by single reverse transcriptionpolymerase chain reaction method (Richardson et al., 2000). Taken together, it can be speculated that these GABA-containing neurons with SPR are most likely interneurons, and may be modulated by SP in the striatal regions.
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Fig. 3. Striatal neurons showing both GABA- (a– c) and SPR- (a'– c') immunoreactivity in the caudate (a, a'), dorsal (b, b') and ventral putamen (c, c') of coronal sections. Representative double-labeled neuronal somas are indicated by arrows and single-labeled somas were indicated with arrow heads. GABA/SPR double-labeled and SPR single-labeled neurons display their smooth or beaded (aspiny) dendrites. Numerous SPR-labeled dendritic processes of various lengths are disconnected and distributed between neuronal somas. The fields of a, b and c are the same as those of a', b' and c', respectively.
Modulation of GABA-containing neurons by tachykinins has been previously documented in the brain (Maubach et al., 2001; Sloviter et al., 2001). For example, co-localization of SPR with GABA was demonstrated in neurons of cortical and hippocampal regions (Acsady et al., 1997; Echevarria et al., 1997; Kaneko et al., 1994; Sloviter et al., 2001). Co-localization of calretinin and SPR was also detected in the striatal neu-
rons, which constitute about 3% of total large and medium-sized striatal neurons (Cicchetti et al., 1996). Furthermore, in situ hybridization study has demonstrated both expression of NKR in preprosomatostatin mRNA containing GABA-ergic striatal interneurons as well as NKR-mediated GABA release in the striatum slice (Preston et al., 2000). Therefore, GABA-ergic interneurons may receive tachykinin modulation via both NKR and/or
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Fig. 4. GABA- (a, b) or SPR- (a', b') immunoreactivity in the GP (a, a') and SNR (b, b'). No double-labeled neurons are detected in GP or SNR, and single-labeled neurons are representatively indicated with arrow heads. The fields of a and b are the same as those of a' and b', respectively.
SPR in the striatum. Evidence has shown that the inhibitory or GABA-ergic interneurons may exert a powerful control on the activity of projection neurons in the striatum (Koo´s and Tepper, 1999). For example, a single inhibitory interneuron may apparently inhibit the firing of over 100 spiny projection neurons. Besides, colocalized neuropeptides were found in GABA-ergic spiny projection neurons, although the function of these peptides remains unexplained. However, the release of neuropeptides from the abundant local collaterals of projection neurons may provide the interacting substrate between GABA-ergic projection neurons and interneurons in the striatum (Cicchetti et al., 2000; Koo´s and Tepper, 1999). Tachykinin receptor-mediated adaptive neural
plasticity was detected in the cortex of ischemic animals (Stumm et al., 2001). It is well known that tachykinins modulate ion channel activity, stimulate both phosphatidylinositol hydrolysis and cyclic AMP intracellular signal cascades via these tachykinin receptors (Helke et al., 1990; Nakajima et al., 1992). Our present result, together with previous observations, also suggests that the effect of SP on the basal ganglia may mostly be via activating interneurons and inducing GABA and acetylcholine release. Further studies are needed to elucidate the intracellular signaling cascades triggered by the functional operation of SP receptors on GABA-containing neurons in the striatum, as well as their pathophysiological implications in the basal ganglia of mammals.
Table 1. GABA single-, SPR single-, and GABA/SPR double-labeled neuronal somas in different regions of the mouse basal gangliaa
CPu GP VP Acb SNR
GABA
SPR
GABA/SPR (% of GABA)
GABA/SPR (% of SPR)
1433⫾124 658⫾38 605⫾77 376⫾64 893⫾118
585⫾39 235⫾43 457⫾51 112⫾34 14⫾8
184⫾27 (13%⫾6%) 0 0 0 0
184⫾27 (31%⫾5%) 0 0 0 0
a The neurons were counted on 12 sections per unilateral region from mouse brains (mean⫾S.E.M.; n⫽6). Positive neurons outside basal ganglia (e.g. cerebral cortex, basal forebrain regions) were not counted.
