The number of striatal cholinergic interneurons expressing calretinin is increased in parkinsonian monkeys

    The number of striatal cholinergic interneurons expressing calretinin is increased in parkinsonian monkeys Sarah Petryszyn, Th´er`ese Di Paolo, Andr´e Parent, Martin Parent PII: DOI: Reference:

S0969-9961(16)30159-0 doi: 10.1016/j.nbd.2016.07.002 YNBDI 3791

To appear in:

Neurobiology of Disease

Received date: Revised date: Accepted date:

15 April 2016 13 June 2016 3 July 2016

Please cite this article as: Petryszyn, Sarah, Di Paolo, Th´er`ese, Parent, Andr´e, Parent, Martin, The number of striatal cholinergic interneurons expressing calretinin is increased in parkinsonian monkeys, Neurobiology of Disease (2016), doi: 10.1016/j.nbd.2016.07.002

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Associate Editor: Dr. Erwan Bezard

Centre de recherche de l’Institut universitaire en santé mentale de Québec, Department of

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Sarah Petryszyn1, Thérèse Di Paolo2, André Parent1, Martin Parent1*

Centre de recherche du CHU de Québec, Faculty of Pharmacy, Université Laval, Quebec City,

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Psychiatry and Neuroscience, Faculty of medicine, Université Laval, Quebec City, QC, Canada

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QC, Canada

Running Title:

Striatal interneurons in a primate model of Parkinson’s disease

Keywords:

Striatum; Interneurons; Parkinson’s disease; Neurodegenerative disorders; Primates.

Address:

Martin Parent, Ph.D. Centre de Recherche, Institut universitaire en santé mentale de Québec 2160, Canardière, Quebec City, Québec, Canada, G1J 2G3 Tel: (418) 663-5747; Fax: (418) 663-8756 E-mail: [email protected]

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Striatal interneurons in MPTP monkeys

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The most abundant interneurons in the primate striatum are those expressing the calcium-binding protein calretinin (CR). The present immunohistochemical study provides detailed assessments

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of their morphological traits, number, and topographical distribution in normal monkeys (Macaca fascicularis) and in monkeys rendered parkinsonian (PD) by MPTP-intoxication. In

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primates, the CR+ striatal interneurons comprise small (8-12 µm), medium (12-20 µm) and large-sized (20-45 µm) neurons, each with distinctive morphologies. The small CR+ neurons

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were 2-3 times more abundant than the medium-sized CR+ neurons, which were 20-40 times more numerous than the large CR+ neurons. In normal and PD monkeys, the density of small

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and medium-sized CR+ neurons was twice as high in the caudate nucleus than in the putamen, whereas the inverse occurred for the large CR+ neurons. Double immunostaining experiments revealed that only the large-sized CR+ neurons expressed choline acetyltransferase (ChAT). The

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number of large CR+ neurons was found to increase markedly (4-12 times) along the entire

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anteroposterior extent of both the caudate nucleus and putamen of PD monkeys compared to controls. Comparison of the number of large CR-/ChAT+ and CR+/ChAT+ neurons together

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with experiments involving the use of showed that it is the expression of CR by the large ChAT+ striatal interneurons,

and not their absolute number, that is increased in the dopamine-depleted striatum. These findings reveal the modulatory role of dopamine in the phenotypic expression of the large cholinergic striatal neurons, which are known to play a crucial role in PD pathophysiology.

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The striatum is the largest and major integrative component of the basal ganglia (Lanciego et al., 2012; Parent and Hazrati, 1995). Like most other forebrain structures, it is

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composed of projection neurons and local interneurons but, at variance with most other brain

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regions, the striatal projection neurons greatly outnumber interneurons. Indeed, striatal projection

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neurons represent about 90-95% of the total neuronal population in rats (Gerfen and Bolam, 2010) and 75-80% in primates (Graveland and DiFiglia, 1985). In contrast to striatal projection

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neurons, striatal interneurons are remarkably heterogeneous. Although they all possess spineless dendrites and an indented nuclear envelope, their size and neurochemical features vary

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considerably. Classically, the striatal interneurons are grouped into two main classes: the large

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aspiny neurons that use acetylcholine as a neurotransmitter, and the small to the medium-sized

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neurons that utilize GABA. The latter class of striatal interneurons can be further subdivided into four distinct groups based on the type of calcium-binding proteins, neuropeptides and enzymes they express: 1) parvalbumin; 2) calretinin (CR); 3) somatostatin, neuropeptide Y and neuronal nitric oxide synthase or nicotinamide adenine dinucleotide phosphate-diaphorase; and 4) tyrosine hydroxylase (TH) (Cicchetti et al., 2000; Kawaguchi et al., 1995; Tepper et al., 2010; Xenias et al., 2015). Of all types of striatal interneurons, those expressing CR are the most abundant in human and nonhuman primates (Aosaki et al., 1995; Bernácer et al., 2012; Inta and Gass, 2015; Kataoka et al., 2010; Wu and Parent, 2000). This neuronal population is composed of a large number of small (8-12 µm) uni- or bipolar neurons that display intense CR immunoreactivity and a moderate number of medium-sized (12-20 µm) triangular or ovoid neurons that are moderately immunoreactive, bot types of CR-positive (+) neurons being uniformly scattered in the striatum.

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It also comprises a smaller number of weakly immunoreactive, large (20-40 µm) and multipolar

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CR+ neurons, the majority of which express the enzyme choline actetyltansferase (ChAT) and thus overlap with the class of large aspiny cholinergic interneurons (Bernácer et al., 2012;

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Kataoka et al., 2010; Petryszyn et al., 2014).

