Neurokinin-1 and neurokinin-3 receptors in primate substantia nigra

Neurokinin-1 and neurokinin-3 receptors in primate substantia nigra

Neuroscience Research 57 (2007) 362–371 www.elsevier.com/locate/neures Neurokinin-1 and neurokinin-3 receptors in primate substantia nigra Martin Le´...

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Neuroscience Research 57 (2007) 362–371 www.elsevier.com/locate/neures

Neurokinin-1 and neurokinin-3 receptors in primate substantia nigra Martin Le´vesque, Marie-Jose´e Wallman, Remy Parent, Attila Sı´k *, Andre´ Parent * Centre de recherche Universite´ Laval Robert-Giffard 2601, Chemin de la Canardie`re, Local F-6500 Beauport, Que´bec, Canada G1J 2G3 Received 20 September 2006; accepted 6 November 2006 Available online 28 November 2006

Abstract Striatonigral axons co-release GABA and substance P (SP) at their target sites, but little is known about the action of SP at nigral level. Therefore, we studied immunohistochemically the cellular and subcellular localization of SP and its high affinity receptors neurokinin-1 (NK1R) and neurokinin-3 (NK-3R) at nigral level in squirrel monkeys. Immunofluorescent studies revealed that, although SP+ fibers arborised more densely in the pars reticulata (SNr) than in the pars compacta (SNc), the two nigral divisions harbored numerous neurons expressing NK1R and NK-3R. Confocal microscopic analyses showed that numerous SNr neurons and virtually all SNc dopaminergic neurons contained both NK-1R and NK-3R. At the electron microscope level, NK-1R and NK-3R were mainly associated with intracellular sites or located at extrasynaptic position on plasma membrane. A small proportion of SP+ boutons also showed NK-3R immunoreactivity. The distribution of NK-1R and NK-3R in SNr and SNc suggests that SP exerts its effect through postsynaptic receptors, as well as via presynaptic autoreceptors and heteroreceptors. These findings indicate that the excitatory peptide SP can modulate the inhibitory action of GABA at nigral level and suggest that the co-release of these two neuroactive substances should be taken into account when considering the functional organization of the basal ganglia. # 2006 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: Basal ganglia; Substance P; Striatum; Electron microscopy; Neuroactive peptides; Co-transmission; Midbrain; Monkey

1. Introduction The substantia nigra is reciprocally linked to the striatum and both structures form a typical neural loop that plays a crucial role in basal ganglia function. Neurons of the pars compacta of the substantia nigra (SNc) project massively to the striatum, where they modulate the entire flow of information that courses through the basal ganglia by releasing dopamine (Anden et al., 1964). In turn, the striatum project back to the substantia nigra and terminates principally in the pars reticulata of substantia nigra (SNr), where it exerts a strong inhibitory action. The importance of these functionally linked nuclei is exemplified by the marked motor disturbances that occur in Parkinson’s and Huntington’s diseases, which are characterized by a selective degeneration of the nigral dopaminergic neurons and striatal projection neurons, respectively. A significant attention has been paid to the mechanisms involved in dopamine regulation at striatal level, but much less * Corresponding authors. Tel.: +1 418 663 5747; fax: +1 418 663 8756. E-mail addresses: [email protected] (A. Sı´k), [email protected] (A. Parent).

