Distribution and morphological characteristics of striatal interneurons expressing calretinin in mice: A comparison with human and nonhuman primates

Journal of Chemical Neuroanatomy 59 (2014) 51–61

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Distribution and morphological characteristics of striatal interneurons expressing calretinin in mice: A comparison with human and nonhuman primates Sarah Petryszyn, Jean-Martin Beaulieu, André Parent, Martin Parent * Department of Psychiatry and Neuroscience, Faculty of Medicine, Université Laval, Quebec City, Quebec, Canada

A R T I C L E I N F O

A B S T R A C T

Article history: Received 23 April 2014 Received in revised form 11 June 2014 Accepted 12 June 2014 Available online 21 June 2014

Striatal interneurons display a morphological and chemical heterogeneity that has been particularly well characterized in rats, monkeys and humans. By comparison much less is known of striatal interneurons in mice, although these animals are now widely used as transgenic models of various neurodegenerative diseases. The present immunohistochemical study aimed at characterizing striatal interneurons expressing calretinin (CR) in mice compared to those in squirrel monkeys and humans. The mouse striatum contains both small (9–12 mm) and medium-sized (15–20 mm) CR+ cells. The small cells are intensely stained with a single, slightly varicose and moderately arborized process. They occur throughout the striatum (77
9 cells/mm3), but prevail in the area of the subventricular zone and subcallosal streak, with statistically significant anteroposterior and dorsoventral decreasing gradients. The medium-sized cells are less intensely immunoreactive and possess 2–3 long, slightly varicose and poorly branched dendrites. They are rather uniformly scattered throughout the striatum and three times more numerous (224
31 cells/mm3) than the smaller CR+ cells. Double immunostaining experiments with choline acetyltransferase (ChAT) as a cholinergic marker in normal and Drd1a-tdTomato/Drd2-EGFP double transgenic mice reveal that none of the small or medium-sized CR+ cells express ChAT or D1 and D2 dopamine receptors. In contrast, the striatum in human and nonhuman primates harbors small and medium-sized CR+/ChAT cells, as well as large CR+/ChAT+ interneurons that are absent in mice. Such a difference between rodents and primates must be taken into consideration if one hopes to better understand the striatal function in normal and pathological conditions. ã 2014 Elsevier B.V. All rights reserved.

Keywords: Basal ganglia Striatum Calretinin Mouse Monkey Human

Introduction The striatum is the major integrative component of the basal ganglia, a set of subcortical structures that plays a crucial role in the control of motor activities and rewarded behaviors (Graybiel, 1998; Parent and Hazrati, 1995; Schultz, 2002). It harbors a multitude of similarly organized spiny GABAergic projection neurons and a smaller number of morphologically and neurochemically highly

Abbreviations: BAC, bacterial artificial chromosome; ChAT, choline acetyltransferase; CR, calretinin; D1/D2, Drd1a-tdTomato/Drd2-EGFP; GABA, g-aminobutyric acid; LV, lateral ventricle; NeuN, neuronal nuclear antigen; PB, sodium phosphate buffer; PBS, sodium phosphate buffer saline; PFA, paraformaldehyde. * Corresponding author at: Centre de Recherche de l'Institut Universitaire en Santé Mentale de Québec (CRIUSMQ), Université Laval 2160, Chemin de la Canardière, Quebec City G1J 2G3, Canada. Tel.: +1 418 663 5747; fax: +1 418 663 8756. E-mail address: [email protected] (M. Parent). http://dx.doi.org/10.1016/j.jchemneu.2014.06.002 0891-0618/ ã 2014 Elsevier B.V. All rights reserved.

diversified aspiny interneurons (Kawaguchi et al., 1995; Tepper and Bolam, 2004). In primates, striatal interneurons constitute about 20% of the total neuronal population (Graveland and DiFiglia, 1985; Graveland et al., 1985), whereas this proportion is of only 2–3% in rodents (Rymar et al., 2004). However, despite their small number, striatal interneurons exert a powerful pre- and post-synaptic striatal modulation (reviewed in Gittis and Kreitzer, 2012; Goldberg et al., 2012) and are thought to be involved in several movement and psychiatric disorders (Ding et al., 2011; Kataoka et al., 2010; Pisani et al., 2007). The vast majority of striatal interneurons uses g-amino-butyric acid (GABA) as a neurotransmitter, but can be distinguished from one another by their morphological features and by the fact that they express different sets of neuropeptides or proteins (Cicchetti et al., 2000; Kawaguchi et al., 1995). Calretinin (CR), a calcium-binding protein of the “EFhand” family, is one of these distinguishing factors. It is expressed by all members of the most abundant class of striatal interneurons in monkeys and humans (Cicchetti et al., 2000; Parent et al., 1995;

