Chemical Anatomy of Pallidal Afferents in Primates

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CHEMICAL ANATOMY OF PALLIDAL AFFERENTS IN PRIMATES

Brain Structure and Function

Lara Eid and Martin Parent*

Centre de recherche de l’Institut universitaire en santé mentale de Québec

Department of Psychiatry and Neuroscience, Faculty of Medicine, Université Laval,

Quebec City, QC, Canada, G1J 2G3

*Correspondance : [email protected] 1 Material and methods

Animals

Two adult male cynomolgus monkeys (Macaca fascicularis, Primus Bio-Ressources Inc,

Montreal, QC, Canada) weighing 3.5 kg and 4.2 kg as well as one adult male squirrel monkey (Saimiri sciureus, Buckshire Corporation, Perkasie, PA, USA) weighing 910 g were used. Animals were housed under a 12 h light-dark cycle with food and water ad libitum. Experimental protocols were approved by the « Comité de Protection des

Animaux de l’Université Laval », and all procedures were made in accordance with the

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

(Ed2).

Tissue Preparation

One macaque monkey was euthanized by pentobarbital overdose (0.6 mg/kg, i.m.). Its brain was dissected out and fixed by immersion in 4 % paraformaldehyde (PFA) diluted in phosphate-buffered saline (PB, 50 mM ; pH 7.4) for 4 days. Other monkeys were deeply anesthesized with a mixture of ketamine (20 mg/kg, i.m.) and xylazine (4 mg/kg, i.m.) along with acepromazine (0.5 mg/kg, i.m.) and transcardially perfused. The macaque monkey was perfused with 300 mL ice-cold sodium phosphate-buffered saline

(PBS, 50 mM ; pH 7.4), 1 L of 4% PFA to which 0.2 % of glutaraldehyde was added, followed by another 1.5 L of 4 % PFA. The squirrel monkey was perfused with 200 mL of PBS, 500 ml of 3 % acrolein diluted in PB followed by 1 L of 4 % PFA. Brains were rapidly dissected out and post-fixed by immersion in 4 % PFA, either for 1 h (squirrel monkey) or for 24 h (macaque monkey). These 2 brains were cut in the coronal plane with a vibratome (Leica) into 50 µm-thick sections that were used for immunofluorescence. From the immersion-fixed macaque brain, two 7 mm-thick blocks were taken from the anterior and posterior pallidum of the left hemisphere and one 4 mm- thick block from the anterior pallidum of the right hemisphere. These blocks were used for Golgi-Cox/immunofluorescence experiments.

1.1 Golgi-Cox impregnation

The FD Rapid GolgiStain™ kit (Cat. No PK 401, FD Neurotechnologies, Inc,

Columbia, MD, USA) was used to perform Golgi-Cox impregnation according to instructions provided. Blocks were then serially cut into 50 µm-thick sections using a cryostat (Leica CM 1900), collected in antifreeze solution and kept at -30 °C for further use.

1.2 Double immunofluorescence on Golgi-Cox impregnated tissue

Four adjacent Golgi-Cox impregnated sections were taken from the mid-pallidal level and processed for double immunofluorescence. Briefly, sections were incubated overnight in a blocking solution composed of 5 % normal donkey serum, 5 % normal horse serum and 2 % Triton X-100 diluted in PBS. Sections were then incubated for 4 days in the following primary antibody combinations: (1) the antibody against the serotonin transporter (SERT) made in goat (1:500 ; Cat. No SC-1458, Santa Cruz biotechnology, Santa Cruz, CA USA) and the tyrosine hydroxylase (TH) antibody made in mouse (1:500 ; Cat. No 22941, ImmunoStar, Hudson, WI, USA) and (2) the choline acetyltransferase (ChAT) antibody made in goat (1:25 ; Cat. No AB144P, EMD,

Millipore Corporation, Billerica, MA, USA) and the TH antibody described above. All sections were rinsed thoroughly in PBS and incubated for 2 h with a biotinylated horse anti-goat antibody (Cat. BA-9500, Vector Laboratories, Burlingame, CA, USA) diluted

