Innervation sérotoninergique des ganglions de la base

Innervation sérotoninergique des ganglions de la base

SEROTONIN INNERVATION OF HUMAN BASAL GANGLIA

Marie-Josée Wallman, Dave Gagnon, Martin Parent
Centre de recherche CERVO 2601, Ch. de la Canardière, Québec, Québec Canada G1J 2G3
European Journal of Neuroscience (2011) 33: 1519-1532

Résumé

Cette étude procure la toute première description détaillée de l’innervation sérotoninergique (5-HT) des ganglions de la base chez l’humain, en condition normale. Nous avons utilisé une approche immunohistochimique sur du tissu post-mortem avec des anticorps dirigés contre le transporteur de la sérotonine et l’enzyme de synthèse de la 5-HT (tryptophane hydoxylase) afin de visualiser respectivement les axones et les corps cellulaires 5-HT. Des sections adjacentes ont été immunomarquées à la tyrosine hydroxylase dans le but de pouvoir comparer la distribution des axones 5-HT avec celle des axones dopaminergiques. Les ganglions de la base chez l’humain sont innervés par des axones 5-HT qui proviennent principalement du noyau raphé dorsal et, de manière moins abondante, du noyau raphé médian. Ces axones forment des faisceaux larges ascendants qui se fragmentent en pénétrant dans la décussation supérieure des pédoncules cérébelleux. Ils se regroupent ensuite au sein de l’aire tegmentaire ventrale et montent le long du faisceau prosencéphalique médian, immédiatement sous les fibres dopaminergiques ascendantes. À des intervalles réguliers, les axones 5-HT se détachent du faisceau prosencéphalique médian et se déplacent latéralement pour s’arboriser à l’intérieur de toutes les composantes des ganglions de la base, où ils montrent des densités et des patrons d’innervations des plus variables. La substance noire est la composante des ganglions de la base la plus densément innervée, tandis que le noyau caudé reçoit une innervation plus hétérogène que le putamen et le globus pallidus. Le noyau subthalamique arbore des fibres immunoréactives 5-HT qui montrent un gradient décroissant mediolatéral. Le fait que toutes les composantes des ganglions de la base reçoivent une innervation 5-HT dense indique que, de concert avec la dopamine, la 5-HT joue un rôle crucial dans l’organisation fonctionnelle de ces structures.

Abstract

This study aimed at providing a first detailed description of the serotonin (5-hydroxytryptamine, 5-HT) innervation of the human basal ganglia under nonpathological conditions. We have applied an immunohistochemical approach to postmortem human brain material, with antibodies directed against the 5-HT transporter and the 5-HT synthesizing enzyme (tryptophane hydroxylase) to visualize 5-HT axons and cell bodies, respectively. Adjacent sections were immunostained for tyrosine hydroxylase to compare the distribution of 5-HT axons with that of dopamine axons. Human basal ganglia are innervated by 5-HT axons that emerge chiefly from the dorsal and, less abundantly, from the median raphe nuclei. These axons form thick ascending fascicles that fragment themselves as they penetrate the decussation of the superior cerebellar peduncle. They regroup within the ventral tegmental area and ascend along the medial forebrain bundle, immediately beneath the dopamine ascending fibers. At regular intervals along their course, 5-HT axons detach themselves from the medial forebrain bundle and sweep laterally to arborize within all basal ganglia components, where they display highly variable densities and patterns of innervation. The substantia nigra is the most densely innervated component of the basal ganglia, whereas the caudate nucleus is more heterogeneously innervated than the putamen and pallidum. The subthalamic nucleus harbors 5-HT immunoreactive fibers that display a mediolateral-decreasing gradient. The fact that all components of human basal ganglia receive a dense 5-HT input indicates that, in concert with dopamine, 5-HT plays a crucial role in the functional organization of these motor-related structures, which are often targeted in neurodegenerative diseases.