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Wu and Parent, 2000). The striatum in human and nonhuman primates contains a large number of medium-sized (9–21 mm) CR positive (+) neurons, which represents a unique class of striatal interneurons, and a smaller number of large (24–42 mm) CR+ neurons that largely belong to the population of giant cholinergic interneurons (Cicchetti et al., 1998). Recently, the presence of smaller (10–12 mm) CR+ neurons have also been detected in the human striatum (Bernácer et al., 2012). All these CR+ interneurons are heterogeneously distributed in the primate striatum and, although the medium-sized CR+ neurons outnumber the large CR+ neurons in the entire striatum, the large CR+ neurons are significantly more numerous in the putamen than in the caudate nucleus (Parent et al., 1995). A recent stereological investigation of the CR+ elements in the human striatum (Bernácer et al., 2012) has revealed that they predominate in the so-called associative territory of the striatum, which comprises chiefly the head of the caudate nucleus and the pre-commissural portion of the putamen (Parent, 1990). The zone of overlap between the associative territory and the sensorimotor territory, which is centered upon the post-commissural putamen (Parent, 1990), is markedly enriched in CR+ neuronal elements (Bernácer et al., 2012). In the rat, where the presence of striatal CR+ neurons was first detected (Jacobowitz and Winsky, 1991; Résibois and Rogers, 1992), these elements were characterized as a distinct subset of GABAergic medium-sized (7–20 mm) aspiny interneurons (Bennett and Bolam, 1993; Kubota et al., 1993), which corresponds to medium-sized CR+ interneurons detected in primates (Cicchetti et al., 2000). Although a few small and large-sized CR+ neurons have been occasionally visualized in the rat striatum, these elements could easily represent the two extremes of a single continuum centered upon the medium-sized neurons (Rymar et al., 2004). A comparative study of the distribution of CR+ striatal cells in rats, monkeys and humans has revealed that the most abundant striatal interneurons in human and nonhuman primates are those expressing CR, whereas, in rats, this position is occupied by neurons displaying parvalbumin immunoreactivity (Wu and Parent, 2000). In the rat, CR+ interneurons are distributed according to a marked anteroposterior decreasing gradient (Bennett and Bolam, 1993; Rymar et al., 2004), with a clear prevalence of such chemospecific elements in the dorsomedial quadrant of the pre-commissural striatum (Figueredo-Cardenas et al., 1996; Rymar et al., 2004). In comparison to the wealth of data on the CR+ striatal interneurons gathered during the last two decades in rats, monkeys and humans, very little is known of such chemospecific striatal elements in the striatum of mice. Recently, the occurrence of very small (8–10 mm) CR+ cells have been reported in the mice striatum, but these elements have been found to be negative for most known markers of striatal neurons, including the neuronal nuclear antigen (NeuN), which is commonly used to ascertain the neuronal nature of a given cell (Revishchin et al., 2010a; Revishchin et al., 2010b). Even more recently, the presence in the mice striatum of CR+ neurons of various sizes have been mentioned, but without details about their morphological features and topographical distribution (Tepper et al., 2010). This lack of information is surprising, particularly because mice have recently become an animal model widely used to study the various neurodegenerative conditions, including Parkinson’s disease. For example, in the Pitx3-deficient aphakia mouse, the lack of the transcription factor Pitx3 results in selective loss of nigrostriatal dopaminergic projections, so that such an animal can be readily used as an experimental model of Parkinson’s disease (see Ding et al., 2011). Furthermore, the recent identification of different genetic mutations (a-synuclein, Parkin, LRKK2, PINK1, DJ-1) has led to the development of highly useful genetic models of Parkinson’s

disease (Dawson et al., 2010). The advent of transgenic mice that express the enhanced green fluorescent protein (EGFP) under the control of endogenous regulatory sequences that code various compounds of interest, such as the dopaminergic receptors D1 and D2 (Gong et al., 2003; Shuen et al., 2008) have opened up new avenues for the morphological and functional study of the rodent striatum. Despite the availability of these powerful transgenic models, our knowledge of the neuronal organization of the striatum in mice is minimal by comparison to that in rats and primates. Yet such information is essential for the correct interpretation of the complex alterations in striatal organization that are likely to occur following various experimental manipulations of these mice, particularly those intended to be used as Parkinson’s disease models. In an attempt to bridge this gap, we initiated a series of morphological and immunohistochemical studies of the CR+ striatal interneurons in C57Bl6 mice and D1/D2 double transgenic mice, and we used the very same methodological approach to directly compare the organization of these chemospecific striatal interneurons in mice with that in humans and nonhuman primates. It is hoped that these normative data will further our understanding of the functional organization of the striatum across species. Material and methods Tissue preparation All animal tissues were obtained according to protocols that had been approved by the Institutional Animal Care and Use Committee (Comité de Protection des Animaux de l'Université Laval, # 2011165-3), and all procedures involving animals and their care were made in accordance with the Canadian Council on Animal Care’s Guide to the Care and Use of Experimental Animals (Ed2). Human post-mortem brain tissues were obtained from the human brain bank of the Centre de Recherche de l'Institut Universitaire en Santé Mentale de Québec (CRIUSMQ), which required informed consent before donation of tissues. The IUSMQ’s Ethics Committee approved the brain collection, storage and handling procedures (#274), which were described in detail elsewhere (Huot et al., 2007). Mice Mouse brains were obtained from 4 C57Bl6 male individuals of 3 months old (Mus musculus; Charles River, Quebec City, Canada) and from 2 Drd1a-tdTomato/Drd2-EGFP (D1/D2) double transgenic mice of 3 months old with a mixed background (C57Bl6xB6SJLF1) (Gong et al., 2003; Shuen et al., 2008) that were raised in our animal facility. These animals were housed in a temperaturecontrolled room (21–25  C) under a 12 h light/dark cycle and had access to food and water ad libitum. Animals were deeply anesthetized with a mixture of Ketamine (100 mg/mg, i.m.) and Xylazine (10 mg/mg. i.m.) and perfused transcardially with an initial wash of 50 ml of ice-cold 0.9% saline followed by 300 ml of 4% paraformaldehyde (PFA) diluted in 0.1 M sodium phosphate buffer (PB; pH 7.4). Brains were dissected from the skulls, postfixed overnight in 4% PFA and cut with a vibratome (Leica) into 50 mm-thick coronal or sagittal sections, which were serially collected in sodium phosphate buffer saline (PBS, 0.1 M, pH 7.4). Monkeys Nonhuman primate brains were obtained from two young adult male squirrel monkeys (Saimiri sciureus; Buckshire Corporation, Perkasie, PA, USA) that were housed under a 12 h light/dark cycle and had access to food and water ad libitum. Animals were deeply anesthetized with a mixture of Ketamine (150 mg/kg, i.m.) and