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Interestingly, new neurons are reportedly generated in the striatum of adult rats under

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both normal and poststroke conditions and these newborn elements were shown to be primarily CR+ interneurons (Ma et al., 2013; Yang et al., 2008). Likewise, new CR+ interneurons are

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alledgdely added to the human striatum at a substantial rate throughout adult life (Ernst et al., 2014; Inta et al., 2015; Inta and Gass, 2015). Conversely, CR+ striatal interneurons seem to be

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specifically targeted in some psychiatric and neurodegenerative diseases (Kataoka et al., 2010;

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Ma et al., 2014; Mura et al., 2000). In Parkinson’s disease (PD), the degeneration of the

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nigrostriatal dopaminergic projection has been shown to affect not only the morphological features of striatal projection neurons but also the number of the various types of striatal interneurons (Gittis and Kreitzer, 2012; Huot et al., 2007; Pisani et al., 2007). In regards to the CR+ striatal interneurons, studies undertaken in rodents in which the nigrostriatal dopaminergic pathway has been lesioned by means of the neurotoxin 6-hydroxy-dopamine (6-OHDA) led to contradictory findings. While some data indicate a significant but transitory increase in the number of striatal CR+ neurons (Mura et al., 2000), others suggest a permanent decrease of the same neuronal elements (Ma et al., 2014). In face of the conflicting results that the 6-OHDA rodent model of PD has yielded and because nothing is known about the CR+ interneurons in the dopamine-denervated striatum of primates, we undertook the present study to characterize changes regarding morphological

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features, topographical distribution and densities of the three major types of CR+ striatal

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interneurons in PD macaque monkeys.

Animals

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Eight, four-year-old, ovariectomized female cynomolgus monkeys (Macaca fascicularis,

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3.33 ± 0.38 Kg) were used. Animals were housed in a temperature-controlled room (21–25°C) under a 12 h light/dark cycle and had free access to food and water ad libitum, in accordance

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with the Canadian Council on Animal Care’s Guide to the Care and Use of Experimental

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Animals. Université Laval’s Institutional Animal Care and Use Committee had approved all our

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protocols.

MPTP intoxication

Four monkeys served as controls, whereas 4 others received injections of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), so as to render them parkinsonian. The MPTP intoxication was initiated 4 months after ovariectomy. MPTP neurotoxin (Sigma-Aldrich Canada Ltd., Oakville, ON, Canada) was administered on a continuous basis via a subcutaneous osmotic mini-pump (model 2ML4, Alzet, Cupertino, CA, USA) filled with 14 mg of MPTP during 2 consecutive weeks. After the removal of the mini-pump, further intra-muscular injections of the toxin were given, as needed, until stabilization of bilateral parkinsonian symptoms. The monkeys received a cumulative dose of MPTP that ranges from 6 to 14.25 mg (see Table S1 as supplementary material for individual values). Mobility, climbing, gait,

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grooming, voicing, social interaction and tremor were used as criteria to define a disability score

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for parkinsonian symptoms (Hadj Tahar et al., 2000; Hadj Tahar et al., 2004; Samadi et al., 2004; Samadi et al., 2006). Each animal was scored twice for 2 hours and the mean of the scores

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obtained was used to define the severity of their parkinsonian syndrome. These animals were

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sacrificed 5 months after the last MPTP injection, as described below.

Bromo-deoxyuridine injections

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All monkeys used in the present study received intravenous injections of Bromodeoxyuridine (BrdU, 100 mg/Kg, Sigma, St.Louis, MO, USA), dissolved in 0.9% NaCl with

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weeks after the last BrdU injection.

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0.007 N NaOH, once a day for 5 consecutive days. The monkeys were allowed to survive 4

Transcardiac perfusions

At the end of the protocol, both controls and MPTP-intoxicated monkeys were deeply anesthetized with a cocktail of ketamine (20 mg/Kg, i.m.) and xylazine (4 mg/Kg, i.m.) and maintained under isoflurane anesthesia (3%). They were perfused transcardially with an initial wash of PBS (0.1M; pH 7.4; 300 mL) followed by 4% paraformaldehyde (PFA; 1 L) to which 0.2% of glutaraldehyde was added, then washed again with 4 % PFA only (1.5 L). Brains were dissected from the skulls, immersed and post-fixed (24 h) in 4% PFA. They were cut with a vibratome (VT1200S; Leica, Wetzlar, Germany) into 50 µm-thick coronal sections, which were serially collected in cold PBS (0.1 M; pH 7.4).

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Nigral

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In each animal, five equidistant (900 µm interval) transverse sections were randomly selected through the anteroposterior extent of the right substantia nigra pars compacta (SNc) and

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immunostained for tyrosine hydroxylase (TH, see Table S2 as supplementary material for details

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on primary antibodies). The segment that was sampled extended from 10 to 5 mm behind the

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anterior commissure (Martin and Bowden, 2000). Sections were first incubated 1 h in a blocking solution (0.1% Triton X-100, 2% of normal horse serum, diluted in PBS) followed by overnight

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incubation with a monoclonal TH antibody (1/1000). The sections were then rinsed in PBS and incubated 2 h with an anti-mouse biotinylated antibody (1/1000, Vector Laboratories). Following

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rinses in PBS, sections were incubated 1 h with the avidin-biotin-peroxydase complex (1/100,

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Vector Laboratories) diluted in the blocking solution. Sections were then rinsed once in PBS,