Introduction

Serotonin (5-hydroxytryptamine, 5-HT) is involved in multitudinous functions, such as the regulation of sleep-waking cycle (Steriade, 2004; Jones, 2005; Monti & Jantos, 2008), the modulation of pain signals (Sommer, 2004) and the pathogenesis of mood disorders (Owens & Nemeroff, 1994; Cools et al., 2008). Such a multifaceted role of 5-HT is possible because this monoamine is produced and released by a widely distributed neuronal system that reaches virtually all major brain structures (Parent, 1996). Endowed with a markedly collateralized axon, 5-HT neurons have their cell body confined to the raphe nuclei, which occur in all vertebrate species (Parent et al., 1984; Parent, 1986). Originally divided into nine entities (groups B1-B9 of Dahlstroem & Fuxe, 1964a), they are actually considered to form a small caudal and a large rostral group having distinct efferent projections (Tork, 1990; Hornung, 2003; Monti & Jantos, 2008). The caudal group comprises medullary raphe nuclei, which project to the spinal cord whereas the rostral group, scattered along the pons and midbrain, contains the dorsal (DRN, B6 and B7) and median (MRN, B8) raphe nuclei, which supply about 85% of the 5-HT forebrain innervation (Parent, 1996; Hornung, 2003; Monti & Jantos, 2008).
Among the various forebrain structures that receive a particularly dense 5-HT innervation are the basal ganglia, which play a crucial role in the control of motor behavior (Di Matteo et al., 2008). The density and distributional pattern of 5-HT axons vary significantly from one basal ganglia component to the other. This has been clearly established by various mapping studies undertaken in rats (Moore et al., 1978; Parent et al., 1981; Steinbusch, 1981; Mori et al., 1985a; Mori et al., 1985b; Mori et al., 1987; Harding et al., 2004; Parent et al., 2010) and monkeys (Schofield & Everitt, 1981; Mori et al., 1985a; Mori et al., 1985b; Azmitia & Gannon, 1986; Mori et al., 1987; Lavoie & Parent, 1990; Parent et al., 2010). In humans, morphological studies of the 5-HT neuronal system have focused principally on the raphe nuclei (Baker et al., 1991a; Baker et al., 1991b; Hornung, 2003). Some biochemical studies have provided useful information on the 5-HT content of human basal ganglia in both health and disease conditions (Lloyd et al., 1974; Walsh et al., 1982; Hornykiewicz, 1998; Kish et al., 2008). However, except for pioneering data on ascending 5-HT axons gathered from embryonic brain tissue (Olson et al., 1973), little information is available on the morphological aspect of the 5-HT innervation of the basal ganglia in man.
This lack of knowledge has prompted us to undertake a detailed immunohistochemical study of the organization of the 5-HT innervation of human basal ganglia using antibodies against the 5-HT transporter (SERT) and tryptophan hydroxylase (TPH), the rate-limiting enzyme in 5-HT synthesis. These antibodies were applied to human postmortem material obtained from healthy adult individuals. Such knowledge will hopefully help us comprehend the role of 5-HT at basal ganglia level and better interpret the complex neurochemical changes that occur within these nuclei in neurodegenerative diseases.

Materials and Methods

Tissue preparation

Morphological descriptions are based on the analysis of postmortem material obtained from 8 men and 1 woman with no clinical or pathological evidence of neurological or psychiatric disorders. The mean age and postmortem delays are 47.1 8.2 years and 11.6 0.7 h, respectively (Table 1). The material was taken from the brain bank established at the Centre de Recherche Université Laval Robert-Giffard (CRULRG). Brain banking and postmortem tissue handling procedures were approved by the Ethic Committee of the Centre Hospitalier Robert-Giffard and by Université Laval. The brains were obtained with written consents and the analyses were performed in conformity with the Code of Ethics of the World Medical Association (Declaration of Helsinki).
The nine brains were sliced in half along the midline; the right side of the brain served for neuropathological examination, while the left side was used for immunohistochemical investigation. The latter hemi-brains were sliced unfixed into 2 cm-thick slabs along the coronal or sagittal plane and the slabs were fixed by immersion in 4% paraformaldehyde at 4°C for 3 days. They were then stored at 4°C in a 0.1M phosphate-buffered saline (PBS, pH 7.4) solution containing 15% sucrose and 0.1% sodium azide. The slabs containing the basal ganglia and adjacent structures were cut with a freezing microtome into 50 µm-thick sections that were serially collected in PBS and stored at –20°C in a solution containing glycerol and ethanediol until immunostaining.
A detailed view of the overall pattern of the 5-HT innervation of the human basal ganglia was obtained by examining serial coronal sections taken at four different caudorostral levels: through the mid portion of the subthalamic nucleus (STN, level 1); through the maximally developed pallidal complex (level 2); through the anterior commissure (level 3); and through the pre-commissural portion of the striatum (level 4). These four levels correspond respectively to levels 21, 27, 35 and 39 of the human brain atlas of Mai and colleagues (Mai et al., 2008). Sagittal sections covering the rostral portion of the pons, the entire midbrain and the caudal portion of the forebrain were also examined to visualize the 5-HT cell bodies of the DRN and MRN and to obtain an overall view of the initial trajectory of the 5-HT ascending axons. Complete sets of coronal sections taken at three caudorostral levels of STN were also used to provide a detailed picture of the 5-HT innervation of this basal ganglia key component.