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Humans The human post-mortem materials used in the present study were gathered from 3 males without a known history of neurological or psychiatric disease. They died of cardiac infarct, polytraumatism and cerebral hemorrhage, respectively and their ages were 65, 71 and 82 years old (X ¼ 72:7years). Brain weights were 1550 g, 1540 g and 1250 g (X ¼ 1446:7g) and the postmortem delays were 24, 20 and 18 h (X ¼ 20:7h). Briefly, the brains were first sliced into 0.5 cm-thick slabs that were fixed by immersion at 4  C in 4% PFA in 0.1 M PB (pH 7.4) for 48 h. The slabs containing the striatum were stored at 4  C in 0.1 M PBS (pH 7.4) with 15% sucrose and 0.1% sodium azide. They were then cut into 50 mm-thick coronal sections using a freezing microtome and the sections were collected in PBS until staining.

placental peptide. The antiserum recognizes a single band of 68– 70 KDa on western blots of rat peripheral nerve (Brunelli et al., 2005) and mouse brain lysates (manufacturer’s datasheet). The staining obtained was identical to that reported previously with other antibodies against choline acetyltransferase (Parent and Descarries, 2008). Striatal sections were first pre-incubated for 1 h in a blocking solution containing 0.1% Triton X-100 and 2% normal rabbit serum diluted in PBS. Sections were then incubated for 48 h in the same solution to which the primary antibody against ChAT (1:25) was added. The sections were then rinsed in PBS and incubated for 1 h in the blocking solution containing biotinylated anti-goat secondary antibody raised in rabbit (1:200, Vector Laboratories, Catalog # BA5000). Sections were rinsed with PBS and incubated for 2 h in the blocking solution containing Cy5streptavidine (1:200, Molecular Probes, Catalog # SA1011). Sections were rinsed, air-dried and coverslipped with fluorescence mounting medium (DAKO, Ontario, Canada). Fluorescent emission of EGFP, tdTomato and Cy5 (peaks at 504, 581 and 670 nm, respectively) were sequentially acquired using the best signal mode of a LSM700 confocal microscope (Zeiss), without any potential bleed-through artifacts.

Single immunostaining for calretinin

Double-immunostaining for calretinin and choline acetyltransferase

The polyclonal antibody against CR used in the present study (Swant, Catalog # 7699/4) was produced in rabbit by immunization with recombinant human CR containing a 6-his tag at the Nterminal. This antibody does not crossreact with calbindin D-28k or other known calcium-binding proteins, as determined by its distribution in the brain, as well as by immunoblots (manufacturer’s datasheet). Immunolabeling of mouse, monkey and human brain sections with this antibody allows for a visualization of cell bodies and neurites in the distribution and density expected from the CR+ neurons only and immunostaining of brain sections from CR knock-out mice reveals the absence of specific staining in the cerebral cortex and the striatum (Schiffmann et al., 1999). On extracts of soluble proteins from mouse and monkey brains separated by SDS-PAGE, the antiserum specifically recognizes a band of 29–30 kDa corresponding to the molecular weight of the CR protein. Six sections taken throughout the entire striatum at a 600 mm interval from 4 C57Bl6 mice were selected and immunostained for CR and counterstained for 40 , 6-diamidino-2-phenylindole (DAPI). Briefly, sections were first incubated for 1 h in a blocking solution containing 1% Triton X-100 and 2% normal donkey serum diluted in PBS and then for 16 h in the same blocking solution to which an antibody against the CR was added (1:500, Swant, Catalog # 7699/ 4). The sections were then rinsed in PBS and incubated for 2 h in the blocking solution to which a secondary antibody against rabbit coupled with Texas Red was added (Jackson Immunoresearch Laboratories, Catalog # 711-075-152). The sections were rinsed in PBS, and incubated for 10 min with 100 ng/ml DAPI diluted in the PBS. Finally, the sections were rinsed in PBS, mounted on gelatincoated slides, air-dried, and coverslipped with fluorescence mounting medium (DAKO, Ontario, Canada). Striatal sections from the D1/D2 mice were also immunostained for the CR as described above, except that normal goat serum and goat Cy5 secondary antibody against rabbit (Jackson Immunoresearch Laboratories, Catalog # 111 175 003) were used.

Striatal transverse sections from 1 C57Bl/6 mouse, 2 squirrel monkeys and 3 post-mortem human brains were pre-incubated for 1 h in a blocking solution composed of 0.1% Triton X-100, 2% normal donkey serum and 2% normal horse serum diluted in PBS, then incubated simultaneously with the antibody against ChAT (1:25, 48 h) and with the antibody against CR (1:500, 16 h). Sections were then rinsed with PBS and incubated for 1 h with biotinylated anti-goat antibody made in horse (1:1,000, Vector Laboratories, Catalog # BA9500). Sections were rinsed with PBS and incubated with a secondary antibody against rabbit, made in donkey, coupled to Texas Red (1:200, Jackson Immunoresearch Laboratories, Catalog # 711-075-152, emission peak at 615 nm) and a green-fluorescent Alexa Fluor 488 streptavidin (Molecular Probes, Catalog #S-11223, emission peak at 519 nm). Sections were rinsed, air-dried and coverslipped with fluorescence mounting medium (DAKO, Ontario, Canada).

Xylazine (10 mg/kg, i.m.), along with Acepromazine (0.5 mg/kg, i. m.), a myorelaxant, then transcardially perfused with and initial wash of 200 ml of ice-cold PBS (50 m M, pH 7.4) followed by 500 ml of 3% acroleine in PB and 1 l of 4% PFA diluted in PB. Brains were dissected out, post-fixed in 4% PFA and cut with a vibratome into 50 mm thick coronal sections collected in PBS.