1:200 in the blocking solution, followed by more rinses in PBS. They were then incubated for 2 h in the same blocking solution containing 1:200 dilutions of Texas Red conjugated donkey anti-mouse antibody (Cat. No 715-075-150, Jackson

ImmunoResearch Laboratories Corporation, West Grove, PA, USA) and Alexa Fluor 488 conjugated streptavidin (Cat. No S11223, Molecular Probes, Life Technologies,

Burlington, ON, Canada). Sections were rinsed in PBS, mounted on gelatin-coated slides, air dried and coverslipped with Vectashield antifade fluorescence mounting medium

(Cat. No H-1400, Vector Laboratories).

SERT/TH and ChAT/TH double immunostained Golgi-Cox impregnated sections were scanned with a confocal microscope (LSM 700, Zeiss Canada). Putative contacts between SERT, TH or ChAT axon varicosities and Golgi-Cox stained pallidal dendrites were identified as such by carefuly examining z-stacks. Confocal images were processed with the Zen 2011 software (Zeiss Canada) for maximum intensity projection. Brightness and contrast were adjusted using the Photoshop software (v. CS2) to faithfully represent our observations.

1.3 Double Immunofluorescence

In a first set of experiments, one brain section from the transcardially perfused macaque was taken at the mid-pallidal level and immunostained for parvalbumin (PV) (Cat. No P3088, Sigma-Aldrich Canada Co., Oakville, ON, Canada). Briefly, the free- floating section was first incubated in a 0.5 % NaBH4 solution diluted in PBS for 30 min.

Following several rinses in PBS, the section was incubated for 1 h in a blocking solution containing 2 % normal donkey serum and 0.3 % Triton X-100 diluted in PBS. The section was then incubated overnight in the same blocking solution to which a 1:500 dilution of the mouse anti-PV antibody was added, rinsed thoroughly in PBS and incubated for 2 h in the 1:200 dilution of Texas Red conjugated donkey anti-mouse antibody. The section was rinsed in PBS, mounted of gelatin-coated slides, air-dried and processed with autofluorescence eliminator reagent (Cat. No 2160, EMD Millipore

Corporation) according to instructions provided by the manufacturer, after which they were coverslipped with Vectashield antifade fluorescent mounting medium with DAPI

(Cat. No H-1200, Vector Laboratories).

In a second set of experiments, two brain sections taken from the mid-pallidal level of the perfused squirrel monkey were immunostained for ChAT (as above) and for calretinin (CR, Cat. No 7699/4, Swant, Swiss antibodies, Marly, Switzerland). The free- floating sections were incubated in the NaBH4 solution, then in the blocking solution containing 2 % normal donkey serum, 2 % normal horse serum and 2 % Triton X-100 and then incubated for 48 h with 1:25 dilution of goat anti-ChAT and a 1:500 dilution of rabbit anti-CR primary antibodies in blocking solution. After several rinses, sections were incubated for 2 h in a 1:1000 biotinylated horse anti-goat secondary antibody (Cat. No

BA-2000, Vector Laboratories) and for 2 h in 1:200 dilutions of FITC conjugated donkey anti-goat (Cat. No 705-095-147, Jackson ImmunoResearch Laboratories

Corporation) and Alexa Fluor 594 donkey anti-rabbit (Cat. No 711-585-152, Jackson ImmunoResearch Laboratories Corporation) in the same blocking solution as above.

Sections were rinsed, mounted on gelatin-coated slides, air-dried, processed with the same autofluorescence eliminator reagent and coverslipped with Vectashield antifade fluorescent mounting medium (H-1400, Vector Laboratories).

Sections processed for PV immunofluorescence with DAPI and for ChAT and CR double immunofluorescence were examined with the LSM 700 confocal microscope

(Zeiss Canada) equipped with four solid-state lasers and a 40X/1.4 oil objective. Images were processed with the Zen 2011 software.