Antibodies

Antibodies raised against 5-HT itself have been successfully used to study the 5-HT system in the brain of rats (Steinbusch, 1981) and monkeys (Lavoie & Parent, 1990), but this approach cannot be applied to human postmortem tissue because 5-HT is rapidly dissipated from neurons after death.
Morphological studies of the human 5-HT system have to rely on the detection of various 5-HT-associated proteins, which are preserved in their natural state and location for many hours after death and are thus much more easily detectable with immunohistochemical procedures than 5-HT (Tork et al., 1992). In the present study, the human 5-HT neuronal system was visualized with the help of two complementary markers: (a) the 5-HT biosynthetic enzyme tryptophan hydroxylase (TPH), which predominates in the somatodendritic domain of 5-HT neurons, and (b) the 5-HT transporter (SERT), which abound in 5-HT axons and axon varicosities (terminals) (Qian et al., 1995). More specifically, we used a mouse monoclonal antibody against TPH (catalog # 10678; Sigma, St-Louis, MO, USA) and a goat polyclonal SERT antiserum (catalog # sc-1458; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The preparation and characterization of the TPH antibody, including the demonstration of its specificity by preabsorption and Western blot, have been described elsewhere (Haycock et al., 2002). The distribution of the immunostaining with this antibody matched that of 5-HT neurons, without any cross-reactivity with midbrain dopamine (DA) neurons (Haycock et al., 2002). The SERT antiserum was raised against 20 amino acids in the C-terminal of the human SERT. It was affinity-purified and characterized by Western blot in brain tissue (Santa Cruz Biotechnology). This anti-SERT is considered a faithful marker of 5-HT neurons at both light and electron microscopic levels (Pickel & Chan, 1999; Huang et al., 2004), and its production and characterization have been reported in detail elsewhere (Zhou et al., 1996). Complementary studies were performed with antibodies against either the enzyme tyrosine hydroxylase (TH, catalog # 22941; ImmunoStar, Hudson, WI, USA), the neuroactive peptide enkephalin (ENK, provided by Dr. Claudio Cuello, McGill University) or the dopamine-β-hydroxylase (DBH, catalog # AB1538; Chemicon, Temecula, CA, USA). The monoclonal antibody against TH was used principally to determine the relative position of the 5-HT and DA axons as they ascend towards the basal ganglia. Because TH, the rate-limiting enzyme in the synthesis of catecholamines, is also expressed by noradrenergic neurons, immunohistochemistry for DBH, the enzyme that transforms DA into noradrenalin, was used to label noradrenergic axons in the human basal ganglia. The rabbit polyclonal antibody against the N-terminal 18 amino acid of the human DBH has been previously well characterized (Nagatsu et al., 1990). The monoclonal antibody against ENK served essentially to visualize the striosomal compartment of the human striatum. This antibody did not distinguish between Met- and Leu-enkephalin and details about its production and characterization can be found elsewhere (Cuello et al., 1984). The TH, DBH and ENK antibodies were routinely used in previous studies of the human basal ganglia (see Prensa et al., 1999; Prensa et al., 2000; Huot et al., 2007).

Immunohistochemistry

Complete series of coronal sections taken at 300 µm interval and covering the entire caudorostral extent of the basal ganglia (levels 1 to 4) of brains 1, 2 and 5-9, were processed for the demonstration of SERT immunoreactivity. After three rinses in PBS, the sections were placed for 30 min at room temperature in hydrogen peroxide (3% H2O2 in ethanol) to eliminate endogenous peroxidase activity. The free-floating sections were then preincubated for 30min at room temperature in a PBS solution containing 2% normal rabbit serum (NRS) and 0.1% Triton X-100, and incubated overnight at 4°C in the same solution to which the goat anti-SERT antibody was added (Santa Cruz Biotechnology; 1:500). The sections were then rinsed, reincubated for 1 h at room temperature with a rabbit anti-goat biotinylated IgG (catalog # BA-5000; Vector Labs, Burlingame, CA, USA; 1:500). After three rinses in PBS, the sections were reincubated for 1 h at room temperature in 2% avidin-biotin-peroxidase complex (catalog # PK-4000; Vector Labs). The bound peroxidase was revealed by placing the sections in a medium containing 0.05% 3,3′-diaminobenzidine tetrahydrochloride (DAB, catalog # D5637; Sigma) and 0.005% H2O2 in 0.05 M Tris buffer, pH 7.6. The reaction was stopped after 4 min by several washes in Tris buffer and PBS. The mapping of 5-HT neuronal profiles was complemented by incubating complete sets of sagittal sections taken from brains 3 and 4 alternatively with either goat anti-SERT or mouse anti-TPH (Sigma; 1:250). The procedure was the same as above except that normal horse serum (NHS) and a horse anti-mouse biotinylated IgG (catalog # BA-1300; Vector Labs; 1:250) were used in cases of sections immunostained for TPH. Furthermore, coronal sections taken through the entire caudorostral extent of the striatum (levels 3 and 4) of brain 1 were incubated with the anti-SERT antibody (same as above), while the adjacent sections were incubated with the mouse anti-ENK antibody (Cuello; 1:50). This set of experiments was designed to compare the density of the 5-HT innervation between the ENK-rich striosomes and the ENK-poor extrastriosomal matrix of the striatum. Coronal sections taken from brain 6 at the locus coeruleus and the post-commissural striatal levels were incubated with the rabbit anti-DBH antibody (Chemicon; 1:250) to label noradrenergic cell bodies and axons, respectively. All immunostained sections were mounted on gelatine-coated slides. Those that were incubated without the primary antibodies remained unstained.

Immunofluorescence

A double immunofluorescence labeling approach was used to compare, on single coronal sections, the location of the SERT and TH immunoreactive axons that both ascend towards the basal ganglia by coursing along the same forebrain pathways. Hence, serial sections from brains 1, 2 and 5, were remove from their storage medium and rinsed in PBS. They were preincubated for 1 h in a PBS solution containing 2% NHS and 0.1% Triton X-100 at room temperature and then incubated overnight at 4°C in the same solution to which two primary antibodies were added, one directed against SERT (goat anti-SERT, Santa Cruz Biotechnology; 1:250) and the other against TH (mouse anti-TH, ImmunoStar; 1:500). After rinses in PBS, sections were incubated at room temperature in a solution containing a rabbit anti-goat biotinylated IgG (Vector Labs; 1:250). After additional rinses in PBS, sections were incubated for 2 h at room temperature in the dark, in a PBS solution containing Alexa 488-conjugated streptavidin (cataolog # S-11223; Molecular Probes, Eugene, OR, USA; 1:200) and Alexa 555-conjugated rabbit anti-mouse (catalog # A-21427; Molecular Probes; 1:200). The sections were finally rinsed three times in PBS and mounted on gelatine-coated slides. Once dried, sections were treated with an autofluorescence eliminator reagent (catalog # 2160; Chemicon). They were then cover slipped with a fluorescent mounting medium (catalog # S3023; DAKO Corporation, Carpinteria, CA, USA). Sections incubated without the primary antibodies remained virtually free of immunofluorescence.

