Animals
Brains were obtained from 8 C57BL/6 male mice of 3-month-old (Mus musculus; Charles River, QC, Canada). 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. All protocols were approved by the Institutional Animal Care and Use Committee (Comité de Protection des Animaux de l’Université Laval, # 2019-287/VRR-18-107) 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).
6-OHDA unilateral injection
Four mice received a unilateral 6-OHDA injection in the right medial forebrain bundle (mfb) to lesion the dopaminergic striatal afferent projections. Thirty minutes before 6-OHDA injections, mice received an i.p. injection of desipramine (25 mg/Kg) diluted in saline (0.9 %) at a concentration of 2 mg/mL. Mice were then anesthetized using 2% isoflurane and their heads were fixed in a stereotaxic apparatus. A hole was drilled and the following stereotaxic coordinates relative to bregma were aimed: antero-posterior = -1.2 mm; mediolateral = 1.1 mm; dorsoventral = -5.0 mm, corresponding to the mfb, according to the mouse brain atlas of Franklin and Paxinos (1997). A glass micropipette containing a freshly prepared solution of 6-OHDA diluted in ascorbic acid (0.02%) at a concentration of 6 µg/µL was slowly introduced in the mfb. The 6-OHDA was then pressure injected (0.125 µL/min, during 2 min). Following injection, the micropipette was left in place for 2 min before being slowly retracted. A total volume of 0.25 µL of 6-OHDA was injected into the mfb, corresponding to 1.5 µg of 6-OHDA. Four other mice served as the sham group : each animal received a unilateral injection of 0.02% ascorbic acid (vehicle), according to the procedure described above. After the surgery, the skin was sutured and the eight mice were allowed to recover.
Behavioral assessment
The severity of the dopamine lesion was assessed 20 days after 6-OHDA or vehicle unilateral injections. Mice from the two experimental groups were introduced in a large glass cylinder and spontaneous motor behavior was recorded for 10 min, using a digital camera. The number of spontaneous rotations ipsilateral and contralateral to the injection side were then counted post-hoc, by an experimenter blinded to the experimental treatment.
Tissue preparation
Forty days following 6-OHDA or vehicle stereotaxic injections, animals were deeply anesthetized with a mixture of ketamine and xylazine (100 mg/kg, 10 mg/kg, i.p.), and perfused transcardially with an initial wash of 0.9% saline (50 mL), followed by 2% acroleine diluted in phosphate buffer (PB, 100 mL) and by 4% paraformaldehyde (PFA; 100 mL, diluted in PB). Brains were dissected out, post-fixed for 1h in a 4% PFA solution and cut with a vibratome (Leica VT1200S) into 50 µm-thick coronal sections, which were serially collected in sodium phosphate buffer saline (PBS, 0.1M, pH 7.4).
Immunostaining of TH and DAT
For each mouse, two coronal sections were selected through the striatum at 1.42 and 0.14 mm relative to the bregma, and one section through the substantia nigra at -3.10 mm from the bregma (Franklin and Paxinos 1997). These sections were immunostained for tyrosine hydroxylase (TH) in order to assess to the DA lesion caused by 6-OHDA injection. Briefly, sections were incubated for 30 min into a 0.5% solution of sodium borohydride (NaBH4) diluted in PBS then rinsed thoroughly in PBS. Sections were then pre-incubated for 1h into a blocking solution composed of 0.1% Triton X-100, 2% of normal donkey serum diluted in PBS. They were then incubated overnight (ON) with a polyclonal rabbit antibody against TH (Catalog # AB152, Millipore) diluted 1:1000 in the blocking solution. Sections were rinsed in PBS and incubated for 2h with an anti-rabbit biotinylated secondary antibody (Catalog # 711-075-152 58142, Jackson Immunoresearch Laboratories) raised in donkey and diluted 1:1000 in the blocking solution. Sections were rinsed and incubated for 1h with the avidin-biotin-peroxidase complex (Catalog # PK4000, Vector Laboratories). Sections were then rinsed once in PBS and twice in Tris-saline buffer (TBS; 50 mM, pH 7.4) and incubated for 3 min in a solution containing 0.05% of 3,3' diaminobenzidine (Catalog #D5637, Sigma) and 0.005% H2O2 diluted in TBS to reveal the bound peroxidase. Finally, sections were rinsed in TBS to stop the reaction and mounted on gelatine-coated slides, air-dried overnight, dehydrated in alcohol grade series, cleared in toluene and coverslipped with Permount.