information is currently available on striatal output functions, especially regarding neuropeptide release by striatal projection neurons. The latter neurons use GABA as their main transmitter, but more than half of them co-release substance P (SP) and neurokinin A. These neuropeptides can act as neurotransmitters, neuromodulators, or neurotrophic-like factors and their effects are mediated by three distinct neurokinin receptors, namely neurokinin-1 (NK-1R), neurokinin-2 (NK-2R) and neurokinin-3 (NK-3R) receptors. SP preferentially binds to NK-1R, but it can also bind to NK-2R and NK-3R with hundred times less affinity that their preferred ligand, which are neurokinin A and neurokinin B, respectively. Although physiological studies have clearly shown that SP acts on nigral neurons (Walker et al., 1976; Humpel et al., 1991; Liminga et al., 1991; Keegan et al., 1992; Overton et al., 1992; Liminga, 1993; Liminga and Gunne, 1993; Stoessl et al., 1995; Nalivaiko et al., 1997), it is still not clear which types of receptors mediate these effects. A mismatch between SP binding density and SP innervation in SN was reported following standard binding studies in rodents (Rothman et al., 1984; Quirion and Dam, 1985; Dam and Quirion, 1986; Mantyh and Hunt, 1986; Maeno et al., 1993; Ribeiro-da-Silva and Hokfelt, 2000). The presence of both NK-1R and NK-3R in

0168-0102/$ – see front matter # 2006 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. doi:10.1016/j.neures.2006.11.002

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substantia nigra was later detected by means of in situ hybridization (Bannon and Whitty, 1995; Whitty et al., 1995) and immunohistochemistry (Chen et al., 1998; Mileusnic et al., 1999; Yip and Chahl, 2001). By comparison, there are only a few studies that looked for the presence of neurokinin receptors in substantia nigra of primates. One of these investigations reported the presence of NK-1R mRNA in human SNc (Whitty et al., 1997) and another detected NK-1R protein in substantia nigra extracts following Western blot analyses of in human postmortem tissue (Mounir and Parent, 2002). Precise information about the topographical and ultrastructural localization of neurokinin receptors in SNc and SNr of primates is thus lacking. This type of knowledge is essential to properly understand the role of tachykinins and their receptors in the functional organization of primate basal ganglia, in health and disease conditions. The first aim of the present study was to determine if SNc and/or SNr expresses neurokinin receptors in primates. Since the substantia nigra is practically devoid of NK-2R binding sites (Saffroy et al., 2003), we focussed essentially on the topographical distribution of NK1R and NK-3R receptors. The second aim was to describe the precise location of NK-1R and NK-3R at the subcellular level and determine whether these receptors are present at the postsynaptic or presynaptic level or both. 2. Materials and methods 2.1. Preparation of tissue sections Three male and two female squirrel monkeys (Saimiri sciureus; 700–900 g) were used in this study. The work was performed in accordance with the Canadian Guide for the Care and Use of Laboratory Animals and all surgical and animal care procedures were approved by the Institutional Animal Care Committee of Laval University. The animals were deeply anesthetised with an overdose of ketamine (75 mg/kg) plus xylazine (5 mg/kg) and perfused transcardially with saline (0.9% NaCl) solution at room temperature followed by cold solution of 4% paraformaldehyde and 15% picric acid in phosphate buffer (PB, 0.1 M, pH 7.4). Three brains were used for standard immunohistochemistry. After their removal from the skull, the brains were postfixed overnight at 4 8C in a fixative solution and cryoprotected in 30% sucrose until immersion. They were then cut with a freezing microtome into 50-mm-thick sections and serially collected in 0.1 M PBS. The two brains that were used for electron microscopy were postfixed for 1 h and were cut with a vibratome into 60-mm-thick sections.

2.2. Antisera Antibodies against NK-1R, NK-3R, tyrosine hydroxylase (TH) and SP were obtained commercially. The high specificity of these antibodies has been demonstrated previously following studies undertaken in several laboratories, including our own (Cuello et al., 1979; Aubert et al., 1997; Tooney et al., 2000; Mounir and Parent, 2002; Cossette et al., 2005). These antibodies gave highly specific labeling consistent with previous anatomical data. The anti-TH antibody was a mouse monoclonal antibody generated against TH isolated and purified from rat PC12 cells (Immunostar, Hudson, WI; #22941). As shown by Western blotting, this antibody does not cross-react with dihydropteridine reductase, dopamine-b-hydroxylase, phenylethanolamine-N-methyltransferase, phenylalanine hydroxylase or tryptophan hydroxylase (manufacturer’s information). The anti-SP was a rat monoclonal antibody (Medicorp, Montre´al, Canada) that did not cross-react with other known mammalian brain peptides (Cuello et al., 1979). The staining pattern obtained with this antibody in primate midbrain was identical to that previously reported (Inagaki and Parent, 1984). The anti-NK-1R polyclonal antiserum was raised against a