Material analysis

The immunostained sections were observed and photographed under both bright and darkfield illuminations with a microscope equipped with a digital camera and a camera lucida (Leitz DM RB; Leica, Onrario, Canada). The location of the 5-HT axons and terminals in each of the various components of the human basal ganglia were precisely mapped on four drawings (levels 1-4) derived from the atlas of Mai and colleagues (Mai et al., 2008). The location of the 5-HT cell bodies in DRN and MRN and the initial trajectory of their ascending axons were further studied by analyzing serial parasagittal sections covering 1,800 µm on each side of the midline. We generated a composite picture of it by drawing a typical section extending from the upper pons to the lower forebrain upon which the distribution of the 5-HT neurons was mapped out following observations made on alternate sections immunostained for either SERT or TPH. The location of the 5-HT cells bodies in the DRN and MRN nuclei and their ascending axonal projections were drawn with the help of a camera lucida. A particularly detailed analysis of the 5-HT innervation of STN was undertaken by examining serial SERT-immunostained coronal sections from brain 1 taken at caudal, middle and rostral thirds of the STN. A camera lucida was used to delineate the STN contours and to draw the 5-HT axonal arborization within this basal ganglia component. All diagrams derived from the analysis of coronal or sagittal sections were generated using Canvas X Software (ACDSystems International Inc,Victoria, Canada). Darkfield photomicrographs of SERT-immunolabeled sections were used to examine the density of 5-HT innervation in striosomes after being delineated on ENK-immunostained adjacent sections. Immunofluorescence sections used to compare the relative distribution of SERT and TH immunoreactive axons were observed with a confocal laser-scanning microscope LSM 5 PASCAL (Zeiss, Oberkochen, Germany). The emission signals of Alexa 488 (SERT) and Alexa 555 (TH) were assigned to green and red, respectively. Photomicrographs were handled with the Adobe Photoshop CS4 software (Adobe, San Jose, CA, USA).

Results

Ascending serotonin pathways

The origin and initial trajectory of the ascending 5-HT fibers were particularly well outlined on human brainstem sections cut along the sagittal plane (Fig. 1). These thick and nonvaricose SERT-immunopositive (+) fibers (Fig. 1B, C) emerge from the cell bodies of the DRN and, less abundantly, from those of the MRN (Fig. 1D, E). They arch rostroventrally and traverse the central portion of the midbrain tegmentum. As they penetrate the decussation of the superior cerebellar peduncles they break out into a multitude of small and compact fascicles that form a strikingly intricate network. More rostrally, these fibers collected themselves dorsomedially to the substantia nigra in the form of a rather diffuse bundle that passes partly through the ventral tegmental area and ascends within the lateral hypothalamic area along the medial forebrain bundle (Figs. 1A, 2A). Despite its overall diffuseness, the core of this bundle is nevertheless composed of two smaller fascicles located one above the other and which are particularly visible in the caudal portion of the bundle (Fig. 3C), as it courses lateral to the mammillary bodies. The bundle of SERT+ fibers tapers as it ascends within the lateral hypothalamic area because several immunoreactive fascicles detach themselves from it at different caudorostral levels. These fascicles sweep laterally to innervate various components of the basal ganglia, such as the STN, globus pallidus and putamen, where both thin and varicose, and thick and beaded SERT+ fibers can be found in unequal number (Fig. 4).

Substantia nigra

The substantia nigra is by far the most densely innervated component of the human basal ganglia (Fig. 2A). The nigral 5-HT innervation derives from axons that reach the structure mainly from its caudal border and arborize profusely immediately upon entering the susbtantia nigra. In fact, very few thick and poorly beaded SERT+ axons occur within the confines of the substantia nigra, although some immunoreactive fiber fascicles course along its dorsolateral surface. Within the substantia nigra, SERT+ axons and terminals are rather uniformly distributed throughout the caudorostral and mediolateral extent of the structure. No significant difference could be noted between the pars compacta and the pars reticulata of the substantia nigra in regards to the density of the 5-HT innervation (Fig. 2A). The 5-HT innervation of the substantia nigra derives principally from thin and varicose axons, which outnumber the thick and poorly beaded axons. Some SERT+ axon varicosities were seen in close apposition to the cell bodies of pars compacta neurons, which could easily be delineated by virtue of their neuromelanine content (Fig. 4E, F).