Single immunostaining for choline acetyltransferase Two striatal sections from D1/D2 transgenic mouse were immunostained for the choline acetyltransferase (ChAT; Millipore, Catalog # AB144P), a specific biosynthetic enzyme of acetylcholine. This antibody was made in goat by immunization with a human

Data analysis A confocal laser scanning microscope (LSM700, Zeiss) equipped with a camera (AxioCam), a motorized stage (X and Y axes) and a Z-axis indicator (Leica Z axis control) controlled by a computer running StereoInvestigator (v. 7.00.3; MicroBrightField, Colchester, VT, USA) and Zen (v.7.1, 2011) software was used. In each of the 4 C57Bl6 mice, 6 equally spaced transverse sections were selected across the entire right striatum (from bregma 1.34 mm to 1.66 mm, according to the stereotaxic atlas of Franklin and Paxinos (1997) at fixed intervals of 600 mm. Sections selected were centered upon the anterior commissure. A precise description of regional distribution of the CR+ cells throughout the striatum was achieved by dividing the structure into eight sectors: antero-dorso-lateral (ADL), antero-dorsomedial (ADM), antero-ventro-lateral (AVL), antero-ventro-medial (AVM), postero-dorso-lateral (PDL), postero-dorso-medial (PDM), postero-ventro-lateral (PVL) and postero-ventro-medial (PVM). To do so, the contour of the striatum was first outlined on each CRimmunostained transverse section using a 4X/0.10 objective, according to the stereotaxic atlas of Franklin and Paxinos (1997). As shown in Fig. 2, a vertical line parallel to the midline and passing by the center of the striatum was first traced, dividing the structure into medial and lateral sectors. A horizontal line, perpendicular to and centered on the vertical line was also traced

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to delineate four sectors on each brain section. The anteroposterior axis was then divided in two by considering the first 3 transverse sections as representative of anterior sectors and the last 3 of posterior sectors. The sampling process leading to estimations of the total number of the CR+ cells in each of the eight sectors began by randomly translating a grid formed by 157  157 mm squares over the sections. At each intersection of the grid that fell into the sector, a counting frame measuring 157  157 mm was drawn and examined with a 40X/1.4 objective, leading to the examination of the entire area of each sector. The CR+ cells were counted whenever they came into focus within a 12 mm-thick optical dissector centered in the section. The thickness of the mounted tissue was measured for each counting frame, yielding mean values between 24.4 mm and 20.8 mm. An average of 15.13
3.18 CR+ cells were counted in each sector of the striatum. For each sector, the density of CR+ neurons was expressed in cells/mm3 of tissue, using the total number calculated by the optical dissector and the volume of the sector estimated by Cavalieri’s method. The same approach was used to estimate the density (cells/mm3) of CR + neurons in the entire striatum of each animal. Statistical analysis Differences in the density CR-immunostained cells between striatal sectors were assessed using Wilcoxon-signed rank test. Differences were considered statistically significant at P < 0.05. Statistical analysis was done using GraphPad Prism software (v.5.0; GraphPad Software, San Diego, CA, USA). Mean and standard error of the mean are used throughout the text as central tendency and dispersion measure respectively. Results Mice Confocal imaging of sections taken through the entire anteroposterior extent of the striatum in 4 C57Bl6 mice revealed the presence of CR+ cells, whose overall density is estimated at

301
38 cells/mm3. Based on their size and morphological features, these immunoreactive cells can be divided in two distinct types. The CR+ cells of the first type are typically small and intensely immunoreactive. Their cell body is round or ovoid, with a maximal diameter that ranges from 9 to 12 mm. These CR+ cells display a single, slightly varicose and moderately arborized process that extends as far as 100 mm within the striatal neuropil (Fig. 1A). Although they occur throughout the striatum (overall density of 77
9 cells/mm3), they prevail in the anterior and dorsal sectors of the structure (Fig. 2A,B), particularly in the areas of the subventricular zone and the subcallosal streak, where they form typical clusters. Isolated CR+ neurons of this type also occurred directly within the subependymal layer, where they display a typical immature aspect (insert in Fig. 1A). Detailed stereological analyses reveal that the small CR+ striatal cells are distributed according to statistically significant anteroposterior and dorsoventral decreasing gradients (Fig. 2B). More specifically, the precommissural portion of the striatum harbors 113
25 CR+ cells/ mm3 compared to 41
16 cells/mm3 for the post-commissural sector (P = 0.0004). Similarly, the densities of the small CR+ cells in the dorsal half compared to that in the ventral half of the dorsal striatum are 134
24 cells/mm3 and 19
6 cells/mm3, respectively (P = 0.0002). In contrast, no significant variation was noted between the lateral versus medial half of the striatum, the values being 97
25 cells/mm3 and 56
18 cells/mm3, respectively (P = 0.058). The CR+ cells of the second type are larger but less intensely immunofluorescent than those of the first type. They possess a bipolar or ovoid cell body, with a maximum diameter ranging between 15 and 20 mm, that typically gives rise to two long, slightly varicose and poorly branched dendrites (Fig. 1D). These mediumsized neurons are rather uniformly scattered throughout the striatum and they outnumber the CR+ cells of the first type by a factor of three (overall density of 224
31 cells/mm3 compared to 77
9 cells/mm3). Despite some slight numerical variations between the various striatal territories, the number of mediumsized CR+ neurons does not vary significantly along the

Fig. 1. Morphological characteristics of striatal cells expressing calretinin (CR) in mice. The presence of the CR (Red) or choline acetyltransferase (ChAT; Green) was detected after confocal microscopic examination of doubly-immunostained striatal sections obtained from C57Bl6 mice. The photomicrographs show a small CR+ cells immunonegative for ChAT (A–C) and a medium-sized CR+ cells, also immunonegative for ChAT (D–F). Note the presence of large ChAT+ interneurons in B and C that are immunonegative for the CR. The insert in A provides examples of two small CR+ cells located in the subventricular zone. Scale bars = 20 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).