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synthetic peptide from the N-terminus fragment of the human NK-1R (residues 1–17; H2N-MDNVLPVDSDLSPNISTC-COOH; Novus Biologicals, Littleton, CO; #NB 300-119). The antiserum stains a single band of 53 kDa molecular weight on Western blot (manufacturer’s information). The anti-NK-3R polyclonal antiserum was raised against a synthetic peptide from the C-terminus fragment of the rat NK-3R that is conserved in primate NK-3R (residues 438–452; H2N-SFISSPYTSVDEYS-COOH; Novus Biologicals; #NB 300-102). The labeling was absent when the antiserum was preadsorbed with the antigenic peptide (NK-3 438–452; manufacturer’s information). This antiserum was selectively localized to fibers and astrocytes in the superficial cortical layers (Tooney et al., 2000; Yip and Chahl, 2001), which are known to express high levels of NK-3 receptors (Mileusnic et al., 1999). The specificity of this antibody has also been verified by using another antibody raised against the N-terminal fragment of the protein (Abcam, Cambridge, MA; #AB13278). The staining pattern obtained with the antibody raised against the N-terminal fragment of the NK-3R peptide was identical to that obtained with the antibody raised against the C-terminal fragment of the same protein.

2.3. Double immunofluorescence Double immunostaining procedures with fluorescent chromogens were used to evaluate the co-localization of NK-1R, NK-3R or SP with the dopaminergic markers TH. Series of sections containing the substantia nigra were placed for 30 min in a solution containing 5% normal serum and 0.2% Triton X-100. The sections were then incubated in a solution containing the primary antibody (Table 1). In these double-labeling experiments, NK-1R, NK-3R and SP were revealed with CY3-conjugated donkey IgG secondary antibodies (Jackson ImmunoResearch, West Grove, PA, 1:200), while TH was visualized with FITC-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, 1:200). Fluorescent signals were imaged by using a Zeiss LSM Pascal confocal laserscanning microscope (Oberkochen, Germany). The emission signals of CY3 and FITC were assigned to red and green, respectively. Sections incubated without the primary antibodies remained virtually free of immunostaining and served as controls.

2.4. Double pre-embedding immunohistochemistry The double pre-embedding method used in the present study was described in details elsewhere (Sik et al., 2000). In brief, sections for pre-embedding immunogold reaction were first cryoprotected in 30% sucrose in 0.1 M PB overnight and freeze-thawed in aluminium boat over liquid nitrogen to enhance the penetration of antisera without destroying the ultrastructure. Sections were then blocked for 30 min in 0.8% bovine serum albumin, 0.1% cold fish skin gelatine (Amersham, Piscataway, NJ) and 5% normal goat serum in TBS, followed by the incubation with NK-1R or NK-3R antisera for 24 h at room temperature. After incubation, sections were placed for 24 h at 4 8C with 0.5 nm gold conjugated secondary antibody (AuroProbe, Amersham, 1:80 in TBS) and immunogold particles were intensified with silver solution (IntenSE, Amersham) for 20 min. After the pre-embedding immunogold staining, sections were incubated in TBS containing the primary antibody against substance P (rat anti-SP; 1:500) for 24 h at 4 8C. Sections were then incubated for 1 h at room temperature in 0.4% biotinylated anti-rat antibody (Vector Laboratories, Burlingame, CA) followed by 1 h incubation at

Table 1 Information on the antibodies used in this study Antibody

Source

Animal source

Dilution

Incubation

NK-1R NK-3R NK-3R TH SP

Novus Biologicals Novus Biologicals Abcam Immunostar Medicorp

Rabbit Rabbit Rabbit Mouse Rat

1:100 1:500 1:500 1:500 1:500

Overnight/RT Overnight/4 8C Overnight/4 8C Overnight/4 8C Overnight/4 8C

Abbreviations: NK-1R, neurokinin-1 receptor; NK-3R, neurokinin-3 receptor; TH, tyrosine hydroxylase; SP, substance P; RT, room temperature.