Subthalamic nucleus

The SERT+ axons observed in STN derive chiefly from one fiber fascicle that detaches itself from the main bundle coursing in the lateral hypothalamic area, to run along the dorsal surface of STN (Fig. 2A). A smaller immunoreactive fascicle that courses along the ventral surface of STN appears also to contribute, albeit less importantly, to the STN 5-HT innervation (Fig. 3). The STN contains a small number of thick and beaded SERT+ varicose fibers and only a few thin and varicose fibers (Fig. 4D). These immunoreactive fibers are distributed according to a clear mediolateral decreasing gradient, whereas the isolated SERT+ axon varicosities present within the nucleus appear equally distributed throughout the structure (Fig. 3). Overall, the density of the 5-HT innervation of STN is about half of that of the substantia nigra (Fig. 2A).

Pallidum

The human pallidal complex receives a moderate 5-HT innervation that derives from several fascicles leaving the main SERT+ fiber bundle at various points along its courses within the lateral hypothalamic area. At caudal pallidal levels, some of the SERT+ fibers that run along the dorsal surface of STN continue their route laterally and pierce the internal capsule to reach the pallidum along its dorsal border (Fig. 2). At more rostral levels, other fascicles sweep laterally and penetrate the pallidum by piercing its ventromedial aspect (Fig. 2B). Other ventrally coursing fibers turn dorsally and invade the various medullary laminae that separate the different components of the lenticular nucleus (Fig. 2B). Some of these medullary SERT+ fibers continue their course dorsally through the internal capsule to finally reach the caudate nucleus, whereas others turn perpendicularly to enter the putamen.
Overall, SERT+ fibers reaching the pallidum arborize more profusely in the internal segment than in the external segment, although much variation exists along the caudorostral axis. In the caudal third of the pallidum (Fig. 2A), the number of SERT+ fibers is similar than in the STN and they appear rather uniformly distributed throughout the structure. Thick and beaded fibers occur as frequently as thin and varicose fibers at this level (Fig. 4B). The medial third of the pallidal complex (Fig. 2B) harbors a much larger number of SERT+ fibers than its caudal third. These fibers are mostly thick and beaded (Fig. 4C) and abound preferentially within the internal segment of the pallidum, where they do not display any preferential orientation. A relatively small number of isolated SERT+ axon varicosities are uniformly distributed throughout the two pallidal segments (Fig. 2B). By comparison, the rostral third of the pallidal complex contains a much smaller number of SERT+ fibers, which are mostly of the thick and weakly varicose type, and only scarce and uniformly distributed isolated axon varicosities.

Striatum

The striatum is by far the most heterogeneously innervated component of the basal ganglia.Overall, the 5-HT innervation of the caudate nucleus is slightly weaker than that of the putamen, but these two components of the striatum share a similar degree of heterogeneity in regard to the distribution of SERT+ fibers and axon varicosities. At caudal levels (Fig. 2A), SERT+ fibers and axon terminals are distributed within the putamen according to a clear dorsoventral gradient, the immunopositive axon profiles being much more abundant ventrally than dorsally in the structure. The tail of the caudate nucleus at the same level harbors a few SERT+ fibers and a moderate number of uniformly distributed SERT+ axon terminals, except for a densely innervated zone at the ventral border of the structure (Fig. 2A). At post-commissural levels (Fig. 2B), SERT+ fibers and axon terminals are much more uniformly distributed within the striatum, except for the dorsal border of the body of the caudate nucleus, where immunopositive axon terminals are less abundant than in the rest of the striatum. The two types of SERT+ fibers occur in about equal proportion in the post-commissural striatum (Fig. 4A). At commissural levels (Fig. 2C), the putamen harbors a large number of rather uniformly distributed SERT+ fibers of both thick/beaded and thin/varicose types. In contrast, isolated SERT+ axon varicosities abound preferentially in the dorsal two-thirds of the putamen. By comparison, the caudate nucleus displays a smaller number of uniformly distributed SERT+ axon varicosities, except for two small zones located within its ventral aspect that are densely innervated (Fig. 2C). At pre-commissural levels (Fig. 2D), only a few SERT+ fibers are homogeneously distributed throughout the striatum, whereas innumerable SERT+ axon varicosities are heterogeneously scattered throughout the structure. These axon terminals abound principally in the dorsal two-thirds of both the putamen and the head of the caudate nucleus, leaving the ventral striatum, including the nucleus accumbens, comparatively less densely innervated (Fig. 2D).
Apart from the striatum, several long, thick and beaded SERT+ fibers also occur throughout the entire caudorostral extent of the basal ganglia within the external medullary lamina that separates the putamen from the claustrum (Fig. 2). They course ventrodorsally in a rather linear fashion within the lamina (Fig. 2C2), spread out in the white matter above the claustrum and finally arborize within the various layers of the cerebral cortex. On the other hand, the detailed examination of adjacent sections stained for enkephalin, a faithful marker of the striosomal compartment of the striatum, and SERT, revealed that the striatal 5-HT innervation do not obey the striosomal-matrix compartmentalization in human. The density and morphological characteristics of SERT+ fibers and axon varicosities are strikingly similar in the two striatal compartments, the striosomes and the extrastriosomal matrix (Fig. 5).