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twice in a Tris-buffered saline (TBS; 50 mM, pH 7.4) and incubated 3 min in a solution containing 0.025% of 3,3' diaminobenzidine tetrahydrochloride (DAB; Sigma) dissolved in TBS to which 0.005% hydrogen peroxide was added to reveal the bound peroxydase. The reaction was stop by TBS and PBS rinses and the sections mounted on gelatin-coated slides, air-dried overnight, dehydrated in 70% ethanol for 10 min, rehydrated in distilled water for 5 min and stained with cresyl violet for 20 min. Finally, the sections were dehydrated in alcool grade series, cleared in toluene and coverslipped with Permount. A stereological approach was used to estimate the number of nigral TH+ neurons. Each immunostained section was examined with a light microscope (Leica, DM 6000B) equipped with a digital camera (Optronics, microfire), a motorized stage (X and Y axes) and a Z-axis indicator (Leica Z axis control) was controlled by a computer running StereoInvestigator software (v.7.00.3; MicroBrightField, Colchester, VT, USA). The contours of the SNc were first outlined

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at a low magnification (2.5X objective). The sampling process leading to estimations of the total

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number of TH+ neurons began by randomly translating a grid formed by 800 x 800 µm squares over the sections. At each intersection of the grid that fell into the contours of the SNc, a

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counting frame measuring 250 x 250 µm was drawn and examined with a 20X objective. Nuclei

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of immunolabelled neurons that fell inside the counting frame and did not contact the exclusion

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lines were counted whenever they came into focus within a 6 µm-thick optical dissector centered in the section. An average number of 91 +/- 12 TH+ neurons were counted in each SNc of MPTP

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monkeys and 341 +/- 38 in control animals, yielding coefficients of error (Gundersen, m = 1 and

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2nd Schmitz-Hof) between 0.05 and 0.12.

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Striatal dopamine depletion following MPTP intoxication

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Three transverse sections were selected for each monkey through the striatum, at 3, 0 and -3 mm from the anterior commissure (Martin and Bowden, 2000). The sections were doubly stained for TH and the dopamine transporter (DAT), using infrared immunofluorescence. First, sections were pre-incubated for 2 h in a blocking solution. They were then incubated with the TH antibody and a monoclonal antibody against DAT (1/500). Sections were then rinsed in PBS and incubated with IRDye 680 anti-mouse (1/1000, Mandel) and IRDye 800 anti-rat (1/1000, Mandel). They were rinsed, mounted on gelatin-coated slides, air-dried and coverslipped with a fluorescence mounting medium (Dako; Mississauga, ON, Canada). These sections were scanned with an infrared imaging system (Odyssey CLx; LI-COR biosciences, Lincoln, NE, USA). Regions of interest in the caudate nucleus and the putamen were scanned at the 3 anteroposterior levels, with 4 optical density readings in the caudate nucleus and the putamen for each monkey.

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CR immunostaining In a first serie of experiments, we used a stereological approach to assess the

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topographical distribution and the density of striatal neurons expressing CR. This was done on 6

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equally spaced coronal sections (1200 µm interval) selected between 4 and -4 mm relative to the

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anterior commissure (Martin and Bowden, 2000). Sections were first incubated for 30 min in a solution of sodium borohydride (5 g/L) and rinsed in PBS. They were then incubated for 1 h in a

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blocking solution and reincubated overnight with a polyclonal anti-CR antibody (1/500). Sections were then rinsed in PBS, incubated with an anti-rabbit biotinylated antibody (1/1000,

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Vector Laboratories) and rinsed in PBS. They were then incubated during 1 h in a solution

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containing ABC, rinsed in PBS and TBS and incubated for 3 min in a solution containing 0.05%

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of DAB (Sigma) dissolved in TBS to which 0.005% hydrogen peroxide was added to reveal the bound peroxydase. The sections were finally rinsed in TBS and PBS to stop the reaction, mounted on gelatin-coated slides, air-dried overnight, dehydrated in alcool grade series, cleared in toluene and coverslipped with Permount. The density of CR+ interneurons in the putamen and the caudate nucleus was estimated by using an unbiased stereological approach, as described above. In this case, the grid was formed by 350 µm x 350 µm squares whereas the counting frame measured 250 x 250 x 12 µm and was examined with a 20X (0.70 N.A.) objective. An average number of 5,939 ± 315 striatal CR+ interneurons were counted in each monkey, yielding coefficients of error (Gundersen, m = 1 and 2nd Schmitz-Hof) between 0.012 and 0.015. Having distinct morphologies and immunostaining intensities, the 3 types of CR neurons could easily be differentiated. When needed, the diameter of CR+ striatal perikarya was measured during the stereological scanning

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using the “quick measure line” tool available in the StereoInvestigator software. The density of

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each type of striatal CR+ interneurons was expressed as number of interneurons per mm3 of tissue, using the estimated number calculated by the optical disector and the volume of the

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striatal territories estimated by the Cavalieri's method. The Cavalieri’s method provided an

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unbiased estimate of the volumes of striatal sectors that were sampeled by using a point grid. The

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points were spaced equally on each section and the number of points contained in a given striatal sector were used by the StereoInvestigator software to compute volume (Garcia-Finana et al.,

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2003).