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Fig. 1. Immunofluorescent confocal images showing the distribution of SP, NK-1R and NK-3R (red, left panels) in SNr and SNc of squirrel monkeys. The SNc dopaminergic neurons are identified by their immunoreactivity for tyrosine hydroxylase (TH) (green, middle panels). The photomicrographs in (a) show that SP+ fibers are more abundant in SNr than in SNc, whereas those in (b) document the presence of NK-1R+ neurons in both SNc and SNr. White arrows indicate double-

M. Le´vesque et al. / Neuroscience Research 57 (2007) 362–371 room temperature in 2% avidin–biotin complex (ABC, Vector). The SP immunoprecipitates were detected using 3,30 -diaminobenzidine (DAB; Sigma, Oakville, Canada) intensified with ammonium nickel-sulfate (DABNi). After osmication (1% osmium tetroxide in PB) for 30 min, sections were washed in PB, dehydrated in a graded series of ethanol, cleared in propylene oxide, subjected to overnight infiltration in Durcupan (Fluka, Buchs, Switzerland), flat embedded and polymerized at 57 8C for 48 h. During dehydration, the sections were treated with 1% uranyl acetate in 70% ethanol for 45 min. Selected parts of SNc and SNr were re-embedded for further ultrathin sectioning. Serial sections were collected onto formvar-coated single slot copper grids and were counterstained with lead citrate before examination on a Philips Tecnai 12 electron microscope (Eindhoven, The Netherlands) equipped with a Megaview II digital camera (Soft Imaging System, Mu¨nster, Germany). A total of 8 blocks containing SNc or SNr from two animals were used for the analysis of the subcellular distribution of NK-1R and NK-3R. Immunolabeling for SP at the fluorescence microscope level was used to delineate SNc from SNr (Fig. 1a). The quantification of the overall distribution of NK-1R and NK-3R labeling within identified presynaptic and postsynaptic elements was ensured by counting every immunogold particle in a total area of 0.5 mm2 for each receptor and for each nigral subregions (SNc and SNr). Our quantitative analysis has focussed exclusively on dendrites and axon terminals. Perikarya were found to contain numerous immunogold particles chiefly associated with membrane-delineated structures, such as the endoplasmic reticulum, transport vesicles and Golgi apparatus. The ultrastructural analysis was carried out by scanning the sections at 16,000–20,000 magnification and profiles containing gold particles were photographed at 43,000–60,000 using multiple images alignment system. Axon terminals were identified by the presence of numerous synaptic vesicles, whereas dendrites were distinguished from axons by their larger diameter and/ or abundance of uniformly distributed microtubules (see Peters et al., 1991). In addition, dendrites were postsynaptic to axon terminals. Neuronal perikarya were identified by the presence of a nucleus, Golgi apparatus, and rough endoplasmic reticulum. Each profile containing gold particle was categorized as either intracellular or associated with the plasma membrane. Gold particles have been considered as associated with the plasma membrane when they were in direct contact with the membrane. In most cases, labeled profiles contained numerous particles, but when only one gold particle was found in a given profile, several adjacent sections were examined to confirm the presence of at least two different gold particles in the same profile. Based on their localization relative to the synapses, gold particles associated with the membrane were also identified as synaptic (localized at the synaptic structure) or extrasynaptic (on membranes, outside of the synapse). In the same series of sections, every bouton containing gold particle was identified as SPimmunopositive (SP+) or SP-immunonegative (SP()). The SP+ terminals were also quantitatively examined to determine the immunogolg labeling frequency. SP+ axon terminals were thus systematically followed on 5–7 adjacent sections to verify the presence of gold particles and the type of synapses they formed. An axon terminal was considered to be selectively SP-immunoperoxidase labeled when it contained DAB precipitates in the cytoplasm that render this profile more electron dense than morphologically similar adjoining profiles. Symmetric synapses were identified by their thin preand post-synaptic specializations by comparison to asymmetric synapses, which had thick postsynaptic densities. Sections were also tilt to visualize parallelapposed membrane and thus determine the exact type of synaptic contact. In most cases, individual gold particles were clearly distinguishable from background immunostaining, but occasionally several gold particles were stuck together and formed a large electron dense proteiform structure. In such cases, if the exact number of particles could not be easily assessed, then the entire structure was counted as one particle. The pre-embedding immunohistochemical protocol used in this study provided good ultrastructural preservation and reliable identification of antigens. This technique consists of several freeze-thaw cycles that helped antibody