Ascending SERT and TH immunoreactive axons

The double-immunofluorescence approach has enabled us to compare the course of the SERT and TH-immunolabeled axons that ascend towards the human basal ganglia on single sections taken through two distinct caudorostral levels. The first caudal level corresponds to the region where STN is maximally developed, defined above as level 1 (Fig. 6A), whereas the rostral level corresponds and to the region where the pallidal complex is maximally developed, defined above as level 2 (Fig. 6E). The more rostral levels, corresponding to that of the anterior commissure (level 3) and the pre-commissural portion of the striatum (level 4), were not analyzed because the SERT+ and TH+ fibers in these rostral regions had already broke out into a multitude of isolated immunostained axon varicosities, the remaining fibers being short, scarce and difficult to trace individually.
The detailed examination of sections taken though the caudal level (Fig. 6A) reveals that the SERT+ and TH+ fibers ascend within the medial forebrain bundle and, in fact, constitute the essential fibrillary component of this weakly myelinated bundle. Within this bundle, the SERT+ fibers tend to be more diffusely distributed then the TH+ fibers, which form a compact entity lying upon the dorsal surface of the substantia nigra (Fig. 6A, B). Both SERT+ and TH+ fiber bundles that ascend within the lateral hypothalamic area give rise to fiber fascicles that sweep laterally to course along the dorsal surface of STN (Fig. 6A, C). More laterally, the immunostained fibers traverse the strongly myelinated internal capsule by coursing in a typical sinuous fashion (Fig. 6A,D) until they reach the caudal portion of the pallidal complex. As they travel dorsal to STN and within the internal capsule, the SERT+ and TH+ fibers are closely intermingled with one another.
At the more rostral level (Fig. 6E), the SERT+ and TH+ fibers still occupy much of the mediolateral and dorsoventral extent of the medial forebrain bundle, in the lateral hypothalamic area. In contrast to the more caudal level, however, SERT+ and TH+ fiber bundles are clearly separated from one another, the former being more ventrally and medially located than the latter (Fig. 6E, F). Here again, the bundles of the SERT+ and TH+ fibers give rise to fascicles that sweep laterally beneath the pallidum and invade the internal and external medullary laminae that separate respectively the internal from the external pallidum and the external pallidum from the putamen (Fig. 6E). Beneath the globus pallidus, SERT+ fibers are again more diffusely distributed than TH+ fibers that tend to closely follow the trajectory of the ansa lenticularis (Fig. 6E, G). Within the internal and external medullary laminae, however, both types of fibers are intimately intermingled (Fig. 6E, H). In summary, as they course within the lateral hypothalamic area, the SERT+ fibers are more diffusely distributed than the TH+ fibers, whereas as they approach the basal ganglia through different routes, both types of fibers tend to be more closely intermingled.

Discussion

Methodological considerations

Since the brains analyzed in the present study belonged to individuals whose age ranged from 20 to 80 years, we looked for possible age-dependent variations in the pattern of basal ganglia 5-HT innervation. A comparison of SERT-immunostained sections from the brains of the older (54-80 years-old) and the younger (20-25 years-old) age group in our sample revealed interesting differences. These included a greater inter-individual variation in the overall immunostaining intensity and a larger number of lipofuscin pigments in the old versus young individuals. Furthermore, SERT immunostaining was generally more intense and less diffuse in the younger individuals. Except for these differences, however, there was no significant variation between the two groups in regards to the density and pattern of distribution of SERT imunoreactive axons and axon terminals in the various basal ganglia components.
In addition to age, other factors inherent to the investigation of postmortem human material, such as variations in postmortem delay and agonal status, might explain inter-individual variability in the quality of the immunostaining. In the present discussion, the 5-HT innervation of the basal ganglia in human is compared with that in other species, particularly nonhuman primates, as reported in the literature. Such a comparison must take into account the superior quality of brain tissue from monkeys that have been fixed by systemic perfusion under ideal conditions. Hence, some of the differences between human and nonhuman primates regarding basal ganglia 5-HT innervation reported in the present study might, at least in part, reflect methodological variations due to tissue fixation. However, a careful comparison of the present human data with materials from a previous study of the 5-HT innervation of the basal ganglia in the squirrel monkey (Saimiri sciureus) (Lavoie & Parent, 1990) has revealed that the density and patterns of distribution of the 5-HT fibers and terminals were fairly similar in both groups. Furthermore, the two morphological types of 5-HT axons encountered in the basal ganglia of squirrel monkeys could be clearly delineated in the present human study. The latter finding reveals that the postmortem material used here was of sufficient quality to allow meaningful comparisons between human and nonhuman primates. The major similarities and differences between human and nonhuman primates in regard to the 5-HT innervation will be dealt with below in the various sections devoted to each basal ganglia component.
Antibodies against TH were used in a set of double (TH/SERT) immunofluorescence experiments designed to compare, on single sections, the location of 5-HT and DA axons innervating the human basal ganglia. Although TH is recognized as a faithful marker of DA, this catecholamine-synthesizing enzyme is also present in fibers using noradrenalin as a transmitter. An antibody raised against DBH was thus used to detect the presence of noradrenalin-producing fibers in human basal ganglia. The clear and intense immunolabeling of neurons of the human locus cœruleus obtained with this antibody (see supporting document) served as a positive control for its immunoreactivity. In sections incubated with this anti-DBH antibody, a small number of immunoreactive fibers and axon terminals were noted particularly in the ventral portion of the striatum (see supporting document), but the bulk of the fibers and axon terminals that pervaded the basal ganglia, as visualized in TH-immunostained sections, remained totally immunonegative for DBH. These results, which are perfectly congruent with the data gathered previously in a different series of human individuals (see Prensa et al., 2000), indicate that the basal ganglia in human do not receive a significant noradrenergic innervation and that TH is a reliable DA marker in the different components of human basal ganglia. However, the possibility that a certain proportion of the TH immunoreactive axons ascending in the lateral hypothalamic area might be noradrenergic instead of DA cannot be ruled out.