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CR and ChAT double-immunostaining

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In each monkey, neurons expressing CR and/or ChAT were visualized by means of

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double immunofluorescence applied to 3 striatal sections taken at precommissural (pre-ac), commissural (ac) and postcommissural (post-ac) levels (corresponding to level ac +2, 0 and - 2 mm in the atlas of Martin and Bowden, 2000). In this case, sections were inubated for 48 h with a primary antibody against ChAT (1/25) to which the antibody against CR (1/500) was added the next day. Sections were then rinsed in PBS and incubated with a biotinylated anti-goat antibody (1/1000, Vector). The sections were rinsed, reincubated with an anti-rabbit antibody coupled to an Alexa Fluor 594 (1/200, Jackson) and a streptavidin coupled to an Alexa Fluor 488 (1/200, Molecular Probes). Sections were washed in PBS, incubated for 10 min with 4’, 6 diamidino 2 phenylindole (DAPI, 100 ng/mL). Finally, sections were rinsed with PBS, air-dried and coverslipped with Dako. To assess the density of CR+/ChAT+ interneurons, the contours of the caudate nucleus and the putamen were first outlined at low magnification (2.5X objective). These striatal regions

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were carefuly and entirely examined with a 40X objective. Every ChAT+ interneuron

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encountered were counted and scanned using a confocal microscope to assess their CR content. An average number of 1,673 ± 50 striatal ChAT+ interneurons were examined in each monkey.

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Area calculation of striatal regions that were examined was determined using the

CR and BrdU double-immunostaining

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StereoInvestigator software and results were expressed in cells/mm2.

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In each monkey, one striatal section taken 2 mm anterior to the anterior commissure was doubly lebeled for CR and BrdU. Sections were incubated for 2 h in the blocking solution. They

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were then incubated for 40 min at 37˚C in a solution containing 2N of hydrochloric acid diluted

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in PBS, rinsed in PBS and incubated overnight in a solution containing a primary antibody

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directed againt BrdU (1/200) and CR (1/500). The sections were then incubated for 1 h with a biotinylated anti-rat antibody (1/1000, Vector), reincubated for 2 h with an anti-rabbit antibody coupled to an Alexa Fluor 594 (1/200, Jackson) and a streptavidin coupled to an Alexa Fluor 488 (1/200, Molecular Probes). Sections were then incubated for 10 min with DAPI, rinsed with PBS, air-dried and coverslipped with Dako. The procedure used to assess the presence of CR in BrdU+ striatal cells was virtually the same than that employed for CR and ChAT double immunostaining analyses. An average number of 74 ± 23 striatal BrdU+ cells were examined in each monkey.

Confocal imaging and statistical analysis All immunofluorescence doubly stained sections were scanned with a Laser Scanning Microscope (LSM700, Zeiss). Images were acquired using the Zen software (v.7.1, 2011) and

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processed with the Adobe Illustrator CS3 (v.13.0.0) or Photoshop CS6 (v.13.0.1 x64) software.

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Significant differences were detected by using either Kruskal-Wallis or Mann-Whitney statistical tests. The Prism software (v.5.0, San Diego, CA, USA) was used to perform statistical analyses.

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Mean and standard error of the mean were used throughout the text as central tendancy and

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dispersion measures.

Clinical and morphological effects of MPTP-induced dopamine denervation

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The four MPTP-injected monkeys developed the core symptoms of parkinsonism. The

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disability scores indicate that they developed a mild to severe parkinsonian syndrome (see Table S1 in supplementary material). These clinical effects were associated with a marked alteration of

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the nigrostriatal dopaminergic pathway (Fig. 1), as revealed by the stereological analysis of THimmunostained sections taken through the substantia nigra of these monkeys. The estimated number of TH+ neurons in the SNc was 77% lower in MPTP monkeys compared to controls (21 053 ± 2387 vs. 91 097 ± 7009, Fig. 1C). This marked nigral cell loss was acompagnied by a severe dopamine denervation of the striatum, as revealed on TH and DAT-immunostained striatal sections (Fig. 1 D-F, see also Fig. S1 as supplementary material for DAT). Optical density measurements of TH and DAT striatal immunoreactivity at precommissural, commissural and postcommissural levels in controls and lesioned monkeys showed that MPTP intoxication induces a statistically significant decrease of the striatal dopaminergic innervation along the entire anteroposterior extent of both the caudate nucleus and putamen. Decreases in TH striatal immunostaining ranged from 72% to 92% (Fig.

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1), whereas the corresponding values for DAT varied from 73% to 92% (supplementary

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material). In contrast, the TH or DAT immunostaining intensity of the nucleus accumbens did

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not differ statistically in monkeys of the two groups.

Figure 1. MPTP administration causes severe lesion of the dopaminergic nigrostriatal pathway. A, B: Transverse sections taken through the substantia nigra pars compacta (SNc), immunostained for tyrosine hydroxylase (TH) and counterstained with Nissl from control (A) and MPTP-intoxicated monkeys (B). C: Unbiased quantifications indicate that the number of TH+ cells located in the SNc is significantly decreased in MPTP monkeys, when compared to controls. D, E: Transverse sections taken through the striatum at the anterior commissure (ac) level and immunostained for TH in control (D) and MPTP monkeys (E). F: Optical density measurements of THimmunostained sections indicate a significant decrease of the dopamine innervation in the caudate nucleus (Cd) and the putamen (Put), and a preservation of this innervation in nucleus accumbens (Acb). * P ˂ 0.05, Mann-Whitney.

Morphological characteristics and numerical densities of striatal CR+ interneurons The analysis of CR-immunostained striatal sections in both control and MPTP monkeys revealed the presence of three types of CR interneurons. The first type consists of small (8-12

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µm) cells with round or ovoid perikarya giving rise to one or two short processes, whereas the

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second type is composed of medium-sized (12-20 µm) bipolar or ovoid perikarya with 2 to 3 slightly varicose and poorly branched processes. The third type consists of a smaller population

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of large (20-45 µm) globular or triangular neurons displaying several thick and short varicose

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dendrites that branch frequently. These giant CR+ neurons were weakly immunostained

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compared to the small and medium-sized CR+ cells. The morphological features of each of these three types of CR+ neurons did not vary between control and MPTP monkeys. Immunostaining

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intensity of the small and medium-sized CR+ neurons was similar between the two experimental groups whereas the large-sized CR+ cells were slightly more immunoreactive in the dopamine-

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depleted striatum.