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Fig. 2. Topographical distribution of striatal cells expressing calretinin (CR) in mice. A: drawings of representative transverse sections comparing the distribution of small (red stars) and medium-sized (green circles) CR+ cells in the anterior (left) and posterior (right) striatal sectors of a C57Bl6 mouse. The anteroposterior stereotaxic coordinate of each section is indicated at the bottom of the drawing. B, C: histograms comparing the mean density (cells/mm3; n = 4) of the small (B) and medium-sized (C) CR+ striatal cells along the three major spatial axes. *P < 0.05; **P < 0.005 by Wilcoxon-signed rank test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).

anteroposterior and mediolateral axes of the striatum (Fig. 2A,C). Their density in the pre-commissural portion of the striatum is 259
43 cells/mm3, compared to 237
40 cells/mm3 for the postcommissural region (P = 0.518), whereas the corresponding values for the lateral and medial halves of the structure are 223
34 cells/ mm3 and 273
47 cells/mm3, respectively (P = 0.4534). However, a statistically significant (P = 0.0090) increasing gradient occurs along the dorsoventral axis, the density of the CR+ neurons being 327
42 cells/mm3 in the ventral half of the dorsal striatum compared to 169
30 cells/mm3 in its dorsal half (Fig. 2C). In an attempt to determine if any of the two types of striatal CR+ cells present in the mouse striatum could be part of a population of cholinergic interneurons, striatal sections of mice were immunostained for both the CR and ChAT, a faithful marker of cholinergic neurons. The detailed scanning of these sections reveals that neither the small nor the medium-sized CR+ cells display ChAT immunoreactivity (Fig. 1A–F). Yet large neurons expressing ChAT only are clearly visible in such preparations (Fig. 1B,C). These neurons have a large (>20 mm) globular or triangular cell body

giving rise to 2–4 thin, smooth and long dendrites that branched frequently (Fig. 1B,C). They appear to correspond to the large cholinergic striatal interneurons detected in rats, monkeys and humans (see Discussion). We also examined the possibility of detecting CR+ elements in the striatum of bacterial artificial chromosome (BAC) transgenic mice expressing fluorescent proteins driven by specific promoters of the D1 and D2 dopamine receptors (Drd1a-tdTomato/Drd2-EGFP double transgenic mice or D1/D2 mice). Despite the fact that these D1/D2 mice have a genetic background significantly different from that of the C57Bl6 mice, which are the main focus of the present study, the small and medium-sized CR+ cells described above are clearly visible in the striatum of D1/D2 mice (Fig. 3A–F). They are scattered among a multitude of medium-sized spiny projection neurons that display red (D1) or green (D2) fluorescence, as well as some yellow (expressing both D1 and D2) fluorescent neurons (Fig. 3F,I). The relative paucity of interneurons by comparison to the multitudinous projection neurons can be readily appreciated in such a material. More importantly, neither the small nor the

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Fig. 3. Neurochemical features of striatal cells expressing calretinin (CR) or choline acetyl-transferase (ChAT) in Drd1a-TdTomato/Drd2-EGFP (D1/D2) mice. A–F: the presence of the CR (blue) was detected after confocal imaging of immunostained striatal sections obtained from D1/D2 mice. Small (A–C) and medium-sized (D–F) CR+ cells were observed and devoid of EGFP (green) and TdTomato (red) fluorescent proteins. G–I: cells immunoreactive for ChAT (white) were observed and contained low level of EGFP (green), indicating that they express the D2 dopamine receptor. Arrowheads indicate striatal cells devoid of CR and ChAT immunoreactivity expressing both D1 and D2 dopamine receptors. Scale bar = 20 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).

medium-sized CR+ cells appear to display fluorescence for D1 or D2 receptors. Attempts were also made to further characterize the large cholinergic striatal interneurons by immunostaining sections of the striatum in D1/D2 mice with ChAT antibodies. The analysis of such material reveals that the large ChAT+ striatal interneurons express D2, as shown by a low level of EGFP, but not the D1 dopamine receptors (Fig. 3G–I). Human and nonhuman primates As in mice, small and medium-sized CR+ cells occur throughout the striatum in humans and monkeys. The small CR+ cells are the most abundant types of striatal CR+ neurons in primates. They occurred in both the caudate nucleus and putamen, but are slightly more numerous in the former than the latter striatal subdivision. Their cell body has a shape that varies from round to oval, with a maximum diameter ranging from 8 to 12 mm (Figs. 4 and 5A). One or two thin and varicose dendrites emerge usually from each pole of the cell body, but their relatively weak immunostaining renders difficult the task of tracing their course within the striatal neuropil. Less numerous than the small CR+ cells, the medium-sized CR+ neurons are rather homogeneously distributed throughout the anteroposterior extent of the primate striatum, with a slight predominance in the caudate nucleus over the putamen. Their cell body is oval or triangular and has a maximum diameter that ranges from 12 to 20 mm. It gives rise to 2 or 3 thin, varicose, poorly arborized and weakly immunostained dendrites (Figs. 4, 5D). In addition to these small and medium-sized CR+ cells, the striatum of humans and monkeys harbors a small population of

large CR+ neurons. These neurons, which are slightly more numerous in the putamen than in the caudate nucleus, possess a very large globular or triangular cell body (20–45 mm), from which emerge several thick and short varicose dendrites that branch frequently (Figs. 4G, 5G). As we did for mice, we examined the possibility that some of the three major types of striatal CR+ cells in the primate striatum might be part a population of cholinergic interneurons by applying a double immunostaining procedure for the CR and ChAT to striatal sections of both humans and squirrel monkeys. The detailed scanning of these doubly-immunostained sections reveals that none of the small or medium-sized CR+ cells are immunoreactive for ChAT in monkeys (Fig. 4A–F) and humans (Fig. 5A–F). In contrast, most large multipolar CR+ neurons display a clear immunostaining for this cholinergic marker in both monkeys (Fig. 4G–I) and humans (Fig. 5G–I). Discussion The present study has provided the first detailed description of morphological features, topographical distribution and relative density of CR+ cells that characterize the mouse striatum. The analysis of the size, shape and distributional pattern of these striatal CR+ neurons has revealed that they form two distinct subpopulations: a group of small CR+ unipolar cells and a larger cohort of medium-sized multipolar CR+ neurons. Despite a marked difference in the distribution and morphological characteristics of their cell bodies, both the small and medium-sized CR+ cells in the mouse striatum display dendrites that are devoid of spines. Such a