Serotonin pathways innervating the human basal ganglia

The organization of the 5-HT afferents to human basal ganglia, as revealed in the present study, shared many similarities with that described previously in rats (Parent et al., 1981; Steinbusch, 1981) and monkeys (Azmitia & Segal, 1978; Azmitia & Gannon, 1986; Lavoie & Parent, 1990). In human, multitudinous immunoreactive fibers originating from 5-HT neurons located in the DRN and less abundantly from those in the MRN formed several small and intertwined fascicles that appeared along the sagittal plane as a complex reticulum occupying much of the core of the midbrain tegmentum. The bulk of these fibers arched ventrally, invade the ventral tegmental area, and ascend to the forebrain by coursing along the medial forebrain bundle in the lateral hypothalamic area. Our double immunofluorescent analysis has revealed that SERT immunoreactive fibers were more diffusely organized and slightly more ventrally located in the lateral hypothalamus than TH-immunostained axons. However, both types of fibers were closely intermingled as they depart laterally in the form of several distinct fascicles that invade the various basal ganglia components. Interestingly, both SERT+ and TH+ fibers reached their target structures by ascending along major output pathways of the basal ganglia, principally the ansa lenticularis ventrally and the lenticular fasciculus dorsally.
In the present study, two distinct types of axons were noted in the human basal ganglia: thick and beaded fibers as well as thin and varicose axons. This observation is in accordance with previous immunohistochemical studies conducted in different mammalian species including rats (Kosofsky & Molliver, 1987), cats (Mulligan & Tork, 1988) and monkeys (Takeuchi & Sano, 1983; Hornung et al., 1990; Lavoie & Parent, 1990). Using anterograde tracer, Kosofsky and Molliver (1987) have shown that each of the two types of 5-HT axons that innervate the cerebral cortex of the rat has a distinct origin in the midbrain raphe nuclei. The MRN neurons emit large varicose axons whereas the DRN neurons give rise to thin and varicose fibers (Kosofsky & Molliver, 1987). However, because the 5-HT fibers arising from the two raphe nuclei closely intermingle as they ascend towards the human basal ganglia, it was not possible to determine the specific origin of these two types of fibers in the present immunohistochemical study.
Electron microscopic examination of 5-HT axon varicosities in the striatum (Soghomonian et al., 1989; Descarries et al., 1992), the substantia nigra (Moukhles et al., 1997) and the subthalamic nucleus (Parent et al., 2010) has revealed that the 5-HT axon varicosities are rather small and apposed almost exclusively upon dendritic spines or shafts. A small proportion of them display asymmetrical membrane differentiation but the majority of these terminals do not form synaptic contacts (reviewed in Descarries et al., 2010b). The largely asynaptic character of these axon varicosities has been viewed as morphological evidence for the existence of diffuse transmission by such neuronal systems, in addition to their synaptic mode of transmission (reviewed in Descarries & Mechawar, 2008). This has also led to the suggestion that a low ambient level of transmitter might permanently exist in the extracellular space, the fluctuations of which could regulate a variety of physiological processes mediated by 5-HT (Descarries et al., 1997). Hence, more than the proximity of presynaptic 5-HT axon varicosities, it is the expression of various types of 5-HT receptors by target neurons that determine the excitatory or inhibitory influence of 5-HT on local neuronal networks.