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The stereological unbiased evaluation of the overall density of the three types of striatal

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CR+ interneurons taken together revealed that the mean density of all CR+ interneurons was significantly higher in the caudate nucleus than in the putamen in both controls (1747 ± 163 cells/mm3 vs. 833 ± 28 cells/mm3) and MPTP-intoxicated monkeys (1812 ± 163 cells/mm3 vs. 1025 ± 132 cells/mm3; Fig. 2). The mean density of all CR+ neurons in the caudate nucleus did not vary significantly along the anteroposterior axis, whereas a slight anteroposterior-decreasing gradient was observed in the putamen (Fig. 2).

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Figure 2. Regional distribution of all CR+ interneurons in the striatum of control and MPTP monkeys. A: Histogram showing that the density of CR+ interneurons is significantly higher in the caudate nucleus (Cd) than in the putamen (Put) in both control and MPTP monkeys. B: Histogram showing an anteroposterior-decreasing gradient of CR+ neurons in the Put but not in the Cd. * P ˂ 0.05, Mann-Whitney.

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The small CR+ striatal neurons were significantly more abundant in the caudate nucleus than the putamen in both experimental conditions (Fig. 3C, see also Table S3 and S4 as

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supplementary material). In normal monkeys, the mean density of these neuronal elements was 1335 ± 96 cells/mm3 in the caudate nucleus compared to 623 ± 23 cells/mm3 in the putamen,

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whereas the corresponding values in MPTP-intoxicated monkeys were 1392 ± 66 and 761 ± 67 cells/mm3. The mean density of the small CR+ neurons did not differ significantly along the anteroposterior axis of the caudate nucleus and the putamen, in control and MPTP monkeys (Fig. 3D), except for an anteroposterior-decreasing gradient in the putamen that reached statistical significance in control monkeys only. By comparing densities of the small-sized CR neurons between MPTP and control animals, a statistically significant 22% increase in the postcommissural putamen was observed in MPTP monkeys (see Table S4 as supplementary material). Overall, the medium-sized CR+ neurons were less numerous than the small-sized immunoreactive cells, but both the small and medium-sized CR+ neurons were more numerous in the caudate nucleus than in the putamen (Fig. 3G). In normal monkeys, the mean density of

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the medium-sized CR+ neurons was 409 ± 76 cells/mm3 in the caudate nucleus compared to 202

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± 32 cells/mm3 in the putamen, whereas the corresponding values in MPTP intoxicated monkeys were 392 ± 117 cells/mm3 and 229 ± 77 cells/mm3. There was no statistical difference between

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the densities of medium-sized CR+ neurons in caudate nucleus and putamen when the values

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were compared between controls and MPTP monkeys. In regards to the mean densities of the

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medium-sized CR+ neurons, as estimated along the anteroposterior extent of the caudate nucleus and the putamen, the values did not differ significantly from one another, although there was a

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clear tendency for an anteroposterior-decreasing gradient of the medium-sized CR+ neurons in the putamen of both control and MPTP monkeys (Fig. 3H).

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Overall, the density of the large CR+ neurons was significantly lower than the densities

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obtained for medium and small CR+ neurons. At variance with small and medium-sized CR+

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neurons, the large CR+ neurons were more numerous in the putamen than in the caudate nucleus (Fig. 3K). In normal monkeys, the mean density of the large CR+ neurons was 3 ± 1 cells/mm3 in the caudate nucleus compared to 8 ± 6 cells/mm3 in the putamen, whereas the corresponding values in MPTP intoxicated monkeys were 29 ± 9 and 35 ± 12 cells/mm3. However, the differences between these figures did not reach statistical significance. In contrast to the small and medium-sized CR+ neurons, the large CR+ neurons tend to follow an increasing anteroposterior gradient in the striatum of both controls and MPTP monkeys (Fig. 3L). Although the mean densities of large striatal CR+ interneurons were 4-10 times greater in MPTP monkeys compared to controls, and this in both the caudate nucleus and putamen, only the values obtained for the caudate nucleus at commissural levels reached statistical significance (Fig. 3L).

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Figure 3. Morphological features and regional distribution of the three types of CR+ interneurons throughout the striatum of control and MPTP monkeys. Examination of CR immunostained sections indicates the presence of small (8-12 µm), mostly unipolar CR+ neurons (A, B), medium-sized (12-20 µm) CR+ triangular or polygonal neurons with 2-3 poorly branched dendrites (E, F) and large-sized (20-45 µm) polygonal and multipolar CR+ interneurons (I, J) in the striatum of control and MPTP monkeys. Stereological quantification indicates that the density of the small CR+ neurons is higher in the caudate nucleus (Cd) than the putamen (Put, C) for both experimental groups, and more numerous in the post-commissural Put of MPTP monkeys when compared to control (D). The density of the medium-sized CR interneurons is also higher in the Cd than the Put (G). The density of the large-sized CR interneurons is significantly lower than the small and medium-sized CR cells. In contrast to the small and mediumsized CR neurons, the density of the large CR cells is higher in the Put than the Cd (K) and they are distributed according to an anteroposterior-increasing gradient (L). Our analysis indicates significant increases in the density of the large CR interneurons in MPTP monkeys compared to control. * P ˂ 0.05, # P = 0.0571, Mann-Whitney.