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EXPERIMENTAL PROCEDURES Animal preparation Adult male mice (C57 black strain, n⫽6) weighing 20 –22 g were used in the present study. All animal procedures conformed with the guidelines of the National Institutes of Health for the care and use of laboratory animals (NIH publications no. 80-23), and all efforts were made to minimize animal suffering and reduce the number of animals used. The animals were anesthetized with an overdose of sodium pentobarbital (100 mg/kg, i.p.) and then perfused transcardially with a volume of 20 ml of saline, followed by a volume of 100 ml of 0.1 M phosphate buffer (PB; pH 7.4) containing 4% paraformaldehyde and 0.05% glutaraldehyde for 30 min. The brains were removed immediately and placed in 0.1 M PB containing 30% sucrose overnight at 4 °C. After that, the parts of brains containing the basal ganglia were serially cut into coronal sections (30 m) on freezing microtome.
Double immunofluorescence The sections were incubated overnight at room temperature with a mixture of mouse anti-GABA immunoglobulin G (IgG; Sigma, USA; at a 1:1000 dilution) and affinity-purified rabbit anti-SPR IgG (Sigma; 1:3000 dilution) in 0.01 M phosphate-buffered saline (PBS; pH 7.4) containing 1% normal goat serum, 3% BSA and 0.1% Triton X-100. Subsequently, the sections were rinsed in 0.01 M PBS (pH 7.4), and then incubated for 4 h at room temperature with a mixture of dichorotriazinylamino-fluorescein (DTAF)conjugated donkey anti-mouse IgG and tetramethyl rhodamine isothiocyanate (TRITC)-conjugated donkey anti-rabbit IgG (Chemicon, USA, 1:200 dilution). After being washed, the sections were mounted on gelatin-coated glass slides, and coverslipped in 0.01 M PBS (pH 7.4) containing 50% glycerine and 2.5% triethylenediamine, and then examined under an Olympus fluorescence microscope (BX-60). In a corresponding protocol, double immunofluorescence of SPR with GAD65 (Sigma; 1:500 dilution), or SPR with parvalbumin (Sigma; 1:1000 dilution), and single immunoenzyme histochemistry for SPR, GABA and GAD were also performed in the sections. Blue filter or green filter was utilized to visualize DTAF-labeled neurons or TRITC-labeled neurons, respectively. The interesting images (fluorescence and bright field) were captured with a digital camera (Leica) for further semiquantification and demonstration.
Controls and data analysis For control experiments, The primary antibody was substituted with normal mouse serum (for GABA, GAD or parvalbumin immunocytochemistry) or normal rabbit serum (for SPR immunocytochemistry). Adsorption test was also performed for the specificity of GABA-immunoreactivity, in which 2 g GABA (Sigma) was added into 0.5 ml of diluted GABA antibody solution, incubated overnight at room temperature, centrifuged at 10,000⫻g for 30 min and supernatant was used for incubation of sections. No GABA-, GAD-, parvalbumin, or SPR-ir neurons were found in these control sections (data not shown). For semi-quantification, the GABA-single, SPR-single, and GABA/SPR double-labeled neuronal somas were counted on 12 sections per unilateral region from mouse brains (mean⫾S.E.M.; n⫽6). The diameters of ir neuronal somas were also evaluated under the microscope by measuring the average of their short and long diameters with the use of a morphometric micrometer and expressed as mean⫾S.E.M. The nomenclature and demarcation of brain structures were adapted with reference to the mouse brain atlas (Franklin and Paxinos, 1997). Acknowledgements—This work was supported by a Joint Research Grant from the National Natural Science Foundation of
China and the Research Grants Council of Hong Kong (NSFCRGC grant no. 30218002).
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(Accepted 4 March 2003)