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Fig. 4. Morphological features of striatal cells expressing calretinin (CR) in the squirrel monkey. The presence of the CR (red) or choline acetyltransferase (ChAT; green) was detected after confocal microscopic examination of doubly-immunostained sections of the caudate nucleus. The photomicrographs show small CR+ cells immunonegative for ChAT (arrows in A–C), medium-sized CR+ cells also immunonegative for ChAT (arrows in D–F) and large CR+ cells coexpressing ChAT ((arrows in G–I). Scale bar = 20 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).

common feature reveals that these two types of CR+ cells differ from the multitude of striatal projections neurons, whose dendrites are typically endowed with spines, and, therefore, most likely belong to the much smaller but chemically highly diversified population of striatal interneurons (Cicchetti et al., 2000). The issue of the functional significance of these two types of CR+ neurons in mice will now be addressed in the light of the information already available for similar striatal interneurons in other species. The small striatal calretinin cells The small CR+ striatal cells detected in mice have a very peculiar distribution: they prevail in the anterior and dorsal sector of the striatum, and many of them are clustered in the areas of the subventricular zone and the subcallosal streak, with a few isolated elements lying within the subependymal layer itself. The abundance of small CR+ cells in the anterior sector of the striatum, which receives its cortical input largely from cortices of the associative type, suggest that these chemospecific interneurons are more actively involved in the integrative role of the striatum than in its sensorimotor or limbic functions, which are dealt with in more posterior and ventral aspects of the structure. The presence of small CR+ interneurons in the subventricular zone, a brain area that retains its neurogenic capacity throughout adult life, raises the possibility that some of them are generated after birth. Indeed, the use of cell-proliferation markers in adult rats and rabbits under normal conditions has revealed the presence of newly generated neurons in the dorsolateral sector of the striatum (Dayer et al., 2005; Luzzati et al., 2006). These

newborn neurons appeared to originate from the adjacent subventricular zone and a significant number of them differentiated into CR+ interneurons (Dayer et al., 2005; Luzzati et al., 2006). Neuroblasts that eventually develop a CR+ interneuron phenotype have also been documented in the striatum of adult rats and monkeys under various pathological conditions. For example, the subventricular zone in adult rats was shown to generate newborn CR+ interneurons that migrate within the striatum after hypoxic/ ischemic lesions, but the number of these striatal neurons in such a pathological condition was found to be much smaller than newborn neurons that invade the rostral migratory stream en route to the olfactory bulb (Yang et al., 2008). In macaque monkeys, the presence of the transcription factor Sox-2, which is expressed in pluripotent and adult stem cells, including neural progenitors, has been recently detected in some CR+ interneurons of the striatum (Ordoñez et al., 2013). Furthermore, the administration of the neurotoxin 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP), which causes a selective lesion of the nigrostriatal dopaminergic pathway and renders monkeys Parkinsonian, led to an increase in the proliferation of striatal Sox-2+ cells and to an acute, concomitant decrease in the percentage of Sox-2+/CR+ neurons, which recovered by 18 months (Ordoñez et al., 2013). Given their responsiveness to dopamine insult, striatal Sox-2+/CR+ cells could be envisaged as part of a putative endogenous regenerative mechanism for Parkinson’s disease. In adult humans, a recent study using carbon-14 dating approaches has revealed that newly subventricular zone-generated neurons, including many which express CR, integrate in the striatum under normal conditions (Ernst et al., 2014). The investigation also showed that the neuronal turnover in human striatum is restricted to

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Fig. 5. Morphological characteristics of striatal cells expressing calretinin (CR) in humans. The presence of the CR (red) or choline acetyltransferase (ChAT; green) was detected after confocal imaging of doubly-immunostained striatal sections. The photomicrographs show small CR+ cells immunonegative for ChAT in the caudate nucleus (A– C), medium-sized CR+ cells immunonegative for ChAT in the putamen (D–F) and large CR+ cells coexpressing ChAT in the caudate nucleus (G–I). Scale bar = 20 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).

interneurons, and that postnatally generated striatal neurons appear to be preferentially depleted in Huntington’s disease patients (Ernst et al., 2014). These findings reveal a unique pattern of neurogenesis in the adult human striatum that involves interneurons expressing CR. Evidence for the existence of small CR+ neurons that are generated during adulthood has also been gathered in mice (Revishchin et al., 2010a; Revishchin et al., 2010b). These CR+ cells, whose perikarya (8–10 mm in diameter) and processes are coated with polymorphous spines, abounded in the anterior and dorsal sectors of the striatum, but also occurred in various other forebrain regions, including the deep layers of the frontal cortex and the anterior olfactory nucleus (Revishchin et al., 2010b). These immature looking cells, which are reportedly generated at 7–20 days postnatally, do not express the neurotransmitter g-aminobutyric acid (GABA) or any of the major neuronal or glial cell markers (Revishchin et al., 2010a). They are said to be absent in the forebrain of rabbits, rats and cats and to differ markedly from the typical GABAergic CR+ aspiny striatal interneurons (Revishchin et al., 2010a; Revishchin et al., 2010b). The functional significance of these CR+ elements in the murine brain remains to be determined, but the presence of spines on their processes suggests that they do not correspond to the type of small CR+ striatal cells described in the present study. Our examination of the striatum of man and squirrel monkeys has revealed the presence of small CR+ neurons in this major basal ganglia component in primates. These findings agree with recent observations made in humans (Bernácer et al., 2012) and macaque monkeys (Ordoñez et al., 2013) and, together, they suggest that this type of CR+ neurons has been conserved throughout phylogeny.