Double-immunostaining for CR and ChAT A double CR/ChAT immunofluorescent analysis of the striatum was undertaken to determine if CR+/ChAT+ neurons might account for the increase in the number of large striatal CR+ neurons that we detected in MPTP-intoxicated monkeys (Fig. 4). Only the large CR+ striatal neurons were found to diplay ChAT immunoreactivity, the small and medium-sized CR+

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neurons being totally devoid of such a staining. The bulk of the population of giant striatal

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interneurons in control monkeys consitsts of singly ChAT-labeled cells that do not show any immunoreactivity for CR. The total number of large striatal neurons that displayed ChAT

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immunoreactivity did not vary significantly between control and MPTP monkeys in either the

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caudate nucleus or the putamen and at any of the three anteroposterior levels examined (Fig. 4E).

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In contrast, the proportion of CR+/ChAT+ neurons relative to the total number of ChAT+ neurons was 5 to 10 times higher in MPTP monkeys compared to controls and this in both the

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caudate nucleus and the putamen and at the three anteroposterior levels examined (Fig. 4F). Differences between control and MPTP monkeys reached statistical significance at the pre- and

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commissural levels of the caudate nucleus and at the commissural level of the putamen.

Figure 4. The number of striatal cholinergic interneurons expressing CR is increased in MPTP monkeys. A-D: Large striatal interneurons immunostained for ChAT (green) and CR (red) in the striatum of control (A, B) and

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MPTP monkeys (C, D). Examples of ChAT+/CR- (A, C) and ChAT+/CR+ (B, D) large interneurons are provided. Our stereological quantification indicates similar densities of ChAT+ interneurons between control and MPTP monkeys (E). However, our analysis reveals a significant increase of the number of ChAT interneurons expressing CR in MPTP, when compared to control (F). * P ˂ 0.05, # P = 0.0571, Mann-Whitney.

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In sections immunostained for both CR and BrdU, none of the CR+ cells present in the caudate nucleus or the putamen were found to contain BrdU in either controls or MPTP monkeys

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(Fig. 5). Yet, a few BrdU+ cells occurred throughout the striatum, being slightly more abundant

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in the caudate nucleus than in the putamen. Our quantitative analyses revealed a decrease of 6 times in the number of BrdU+ cells in the caudate nucleus of MPTP-intoxicated monkeys (0.37 ±

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0.12 cells/mm2) compared to controls (2.09 ± 0.30 cells/mm2). A decrease of 8 times was

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observed at the putamen level, with a density of BrdU+ cells of 0.14 ± 0.02 cells/mm2 for MPTP

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monkeys compared to 1.14 ± 0.06 cells/mm2 for controls.

Figure 5. A few BrdU-lebeled cells are found within the striatum of normal and MPTP monkeys. A: A BrDU+ cell (green) devoided of CR lying near a small-sized CR+ neuron (red) in the caudate sucleus of a control monkey. B: A BrdU+ cell adjacent to two small-sized CR+ neurons in the putamen of a MPTP-trated monkey.

The present study has provided the first detailed account of the morphological characteristics, topographical distribution and numerical densities of the CR+ interneurons in the

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striatum of normal and MPTP-intoxicated macaque monkeys. In normal monkeys, significant

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variations were noted between the caudate nucleus and the putamen in regards to the number and distribution of the small, medium and large CR+ interneurons along the anteroposterior axis. In

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MPTP-intoxicated monkeys, we detected a marked increase in the number of the large striatal

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CR+ interneurons compared to controls, together with a smaller augmentation of the small CR+

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interneurons, particularly noticeable in the posterior portion of the putamen. Furthermore, our double immunofluorescent experiments revealed that the vast majority of large CR+

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interneurons, whose number increases in dopamine-depleted striatum, express ChAT and thus belong to the group of giant striatal cholinergic interneurons. Although the total number of large

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ChAT+ striatal neurons was similar in controls and MPTP, the proportion of CR+/ChAT+

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neurons relative to the total number of ChAT+ neurons was significantly increased in MPTP

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monkeys compared to controls.

The striatal CR+ interneurons in normal condition Our investigation of the striatum of normal and MPTP-intoxicated cynomolgus monkeys has confirmed the existence of three types of CR+ neurons detected recently in human and nonhuman primates (Bernácer et al., 2012; Petryszyn et al., 2014). In regard to their density, the small CR+ neurons were found to be overall 3-4 times more abundant than the medium-sized CR+ neurons, which were themselves 20-40 times more numerous than the large CR+ neurons. Our results regarding the topographical distribution of the CR+ interneurons reveal that the small and medium-sized CR+ neurons abound principally in the head of the caudate nucleus, which corresponds to the major portion of the associative striatal territory, whereas the large CR+ neurons are preferentially located in the caudal two-thirds of the putamen, which form the core

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of the sensorimotor striatal territory (Parent, 1990). Such a topographical difference suggests that

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the small and medium-sized CR+ neurons are involved in a different aspect of striatal

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functioning than the large CR+ neurons.