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penetration in thick vibratome sections. In order to avoid underestimation of positive profiles, we analysed ultrathin sections taken from the border of the resin-embedded tissue, where the access to immunoreagents is maximal. However, the absence of triton might have led to an underestimation of the number of labeled profiles.

3. Results 3.1. Double immunofluorescent labeling Double immunolabeling for SP and the dopaminergic marker TH revealed a dense SP innervation in SNr, with a much less abundant number of SP+ terminals in SNc (Fig. 1a). A careful comparative analysis of the NK-1R and NK-3R immunostaining revealed the presence of labeled neurons in both SNc and SNr (Fig. 1b–d). The two receptors were distributed according to a similar topographic pattern in the substantia nigra and most of SNc neurons displayed immunostaining for TH and NK-1R (Fig. 1c) or TH and NK-3R (Fig. 1e). Immunolabeled perikarya and dendrites were distributed rather uniformly throughout the anteroposterior axis of SNc. Only a few TH+ neurons were devoid of either NK-1R or NK-3R and these neurons were morphologically similar to the doubly labeled cells. In SNr, neurons expressing neurokinin receptors were dispersed in the whole extent of the structure and were devoid of TH immunoreactivity (Fig. 1b and d). In both SNr and SNc, a faint fiber labeling could be observed. 3.2. Subcellular localization of NK-1R and NK-3R in SNr Electron microscopic examination of NK-1R and NK-3R immunogold labeling revealed that the majority of neurokinin receptors, located in perikarya or dendrites were mainly localized intracellularly (Figs. 2–4). Indeed, about 86% (n = 200) of NK-1R and 90% (n = 335) of NK-3R immunogold particles were associated with intracellular sites, while the remaining gold particles were associated with the plasma membrane. Virtually all immunosignal on plasma membrane occurred at the edge or far from the postsynaptic membrane specialization (Figs. 2c, d, and 3d, f). In addition, immunogold labeling for both NK-1R and NK-3R was found in presynaptic elements forming symmetric synapses (Figs. 2b, d, f, and 3e, f). Analysis of SP+ boutons revealed that these terminals rarely contained immunogold labeling for NK-1R. Less than 1% (n = 200) of SP+ terminals expressed NK-1R, whereas NK-3R was present in more than 13% (n = 173) of them. In all cases, these SP+ terminals were making symmetrical synapses with dendrites or perikarya (Figs. 2 and 3). Immunogold particles in presynaptic axon terminals were located either inside the terminals (Figs. 2b, d, e, and 3f) or on the plasma membrane at a certain distance from the synaptic specialization (Figs. 2f, and 3e, f).

labeled neurons in SNc. High power views of SNc dopaminergic neurons expressing NK-1R are provided in (c), while the presence of NK-3R+ neurons in both SNc and SNr is documented in (d). High power views of SNc dopaminergic neurons expressing NK-3R are provided in (e). Dashed lines in (b) and (d) are landmarks that trace the border between SNc and SNr. Abbreviations: D, dorsal; M, medial. Scale bars: (a) 500 mm, (b) and (d) 100 mm, (c) and (e) 50 mm.