However, detailed knowledge about the morphological characteristics and distributional patterns of the small CR+ striatal neurons in both monkeys and humans is still lacking. Furthermore, in contrast to the information that is already available on the behavior of the medium and large-sized CR+ striatal neurons in various pathological conditions affecting the striatum, such as Huntington's disease (Cicchetti and Parent, 1996; Massouh et al., 2008), virtually nothing is known of the state of the small CR+ striatal neurons in neurodegenerative diseases. This type of information would greatly help our understanding of the role of this type of chemospecific neurons in the functional organization of the primate striatum. The medium-sized striatal calretinin interneurons Based on their morphological and distributional characteristics, the medium-sized CR+ neurons that we have visualized in the mouse striatum correspond to the typical medium-sized CR+ striatal interneurons that were first observed in rats (Bennett and Bolam, 1993; Jacobowitz and Winsky, 1991; Résibois and Rogers, 1992). Their presence was later confirmed in monkeys (Fortin and Parent, 1994) and humans (Parent et al., 1995; Prensa et al. , 1998). Colocalization studies in rats have revealed that, although they express GABA (Kubota et al., 1993), the medium-sized CR+ interneurons do not stain for markers of the other three types of striatal interneurons (nitric oxide synthase or NOS, parvalbumin, and ChAT), as well as for markers of striatal projection neurons (e.g., calbindin) (Figueredo-Cardenas et al., 1996; Kawaguchi et al., 1995). Investigations using double immunostaining of human striatal sections have confirmed the uniqueness of this

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population of striatal interneurons in primates as well (Cicchetti et al., 1998). A detailed stereological investigation of the CR+ striatal neurons in rats has shown that they represent only 0.5% of the total neuronal population of the striatum and that 90% of them are of medium-size (Rymar et al., 2004). A smaller number of small (7 mm) and large (>20 mm) CR+ neurons also occur, but these elements were said to be part of the same morphological continuum (6.2–26.5 mm) (Rymar et al., 2004). In more recent analyses of the rat striatum, only small (6–10 mm) CR+ neurons have been detected (Ma et al., 2014; Matamales et al., 2009) and some authors have argued that the variations noted previously in the size of CR+ neurons might be related to age and physiological conditions of the animals (Ma et al., 2014). Our own comparative study in rats, squirrel monkeys and humans has shown that the medium-sized CR+ neurons are the most abundant striatal interneurons in primates but not in rats (Wu and Parent, 2000). In primates, we found the CR+ neurons to be 2–4 times more numerous than the others medium-sized striatal interneurons (containing parvalbumin or NOS), whereas the most frequently encountered interneurons in the rat striatum were those expressing parvalbumin (Wu and Parent, 2000). Furthermore, the medium-sized CR+ neurons displayed a striking anteroposterior decreasing gradient in rats (Bennett and Bolam, 1993; Wu and Parent, 2000), but such a declivity is absent in human and nonhuman primates (Wu and Parent, 2000). In the striatum of squirrel monkeys and humans, the medium-sized CR+ neurons are rather uniformly distributed along the anterior–posterior axis, as it is also appears to be the case in mice (see above). Species differences in regards to the relative density and pattern of distribution of striatal CR+ interneurons should be taken into account when evaluating the effect of neurodegenerative diseases on cell densities in the human striatum or when studying animal models of such pathological conditions. Our previous post-mortem investigations of the human brain under pathological conditions have revealed that the mediumsized CR+ striatal interneurons are selectively preserved in patients suffering from Huntington’s disease (Cicchetti et al., 1996; Cicchetti and Parent, 1996). Pathologically, Huntington’s disease is characterized by a marked atrophy of the striatum due to losses of striatal projection neurons, while interneurons are relatively spared (Kowall et al., 1987; Massouh et al., 2008). We still ignore why and how neurodegenerative processes at play in Huntington’s disease selectively target the projection neurons instead of interneurons in the striatum, but experimental studies on animal models of the disease could help decipher the pathogenesis of this devastating neurodegenerative disease. In contrast to Huntington’s disease, nothing is known on the state of the human CR+ striatal neurons in Parkinson’s disease, another major neurodegenerative disease that affects the striatum. Experimental studies in rats in which the dopaminergic nigrostriatal pathway had been unilaterally lesioned by injection of the neurotoxin 6-hydroxydopamine (6-OHDA) have reported a transient increase in the number of CR+ striatal neurons ipsilaterally to the injection site (Mura et al., 2000). Treatment with L-Dopa was said to abolish such an increase, which, in any cases, did not last more than 18 weeks post-lesion (Mura et al., 2000). The authors have interpreted this set of data as evidence for the fact that striatal CR+ neurons are sensitive to dopamine depletion and that increased CR expression in these neurons might be due to enhanced striatal excitatory input from cerebral cortex (Mura et al., 2000). Although a possible influence of dopamine on CR+ striatal neurons has been alluded to previously (Kawaguchi et al., 1995), the mechanism whereby such a putative dopaminergic action might be exerted has remained elusive. The present characterization of the CR+ cells in the striatum of Drd1a-