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The striatal CR+ interneurons in parkinsonian condition

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The number of small CR+ striatal neurons was found to be increased by about 20% in MPTP monkeys compared to controls. The functional significance of such a change is difficult to

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assess, however, since the augmentation was present solely in the putamen (the caudate nucleus being unaffected) and reached statistical significance only in the post-commissural portion of the

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structure. This increase might be related to the marked augmentation of the small TH+ striatal

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interneurons noted previously in dopamine-depleted striatum in rats and monkeys (Betarbet et

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al., 1997; Cossette et al., 2005; Huot et al., 2008; Tandé et al., 2006; Ünal et al., 2015). In contrast, a recent investigation reported a significant decrease in the number of TH+/CR+ cells in the striatum of MPTP-intoxicated monkeys that were chronically treated with L-Dopa (DiCaudo et al., 2012). The authors suggested that L-Dopa modifies the phenotype of these small striatal neurons with a decrease of the TH+/CR+ phenotype in favor of the TH+/GAD+ phenotype. The fact that the small CR+ striatal neurons display a somewhat immature appearance and prevail in the dorsolateral portion of the striatum, at the border of the neurogenetically active subventricular zone (SVZ) in mice (Petryszyn et al., 2014), raises the possibility that these elements might belong to a population of newly generated cells. Along this line, a recent study in monkeys has reported the presence of some CR+ striatal neurons that display Sox2, a transcription factor expressed in pluripotent and adult stem cells, including neuronal progenitors (Ordoñez et al., 2013). Furthermore, the number of these Sox2+ cells was markedly increased in

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MPTP-intoxicated monkeys compared to controls, with a temporary decrease in the level of CR

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expression (Ordoñez et al., 2013). However, dopamine is known to stimulate the production of new neurons in adult SVZ so that lesion of the dopaminergic nigrostriatal pathway in a rodent

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model of PD decreases precursor cell proliferation in the adult SVZ (Höglinger et al., 2004; see

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also Parent et al., 2013). Likewise, the fact that none of the small CR+ neurons had incorporated

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the thymidine analogue BrdU in the present study suggests that the increase in the number of small CR+ striatal neurons in MPTP monkeys does not result from the addition of newly

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generated neurons but most likely comes from a phenotypic change of the chemical makeup of preexisting neurons. A similar conclusion has been reached following a study of TH+ neurons in

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the striatum of MPTP monkeys (Tandé et al., 2006). Altogether, these findings suggest that,

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instead of affecting the number of striatal neurons itself, dopamine modulates, in a

interneurons.

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complementary fashion, the levels of CR expressed by the small GABAergic striatal

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As for the large CR+ neurons, the amplitude of changes noted in MPTP monkeys was

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much more significant than that of the small CR+ neurons discussed above. The density of the large CR+ neurons was 4 times higher in the putamen of MPTP monkeys than in that of control

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monkeys, whereas it was 12 times higher in the caudate nucleus of lesioned versus normal

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animals, and these augmentations were of the same amplitude throughout the anteroposterior

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extent of both striatal components. Our double CR/ChAT immunostaining analysis consolidated the notion that the large CR+ neurons of the primate striatum are part of the population of giant

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cholinergic (ChAT+) striatal interneurons, as suggested previously on the basis of data gathered in squirrel monkeys and humans (Cicchetti et al., 1998; Massouh et al., 2008; Petryszyn et al.,

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2014). The quantitative analysis of the double-immunostained sections further revealed that it is

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the CR+/ChAT+ neurons that increase in number in PD monkeys, whereas the total number

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number of ChAT+ neurons remained relatively constant. Hence, as it is the case for the small CR+ neurons, dopamine, or lack thereof, appears to exert a potent influence on the expression of the CR phenotype by the large striatal interneurons. The functional significance of such an increase in CR expression by the large striatal interneurons in a dopamine-poor environment is unclear. The same can be said of the role that CR plays in the overall functioning of these cholinergic striatal interneurons, the activity of which is markedly increased in the PD state. Our recent study with Drd1a-tdTomato/Drd2-EGFP double transgenic mice (Petryszyn et al., 2014), together with previous data gathered in different species by means fo various methodological approaches (Bergson et al., 1995; Dawson et al., 1988; Goldberg et al., 2012; Yan et al., 1997), indicate that dopamine exerts its influence upon the large cholinergic striatal neurons mainly through D2 dopamine receptors. The large striatal cholinergic interneurons, which are often referred to as tonically active neurons (TAN), also

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Striatal interneurons in MPTP monkeys

appear to express the D5 receptor subtype, a member of the D1 family of dopamine receptors

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(Bergson et al., 1995; Yan and Surmeier, 1997). However, whole cell recording experiments in rats indicated that the dopamine major influence upon large cholinergic interneurons is exerted

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via a reduction of calcium currents resulting from the activation of the D2 receptor subtype (Yan

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et al., 1997).

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Altogether, these findings support the crucial role that the interaction between dopamine and acetylcholine plays in the functional organization of the striatum, particularly in relation to

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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

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pathogenesis of various neurodegenerative diseases, particularly PD, which has long been

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considered as an hypercholinergic condition (Barbeau, 1962; Duvoisin, 1967; Hornykiewicz and

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Kish, 1987). The decrease in striatal dopaminergic innervation that characterises PD leads to an augmentation of acetylcholine release 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 6OHDA-lesioned and genetically modified mice (Ding et al., 2011). Recently, optogenetic inhibition of striatal cholinergic interneurons was shown to alleviate motor deficits in mouse models of PD (Maurice et al., 2015). More specifically, this type of inhibition apparently affects specifically the excitability of striatal D1 medium spiny neurons, normalizes pathological bursting activity in the substantia nigra pars reticulata, one of the two main basal ganglia output structures, and increases the functional weight of the direct striatonigral pathway in cortical information processing (Maurice et al., 2015). The fact that these effects do not occur when the

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inhibitory procedure is applied to nonlesioned mice suggests that the role of the large cholinergic

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striatal interneurons in motor function is highly dependent on dopamine tone (Maurice et al.,

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2015).