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Fig. 2. Electron photomicrographs showing NK-1R immunogold labeling and SP+ terminal boutons in SNr. The SP immunolabeling was revealed with immunoperoxidase method and appears as a dark, homogeneous precipitate. The small black arrows in all pictures point to SP+ terminal boutons forming symmetric synapses on dendrites. The gold particles indicative of the presence of NK-1R are more numerous in the cytoplasmic compartment (see large arrows in a– e), whereas the gold particles associated with the membranes occupy extrasynaptic positions (see arrowheads in c, d and f). Axonal terminals occasionally contained gold particles (white arrows in b, d, e and white arrowhead in f), but only a few SP+ terminals contain presynaptic immunogold labeling. Abbreviations: B, axonal bouton; D, dendrite; SP, substance P+ bouton. Symbols: thin black arrows, symmetric synapse; large black arrows, intracellular gold particle in dendrite; large white arrows, intracellular gold particle in axonal bouton; black arrowhead, gold particle associated with dendritic membrane; white arrowhead, gold particle associated with axonal membrane. Scale bars: 500 nm.

3.3. Subcellular localization of NK-1R and NK-3R in SNc Overall, the immunogold labeling for NK-1R and NK-3R in SNc followed a pattern similar to that seen at the SNr level (Figs. 5 and 6). Immunostaining for NK-1R and NK-3R occurred in perikarya (not shown) and dendritic processes. Here again, the immunogold particles were preferentially associated with intracellular sites (Figs. 4–6) or located at extrasynaptic position in the plasma membrane (Figs. 5 and 6b). Axon terminals forming symmetric contact with SNc neurons also displayed immunostaining for NK-1R and NK-3R (Figs. 5 and 6c, d). Immunogold particles were found inside the terminals or

located on plasma membrane at extrasynaptic position. Less than 1% (n = 120) of SP+ terminals expressed NK-1R, whereas NK-3R gold particles were seen in more than 4% (n = 111) of SP+ terminal boutons. 4. Discussion The substantia nigra is the brain structure that is most densely innervated by SP+ fibers (Ribeiro-da-Silva and Hokfelt, 2000). Despite numerous physiological studies indicating that SP exerts significant effects upon nigral neurons, several studies reported the lack of SP receptors at

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Fig. 3. Electron photomicrographs depicting NK-3R immunogold labeling and SP+ terminal boutons in SNr. Some SP+ terminal boutons in contact with NK-3R+ perikarya are illustrated in (a and b), while the remaining photomicrographs depict contacts with dendrites (c–f). In all cases, the majority of gold labeling occurred intracellularly (see examples in a–c and e), and in cases where immunogold signal was associated with the membrane, gold particles occurred at extrasynaptic position (see arrowhead in d and f). Occasionally both SP+ and SP() boutons contained gold particles (white arrowheads and arrows in e and f). Abbreviation: P, perikarya; see Fig. 2 for other abbreviations and symbols. Scale bars: 500 nm.

nigral level (for review, see Ribeiro-da-Silva et al., 2000). More recently, in situ hybridization and immunocytochemical investigations have provided evidence for the existence of SNc neurons expressing NK-1R and NK-3R receptors in rodents (Whitty et al., 1995; Chen et al., 1998; Futami et al., 1998; Mileusnic et al., 1999; Yip and Chahl, 2001). In primates, however, only one study reported that neurons in human SNc express NK-1R mRNA (Whitty et al., 1997). The authors of the latter study were unable to detect NK-3R and could not determine if SNr neurons express neurokinin receptors in humans. In the present study, the use of highly specific antibodies has allowed us to document the cellular and subcellular localization of tachykinin NK-1 and NK-3 receptors in the substantia nigra of squirrel monkeys. The two receptors

were found in neurons of both SNc and SNr, but they also occurred on axons terminals making symmetrical synapses, including some that contained SP. Our results are summarized in Fig. 7. 4.1. Receptor internalization As many G-protein-coupled receptors, neurokinin receptors have been reported to be internalized after binding to their ligands (Mantyh et al., 1995a,b). The receptor is recycled to the plasma membrane or shuttled to different intracellular compartments, in which it might influence a variety of biological processes. In the present study, the neurokinin labeling for either NK-1 or NK-3 receptors occurred chiefly at