Table des matières

CHAPITRE 1 – INTRODUCTION GÉNÉRALE
1.1. Préambule
1.1.1. Présentation des chapitres
1.2. Les ganglions de la base
1.2.1. Historique
1.2.2. Organisation anatomique
1.2.3. Le striatum
1.2.4. Le globus pallidus
1.2.5. Le noyau subthalamique
1.2.6. La substance noire
1.3. Innervation sérotoninergique des ganglions de la base
1.3.1. La sérotonine
1.3.2. Morphologie générale
1.4. La maladie de Parkinson
1.4.1. Historique
1.4.2. Étiologie
1.4.3. Pathologie
1.4.4. Traitements
1.5. Le système sérotoninergique dans un contexte parkinsonien
1.5.1. La sérotonine dans la maladie de Parkinson
1.5.2. La sérotonine et les dyskinésies induites par la L-DOPA
1.5.3. Les changements du système sérotoninergique dans les modèles parkinsoniens
1.5.4. La sérotonine et les symptômes non moteurs
1.6. Problématique de recherche
1.7. Objectifs de recherche
1.7.1. Objectifs généraux
1.7.2. Objectifs spécifiques
1.8. Approches méthodologiques
CHAPITRE 2- SEROTONIN INNERVATION OF HUMAN BASAL GANGLIA
2.1. Résumé
2.2. Abstract
2.3. Introduction
2.4. Materials and Methods
2.4.1. Tissue preparation
2.4.2. Antibodies
2.4.3. Immunohistochemistry
2.4.4. Immunofluorescence
2.4.5. Material analysis
2.5. Results
2.5.1. Ascending serotonin pathways
2.5.2. Substantia nigra
2.5.3. Subthalamic nucleus
2.5.4. Pallidum
2.5.5. Striatum
2.5.6. Ascending SERT and TH immunoreactive axons
2.6. Discussion
2.6.1. Methodological considerations
2.6.2. Serotonin pathways innervating the human basal ganglia
2.6.3. Patterns and densities of serotonin innervation in distinct components of human basal ganglia
2.7. Acknowledgements
2.8. Figures
CHAPITRE 3 – DISTRIBUTION OF VGLUT3 IN HIGHLY COLLATERALIZED AXONS FROM THE RAT DORSAL RAPHE NUCLEUS AS REVEALED BY SINGLE-NEURON RECONSTRUCTIONS
3.1. Résumé
3.2. Abstract
3.3. Introduction
3.4. Material and methods
3.4.1. Animals
3.4.2. Stereotaxic injections
3.4.3. Tissue processing for axonal reconstructions
3.4.4. Immunofluorescence
3.4.5. Confocal image analysis
3.5. Results
3.5.1. General labeling features and somatodendritic arborization
3.5.2. Axonal trajectory
3.5.3. Distribution of VGLUT3, VMAT2, 5-HT and SERT within the BDA-filled axons
3.6. Discussion
3.6.1. Organization of the somatodendritic domain as an indication of integration capacity
3.6.2. A highly collateralized axon as the morphological substratum of functional diversity
3.6.3. A broad axon terminal domain to influence wide neuronal populations
3.6.4. Number of axon varicosities as an indication of input strength 134
3.6.5. VGLUT3 content of 5-HT axon varicosities as a factor that favors neuroplasticity
3.7. Acknowledgments
3.8. Figures
CHAPITRE 4 – SEROTONIN HYPERINNERVATION OF THE STRIATUM WITH HIGH SYNAPTIC INCIDENCE IN PARKINSONIAN MONKEYS
4.1. Résumé
4.2. Abstract
4.3. List of abbreviations
4.4. Introduction
4.5. Material and methods
4.5.1. Animals
4.5.2. Parkinsonian syndrome induction
4.5.3. Immunohistochemistry
4.5.4. Material analysis
4.6.2. Neuronal density of TpH immunoreactive neurons was unchanged in the DRN of MPTP monkeys
4.6.6. SERT immunoreactive axon varicosities established more synaptic contacts in the DA-denervated striatal area
4.7.1. Topographical distribution of 5-HT axon varicosities in the striatum of normal cynomolgus monkeys
4.7.2. Density of 5-HT axon varicosities in the striatum of MPTP-intoxicated cynomolgus monkey
4.7.5. Conclusion
4.8. Figures
CHAPITRE 5 – EVIDENCE FOR SPROUTING OF DOPAMINE AND SEROTONIN AXONS IN THE PALLIDUM OF PARKINSONIAN MONKEYS
5.1. Résumé
5.2. Abstract
5.3. Abbreviations
5.4. Introduction
5.5. Material and methods
5.5.1. Animals and behavioural assessment
5.5.2. Tissue preparation and immunohistochemistry
5.5.3. Stereology
5.5.4. Ultrastructural analysis of SERT+ and TH+ axon varicosities
5.5.5. Statistical analysis
5.6. Results
5.6.1. MPTP intoxication induces a significant DA lesion and motor impairments
5.6.2. TH+ innervation of the pallidum in normal condition
5.6.3. TH+ innervation of the pallidum in parkinsonian monkeys
5.6.4. Ultrastructural features of TH+ axon varicosities
5.6.5. 5-HT innervation of the pallidum in normal condition
5.6.6. 5-HT innervation of the pallidum in parkinsonian monkeys
5.6.7. Ultrastructural features of SERT+ axon varicosities
5.7. Discussion
5.7.1. The morphological characteristics of dopamine and serotonin pallidal afférents
5.7.2. The DA pallidal innervation is significantly increased in parkinsonian monkeys
5.7.3. The 5-HT pallidal innervation is significantly increased in parkinsonian monkeys
5.8. Conclusion
5.9. Acknowledgments
5.10. Figures
CHAPITRE 6 – Striatal neurons expressing D1 and D2 receptors are morphogically distinct and differently affected by dopamine denervation in mice
6.1. Résumé
6.2. Abstract
6.3. Introduction
6.4. Results
6.5. Discussion
6.6. Material and methods
6.6.1. Animals
6.6.2. Stereotaxic injections
6.6.3. Immunohistochemistry
6.6.5. Single-cell injections of identified MSNs
7.1 L’innervation sérotoninergique en conditions normales
7.1.2. La colibération de neurotransmetteurs
7.2. Changement de la microcircuiterie des ganglions de la base dans la maladie de Parkinson
7.3. Conclusion
8. Bibliographie

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