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tdTomato/Drd2-EGFP double transgenic mice has allowed us to demonstrate, for the first time, that the CR+ striatal interneurons do not express D1 or D2 dopamine receptor. Hence, dopamine is likely to exert its influence upon CR+ striatal interneurons indirectly, either by modulating the glutamatergic excitatory striatal projections of cortical or thalamic origin, or by stimulating the large cholinergic interneurons, which express dopamine receptors (see below). The large striatal calretinin interneurons The presence of large or ‘giant’ (>20 mm) striatal neurons has been recognized in rats, monkeys and humans (Parent and Hazrati, 1995). They are among the largest elements of the striatum and correspond best to the aspiny type II cells characterized in monkeys (DiFiglia et al., 1976). These voluminous cells from which emerge several long, thick and aspiny dendrites were originally regarded as the principal projection neurons of the striatum, but retrograde cell labeling studies have revealed that their axons do not project outside the striatum but instead contribute heavily to the intrinsic striatal neuropil (Parent and Hazrati, 1995). Immunohistochemical studies using anti-ChAT as a marker of cholinergic neurons have revealed that, in rats, monkeys and humans, the large striatal interneurons belong to a single population of cholinergic interneurons, which is likely the major source of striatal acetylcholine (Cicchetti et al., 2000). In contrast to the other two types of striatal interneurons, which contain respectively parvalbumin and NOS/somatostatin/neuropeptide Y, as well as the medium-sized spiny projection neurons, which express calbindin, the large cholinergic interneurons were thought to be unique because they did not express neuroactive peptides or proteins (Kawaguchi et al., 1995). However, this unitary view of the chemical phenotype of the large striatal interneurons has been challenged when large CR+ aspiny neurons were detected in squirrel monkey (Fortin and Parent, 1994) and human (Parent et al., 1995) striatum. The morphological features and regional distribution of these large CR+ neurons were similar to those of the large cholinergic interneurons (Parent et al. , 1995). Furthermore, CR/ ChAT colocalization studies in humans revealed that the vast majority of large CR+ neurons in the human striatum also express ChAT (Cicchetti et al., 1998; Massouh et al., 2008). The data gathered in the present study confirms these findings in humans, and further revealed that CR/ChAT colocalization in large striatal interneurons also exists in monkeys. Altogether, these findings indicate that most large striatal neurons that express CR belong to the population of large cholinergic striatal neurons in human and nonhuman primates. The situation in primates appears to be unique compared to that in rodents, where CR+ striatal neurons expressing ChAT have never been detected. The presence of some large CR+ striatal neurons has been alluded to in rats (see above), but none of these elements were found to be ChAT immunoreactive (Rymar et al., 2004). The present investigation of the mouse striatum clearly revealed that neither the small nor the medium-sized CR+ striatal neurons are ChAT immunoreactive. This finding underlines a fundamental difference that exists between rodents and primates in regards to the chemical anatomy of the striatum, but whose functional significance is still not understood. Why would large striatal cholinergic neurons express CR in primates but not in rodents? Is this a newly acquired cytological feature that would allow striatal interneurons to resist pathological insults that are specific to primate brains? Indeed, though its capacity of maintaining intracellular calcium homeostasis, CR might confer to the large cholinergic neurons a certain protection against neurodegenerative processes, particularly glutamatergic excitotoxicity, which is seen as a major pathogenic component of several

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neurodegenerative disorders (Choi, 2005). However, this putative protection mechanism is certainly not entirely effective, at least in Huntington's disease. Data gathered in a transgenic mouse model of Huntington's disease and in post-mortem brain tissue from patients have revealed that, although not physically lost, the large cholinergic striatal neurons display severe dysfunctions in regards to the expression of various cholinergic markers, as well as that of the CR (Massouh et al., 2008; Smith et al., 2006). These findings raise the possibility that CR exerts its protective effect mainly in the early phases of the disease, and becomes less effective in the late phases. The use of the Drd1a-tdTomato/Drd2-EGFP double transgenic mice has also allowed us to demonstrate, for the first time, that the large cholinergic striatal neurons in mice express D2 but not D1 dopamine receptors. This finding is largely congruent with previous data gathered in different species by means of electrophysiological, biochemical, pharmacological and binding approaches (Bergson et al., 1995; Dawson et al., 1988; Goldberg et al., 2012; Yan et al., 1997). Large striatal interneurons also appear to express the D5 receptor subtype, a member of the D1 family of dopamine receptors, as determined by single-cell mRNAs detection in rats (Yan et al., 1997), and immunohistochemical approach in monkeys (Bergson et al., 1995). However, whole cell recording experiments in rats indicated that the dopamine major influence upon large cholinergic interneurons is exerted via a reduction of calcium currents resulting from the activation of the D2 receptor subtype (Yan et al., 1997). Altogether, these findings support the crucial role that the dopamine-acetylcholine interaction plays in the functional organization of the striatum, particularly in relation to the progressive transformation of motivational states into specific motor performances (Schultz, 2002). A disturbance or imbalance of this dual interaction is believed to be central to the pathogenesis of various neurodegenerative diseases. In Parkinson’s disease, for instance, a decrease in striatal dopaminergic innervation leads to an augmentation of the release of acetylcholine by the large striatal interneurons, a phenomenon that disrupts functionally and morphologically the striatal network and contributes heavily to motor symptoms (Pisani et al., 2007). Enhanced striatal cholinergic activity is also believed to be a major actor in the development of L-Dopa-induced dyskinesia, as revealed by the use of 6-OHDA-lesioned and genetically modified mice (Ding et al., 2011). Obviously, such animal models of Parkinson’s disease offer unique opportunities to further our understanding of the pathogenesis of neurodegenerative disorders. However, close attention will have to be paid to species differences between rodents and primates in regards to the anatomical and functional organization of the striatum if one hopes to correctly interpret the changes observed in experimental animals and to translate usefully this type of information to human pathologies. Acknowledgments This study was supported by research grants from the Canadian Institutes of Health Research (CIHR MOP-115008 to M.P.) and the Natural Sciences and Engineering Research Council of Canada (NSERC 401848 to A.P and NSERC 386396 to M.P.). References Bennett, B.D., Bolam, J.P., 1993. Characterization of calretinin-immunoreactive structures in the striatum of the rat. Brain Res. 609, 137–148. Bergson, C., Mrzljak, L., Smiley, J.F., Pappy, M., Levenson, R., Goldman-Rakic, P.S., 1995. Regional, cellular, and subcellular variations in the distribution of D1 and D5 dopamine receptors in primate brain. J. Neurosci. 15, 7821–7836. Bernácer, J., Prensa, L., Giménez-Amaya, J.M., 2012. Distribution of GABAergic interneurons and dopaminergic cells in the functional territories of the human striatum. PLoS One 7, e30504.

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