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stimulation. Indeed, a recent study performed in rat has demonstrated a shift in the presence of NK-1R located at intracellular or at plasma membrane position on midbrain neurons following auditory stimulation or after injection of apomorphine, a dopaminergic agonist (Lessard and Pickel, 2005). These data suggest that the neurokinin receptor system could easily adapt itself to environmental changes. A similar degree of plasticity may also characterize the subcellular localization of neurokinin receptors in primate substantia nigra, which could lead to a marked change in the response to SP and other tachykinins. 4.2. Substance P labeling in substantia nigra The topographical distribution of SP immunoreactivity in the squirrel monkey substantia nigra reported here is congruent with the results of previous studies on axonal and subcellular distribution of SP in the substantia nigra of non-human primate (Bolam and Smith, 1990). Most SP+ axon terminals formed symmetric synapses with postsynaptic nigral neurons, many of which contain either NK-1R or NK-3R. Striatonigral fibers are known to form symmetric synapses with nigral neurons and to contain both GABA and SP (Graybiel, 1990). The nigral release of SP from GABAergic terminals might thus activate NK-1 and/or NK-3 receptors located on nigral neurons and modulate their postsynaptic inhibitory responses to GABA. 4.3. Substance P autoreceptor on striatonigral afferent

Fig. 4. Histograms showing the distribution of immunogold particles indicative of the presence of NK-1R or NK-3R in postsynaptic dendrites, presynaptic SP+ axon terminals and presynaptic SP() terminals. Black and light grey bars indicate gold particles in the cytoplasm and gold particles associated with plasma membrane, respectively. The analysis was carried out on SNr (a) and SNc (b) from two monkeys and a total of 460 and 657 gold particles were counted, respectively.

the intracellular level. This high intracellular labeling might reflect high levels of internalization triggered by a massive release of neurokinins. This could explain the mismatch between SP innervations and SP receptors localization described in the initial studies undertaken with radiolabeled SP (Shults et al., 1982; Mantyh et al., 1984; Rothman et al., 1984; Dam and Quirion, 1986). Moreover, when delivered directly into the striatum SP was found to induce a massive internalization of SP receptors followed by morphological reorganization of cells; for example, distal dendrites stimulated by SP underwent a morphological change from a tubular to a varicose shape (Mantyh et al., 1995a). The high degree of receptor internalization reported in the present study provides an anatomical basis for future studies aiming at defining the role of neurokinin receptors in the regulation of signalling and gene expression in nigral neurons. The relative number of neurokinin receptors present at the plasma membrane or within the cytosol has also been reported to change in response to environmental

The presence of NK-1R and NK-3R on some axon terminals forming symmetric synapses with nigral neurons, as demonstrated in the present study, strongly suggests the existence of neurokinin receptors on nigral GABAergic afferents. A large number of striatal neurons projecting to the substantia nigra are known to contain both GABA and SP (Bolam and Smith, 1990). We report here that a small proportion of SP+ axon terminals in the substantia nigra express NK-3R and, to a lesser extent, NK1R immunoreactivity. This implies that SP of striatal origin may regulate its own transmitters release at nigral level mainly through NK-3R. This finding also suggests that striatonigral SP terminals could be regulated by other neurokinin inputs, such as NKA or NKB. However, early studies on NKB localization in the brain have reported only few NKB containing fibers at the nigral level (Lucas et al., 1992; Marksteiner et al., 1992). Because of the lack of physiological studies on co-release of SP and GABA, it is hard to speculate on the effect of SP release on presynaptic striatal terminals at nigral level. 4.4. Substance P heteroreceptor Besides the demonstration of presynaptic neurokinin receptors on SP afferents, our results also reveal that neurokinin receptors are present on SP-immunonegative terminals that form symmetric synapses in both SNc and SNr. These GABAergic axon terminals could correspond to pallidal inputs, which are known to contact neurons in both SNc and SNr (Bolam and Smith, 1990). A proportion of the GABAergic axon