To assess quantitatively the DA lesion, two coronal sections per mouse were selected through the striatum (bregma = 0.14 mm) and the nucleus accumbens (NAc, bregma = 1.42 mm) and doubly stained for TH and the dopamine membrane transporter (DAT) with infrared immunofluorescence. Sections were first incubated for 30 min with 0.5% NaBH4, then rinsed thoroughly in PBS. They were then pre-incubated for 1h in 0.5% Triton X-100 in PBS containing 2% of normal goat serum and normal donkey serum (blocking solution). Sections were incubated ON in the blocking solution containing the same TH antibody as above (Catalog # AB152, Millipore, 1:1000) and a monoclonal antibody against DAT (Catalog # MAB369, Millipore, 1:1000). Then, sections were rinsed in PBS and incubated for 2h in the blocking solution to which a secondary antibody IRDye 680 anti-rabbit (Catalog # LIC-926-32223, Mandel, 1:1000) raised in donkey and an IRDye 800 anti-rat (Catalog #LIC-926-32219, Mandel, 1:1000) raised in goat were added. Sections were then rinsed and mounted on gelatine-coated slides, air-dried, and coverslipped with Dako fluorescence mounting medium (Catalog # S-3023, Mississauga, ON, Canada).
Immunostaining of CR
To assess the regional distribution of CR+ interneurons in the striatum, 6 equally-spaced coronal sections (bregma 1.30 mm to -1.70 mm, interval of 600 µm) were randomly selected throughout the entire striatum from the 4 6-OHDA-lesioned mice and the 4 sham-lesioned animals. These sections were singly labeled for the calcium binding protein CR. Briefly, the 6 striatal sections per animal were preincubated for 1h in a blocking solution composed of 0.1% Triton X-100 and 2% of normal donkey serum diluted in PBS. They were then incubated ON with an antibody against CR (Catalog # 7699/4, Swant) diluted 1:500 in the blocking solution. Sections were then rinsed in PBS and incubated for 2h with a secondary antibody against rabbit coupled to Alexa Fluor 594 and raised in donkey (Catalog # 711-075-152; Jackson Immunoresearch Laboratories, 1:200). Sections were rinsed in PBS, incubated for 10min with a 100 ng/mL dilution of 4’, 6-diamidino-2-phenylindole (DAPI) in PBS, used as counterstaining. Sections were finally rinsed in PBS and mounted on gelatine-coated slides, air-dried, and coverslipped with Dako fluorescence mounting medium (Catalog # S-3023, Mississauga, ON, Canada).
Double immunostaining of CR and CB
To determine the regional distribution of CR+ interneurons in the shell and core compartments of the NAc, 3 more sections (bregma 1.30, 1.10 and 0.90 mm) were selected throughout the NAc from the 4 6-OHDA-lesioned mice and the 4 sham-lesioned animals. These sections were doubly stained for CR and calbindin-D-28K (CB). Briefly, the same protocol described above was used, except that normal goat serum was added to the blocking solution as well as a monoclonal primary antibody against CB (Catalog# C9848, Sigma, 1:500). In addition, a secondary antibody against mouse and coupled to Alexa Fluor 488 and raised in goat was used at a dilution 1:200 (Catalog #A11001, MolecularProbes).
Data analysis
Assessment of the dopamine lesion
In each mouse, the dopamine lesion induced by 6-OHDA stereotaxic injections was assessed from sections of the striatum and the NAc stained for TH and DAT using the IRDye 680, and the IRDye 800 respectively, as described above. Sections were scanned using the Odyssey imager (LiCor biotechnology). For each mouse, regions of interest were randomly selected by drawing 6 boxes in the striatum and 6 boxes in the NAc (3 in the core and 3 in the shell) of 150 µm2 on both the intact (left) and lesioned (right) side. Mean TH and DAT immunoreactivity values obtained from the lesioned striatum or NAc, divided by immunoreactivity measured from the intact side x100 was used to report the residual immunoreactivity.