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Concluding remarks

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We have shown that the density of the large CR+ neurons is significantly increased in the dopamine-denervated striatum of MPTP monkeys. By comparing the number of large CR+,

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ChAT+ and CR+/ChAT+ neurons

that the number of large striatal ChAT+

interneurons expressing CR is increased in PD monkeys. Altogether, these findings indicate a

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modulatory role of dopamine on CR content of the large cholinergic striatal neurons. The role

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that CR might play in the physiopathology of the primate striatum is still unclear. Some insights

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along this line could come from a detailed study of the level of CR expression by striatal cholinergic neurons in L-Dopa-treated PD monkeys compared to untreated monkeys. Such an investigation would allow us to see if an augmented CR expression can account for the increased excitability/activity of striatal cholinergic interneurons in monkeys as it appears to be the case in mice (Ding et al., 2011). It would also be important to determine if the increase in CR expression by striatal cholinergic neurons in PD monkeys, as reported here for the first time, is the result of permanent adaptation of these neurons to a striatal dopamine denervation or an acute phenomenon likely to be normalized with time.

ACKNOWLEDGEMENTS

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The authors express their sincere gratitude to Dave Gagnon and Marie-Josée Wallman for

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their technical help with various aspects of this work. The study was supported by research grants from the Canadian Institutes of Health Research (CIHR MOP-115008) to Martin Parent.

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The authors have no conflict of interest to declare.

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Figure legends

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Figure 1. MPTP administration causes severe lesion of the dopaminergic nigrostriatal pathway. A, B: Transverse sections taken through the substantia nigra pars compacta (SNc),

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immunostained for tyrosine hydroxylase (TH) and counterstained with Nissl from control (A)

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and MPTP-intoxicated monkeys (B). C: Unbiased quantifications indicate that the number of

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TH+ cells located in the SNc is significantly decreased in MPTP monkeys, when compared to controls. D, E: Transverse sections taken through the striatum at the anterior commissure (ac)

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level and immunostained for TH in control (D) and MPTP monkeys (E). F: Optical density measurements of TH-immunostained sections indicate a significant decrease of the dopamine

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innervation in the caudate nucleus (Cd) and the putamen (Put), and a preservation of this

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innervation in nucleus accumbens (Acb). * P ˂ 0.05, Mann-Whitney.

Figure 2. Regional distribution of all CR+ interneurons in the striatum of control and MPTP monkeys. A: Histogram showing that the density of CR+ interneurons is significantly higher in the caudate nucleus (Cd) than in the putamen (Put) in both control and MPTP monkeys. B: Histogram showing an anteroposterior-decreasing gradient of CR+ neurons in the Put but not in the Cd. * P ˂ 0.05, Mann-Whitney.

Figure 3. Morphological features and regional distribution of the three types of CR+ interneurons throughout the striatum of control and MPTP monkeys. Examination of CR immunostained sections indicates the presence of small (8-12 µm), mostly unipolar CR+ neurons (A, B), medium-sized (12-20 µm) CR+ triangular or polygonal neurons with 2-3 poorly branched dendrites (E, F) and large-sized (20-45 µm) polygonal and multipolar CR+ interneurons (I, J) in

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Striatal interneurons in MPTP monkeys

the striatum of control and MPTP monkeys. Stereological quantification indicates that the

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density of the small CR+ neurons is higher in the caudate nucleus (Cd) than the putamen (Put, C) for both experimental groups, and more numerous in the post-commissural Put of MPTP

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monkeys when compared to control (D). The density of the medium-sized CR interneurons is

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also higher in the Cd than the Put (G). The density of the large-sized CR interneurons is

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significantly lower than the small and medium-sized CR cells. In contrast to the small and medium-sized CR neurons, the density of the large CR cells is higher in the Put than the Cd (K)

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and they are distributed according to an anteroposterior-increasing gradient (L). Our analysis indicates significant increases in the density of the large CR interneurons in MPTP monkeys

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compared to control. * P ˂ 0.05, # P = 0.0571, Mann-Whitney.

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Figure 4. The number of striatal cholinergic interneurons expressing CR is increased in MPTP monkeys. A-D: Large striatal interneurons immunostained for ChAT (green) and CR (red) in the striatum of control (A, B) and MPTP monkeys (C, D). Examples of ChAT+/CR- (A, C) and ChAT+/CR+ (B, D) large interneurons are provided. Our stereological quantification indicates similar densities of ChAT+ interneurons between control and MPTP monkeys (E). However, our analysis reveals a significant increase of the number of ChAT interneurons expressing CR in MPTP, when compared to control (F). * P ˂ 0.05, # P = 0.0571, Mann-Whitney.

Figure 5. A few BrdU-lebeled cells are found within the striatum of normal and MPTP monkeys. A: A BrDU+ cell (green) devoided of CR lying near a small-sized CR+ neuron (red) in the caudate sucleus of a control monkey. B: A BrdU+ cell adjacent to two small-sized CR+ neurons in the putamen of a MPTP-trated monkey.

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Supplementary figure S1. MPTP administration causes severe lesion of the dopaminergic nigrostriatal pathway. Optical densty measurements of DAT-immunostained sections indicate a

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significant decrease of the dopamine innervation in the caudate nucleus (Cd) and the putamen

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(Put), and a preservation of this innervation in the nucleus accumbens (Acb). * P ˂ 0.05, Mann-

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Whitney.

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ACCEPTED MANUSCRIPT Petryszyn et al.

Striatal interneurons in MPTP monkeys

Research Highlights: Based on size and morphology, 3 types of striatal CR interneurons exist in monkeys.

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The number of large CR striatal interneurons is markedly increased in PD monkeys.

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Expression of CR by the large ChAT striatal interneurons is increased in PD monkeys.

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