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Fig. 5. Electron photomicrographs depicting NK-1R immunogold labeling and SP+ terminal boutons in the SNc. Example of intracellular gold particles in dendrite (a), gold particle associated with membrane at extrasynaptic position (arrowhead) (b), and presynaptic gold particle in axonal boutons (c and d). See Fig. 2 for abbreviations and symbols. Scale bars: 500 nm.

Fig. 6. Electron photomicrographs illustrating NK-3R immunogold labeling and SP+ terminal boutons in SNc. NK-3R labeling was observed intracellularly in the majority of cases (large arrows in a, c and d). Both SP+ and SP() axon terminals contained gold particles (large white arrows in c and d). Note that gold particles associated with membranes occurred at extrasynaptic sites (arrowhead in b). See Fig. 2 for abbreviations and symbols. Scale bars: 500 nm.

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the significance of the co-release of an inhibitory neurotransmitter and an excitatory neuropeptide, a phenomenon that might play a crucial role in the functional organization of basal ganglia. Acknowledgements The authors express their gratitude to Mr. Philippe Lemieux and Ms. Mia Brockop for skilful technical assistance. This study was supported by grant MT-5781 of the Canadian Institutes of Heath Research to A.P. Fig. 7. Schematic drawing depicting the localization of neurokinin receptors in primate substantia nigra. NK-1R and NK-3R occurred chiefly within the cytosol or at extrasynaptic position on the plasma membrane at both dendrite and axon terminal levels. Neurokinin receptors are also present on afferent axon terminals suggesting that, in addition to its action at the postsynaptic level, SP also exerts its nigral influence through presynaptic autoreceptors and heteroreceptors by acting on nigral afferent, including through GABAergic local axon collaterals. Abbreviation: GPm, Globus pallidus medialis.

terminals in the substantia nigra could also originate from SNr neurons, which are known to heavily innervate neurons in both SNr and SNc (Juraska et al., 1977; Karabelas and Purpura, 1980; Grofova et al., 1982; Mailly et al., 2003). The localization of neurokinin receptors on these terminals might indicate that SP influences GABA release from pallidal input as well as from nigral local network. The existence of postsynaptic neurokinin receptors at extrasynaptic sites on nigral neurons suggests that SP (and/ or NKA) could act at distance from their release site providing slow response at postsynaptic sites. Detailed physiological studies are obviously required to validate this hypothesis. Several investigations have reported that neurokinins mediate excitatory effects in both SNc and SNr (Reid et al., 1990; Humpel and Saria, 1993; Nalivaiko et al., 1997; Levesque et al., 2003), but the lack of studies on GABA and neurokinin peptides co-transmission opens the door to different interpretations. Even if they occurred in the same terminals, GABA and SP are not stored in the same type of vesicles. Neuropeptides such as SP are stored in large dense core vesicles, whereas GABA is sequestered in small clear vesicles (Lundberg, 1996). Although these two types of vesicles have similar secretory machinery, they differ by their release kinetics (Martin, 1994; Bruns and Jahn, 1995; Park et al., 2006). Indeed, peptides like SP packaged in large dense core vesicles undergo slow release upon prolonged stimulation, whereas secretion of amino acids like GABA occurs rapidly in response to a single action potential. Thus, slow and phasic neuronal firing could led to the release of GABA, which would result in the generation of inhibitory potentials at the postsynaptic target. In contrast, high tonic neuronal firing could led to the release of both GABA and SP, the latter being able to reverse the GABAergic postsynaptic inhibition. Interestingly, these two types of firing mode have been reported in studies with freely moving animal, where striatal neuron fires at low frequency when the animal is at rest but produce burst of discharge during movements (Patino et al., 1995). Obviously, much more studies are needed to understand

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