Stereological counting of CR+ cells
In each mouse, the 6 equally-spaced sections selected across the entire striatum (600 µm interval) were examined with 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) and controlled by a computer running StereoInvestigator software. A detailed scanning of the regional distribution of CR+ interneurons throughout the striatum was achieved by dividing the structure into 8 distinct sectors: antero-dorso-lateral (ADL), antero-dorso-median (ADM), antero-ventro-lateral (AVL), antero-ventro-median (AVM), postero-dorso-lateral (PDL), postero-dorso-median (PDM), postero-ventro-lateral (PVL) and postero-ventro-median (PVM). The contour of the striatum was first outlined on each CR-immunostained coronal section using a 4X/0.10 objective, according to the stereotaxic atlas of Franklin and Paxinos (1997). 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 (Fig. 1A). A horizontal line, perpendicular to and centered on the vertical line was added to delineate four sectors on each brain section. The anteroposterior axis was then divided in two by considering the first 3 coronal sections as representative of anterior (pre-commissural) sectors and the last 3 of posterior (post-commissural) sectors. The 3 sections (200 µm interval) taken trough the NAc of each mouse and doubly stained for CR and CB were also analyzed stereologically, the CB immunostaining being used to delineate the core from the shell. The number of doubly stained cells encountered was counted.
The sampling process leading to the estimation of the total number of CR+ interneurons in each of the striatal sectors and in the core and the shell of the NAc began by using a grid formed by 157 x 157 µm squares placed over each section. At each intersection of the grid that fell into the sector, a counting frame of the same size was drawn and examined with a 40X/1.4 objective leading to the examination of the entire area of each sector (Fig. 1B). On each selected section, a mean number of 48.9 + 4.7 sampling sites in each striatal sector, 42.1 + 4.2 in the shell and 39.4 + 4.0 in the core, were examined. Neurons that fell inside the counting frame and did not contact the exclusion lines were counted whenever their nucleus came into focus within a 12 µm-thick optical disector centered in the section (Fig. 1C). An average of 202 ± 29 CR+ cells were counted in each striatum ipsilateral to the sham or 6-OHDA lesion whereas 181 ± 24 were counted in each striatum contralateral to the lesion, yielding coefficients of error (Gundersen, m = 1 and 2nd Schmitz-Hof) ranging from 0.06 to 0.12 and from 0.06 to 0.14, respectively. In the NAc, an average of 103 ± 21 CR interneurons were counted ipsilateral to the sham or 6-OHDA lesion, while 99 ± 19 were counted contralateral to the lesion, yielding coefficients of error (Gundersen, m = 1 and 2nd Schmitz-Hof) between 0.08 and 0.23, and between 0.07 and 0.22, respectively. These coefficients of error are used to assess the precision of the estimation by taking into account the shape of the region of interest, the distribution of neurons within this region and the sampling criteria, predicting the accuracy of stereological procedure (Gundersen et al. 1999; Schmitz and Hof 2000). For each striatal and NAc region, the density of CR+ cells was expressed in number of CR+ neurons per mm3 of tissue, using the total number estimated by the optical disector and the volume of each sector, as estimated by Cavalieri’s method (García-Fiñana et al. 2003). The proportion of CR+ cells expressing CB was determined from doubly stained sections (as described above) by using two different stereological markers for CR+/CB- cells and for CR+/CB+ cells.
Statistical analysis
Differences in the density of CR cells among the striatal regions (anterior vs posterior, dorsal vs ventral and medial vs lateral) as well as between treatment (6-OHDA vs sham-lesioned animals) were assessed by two-way analysis of variance (two-way ANOVA). The dependent variables considered were the regions and the treatments. The Tukey multiple comparison test was applied post-hoc. Differences in spontaneous motor behavior, TH and DAT immunoreactivity between sham and 6-OHDA-lesioned mice were assessed using the Mann-Whitney nonparametric statistical test. Differences were considered statistically significant at P < 0.05 (*). Statistical analyses were performed by using sample sizes of 4 animals per group and the GraphPad Prism software (v.9.0, San Diego, CA, USA). Mean and standard error of the mean are used throughout the text as central tendency and dispersion measure.