Ten male Sprague–Dawley rats, weighing approximately 250–300 g, were used in this study. Procedures were conducted in accordance with the Animals (Scientific Procedures) Act of 1986 (United Kingdom) and the Society of Neuroscience policy on the use of animals in neuroscience research. Every effort was made to use the minimum number of animals and to minimize suffering.
Preparation of brains for in situ hybridization and immunohistochemistry
Animals were deeply anesthetized using a combination of ketamine (70 mg/kg) and medetomidine (0.5 mg/kg). They were transcardially perfused with a phosphate-buffered paraformaldehyde fixative (4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4). Brains were removed and post-fixed for 12 h in the same solution at 4 °C. They were sectioned in the coronal plane at 40 μm using a vibrating microtome (Vibratome 1500; Vibratome, St. Louis, MO, USA). Free floating sections were rinsed in phosphate-buffered saline (PBS), cryoprotected in a series of sucrose–ethylene glycol solutions (15% followed by 30% sucrose–ethylene glycol) and stored frozen in 30% sucrose–ethylene glycol at −20 °C until use. Solutions used for the preparation of brains and subsequent in situ hybridization steps were treated with an RNase inhibitor (0.1% DEPC).
Riboprobes for in situ hybridization
Sense and antisense digoxigenin-labeled RNA probes for VGluT2, GAD 65 kDa isoform and GAD 67 kDa isoform were prepared using reverse transcriptase–polymerase chain reaction (RT-PCR) and in vitro
transcription. cDNA transcript sequences were identified and downloaded from the Ensembl Rat Genome server (http://www.ensembl.org/Rattus_norvegicus/index.html
). Clustal multisequence alignment analysis (Chenna et al., 2003
) and Blast2-NCBI (http://www.ncbi.nlm.nih.gov/blast/
) searches were used to identify 300–600 bp gene-specific regions within each transcript. Primers flanking target transcript regions were designed using the Vector NTI software package (Invitrogen). Details of primer sequences are shown in .
Two male Sprague–Dawley rats were deeply anesthetized with an overdose of sodium pentobarbital. The brains were carefully removed and the VTA, SN, striatum, cortex and cerebellum were rapidly dissected out. Tissue samples were snap-frozen in liquid nitrogen and stored at −80 °C until use. Frozen tissue samples were thawed in Trizol for total RNA extraction using Invitrogen’s Trizol system.
cDNA transcripts were produced from total RNA extracts using a reverse transcriptase (Superscript First-strand synthesis system, Invitrogen). Gene-specific cDNA sequences were selectively amplified from total cDNA preparations through PCR primed with the appropriate gene-specific primers (Taq polymerase PCR Kit, Qiagen). PCR products were analyzed on a 1% agarose gel. Comparison of actual product size with predicted size was used to establish that successful amplification of the desired target sequence had occurred.
Synthesis of labeled riboprobes
PCR products were used as templates for riboprobe synthesis using an in vitro transcription kit (Digoxigenin RNA Labeling kit; Roche Diagnostics, UK). Labeled RNA products were subjected to alkaline hydrolysis in 0.1 M carbonate buffer (40 mM NaHCO3, 60 mM Na2CO3) to yield approximately 100 bp length riboprobes.
In situ hybridization
A 1:6 series of coronal sections (40 μm) through the midbrain was hybridized for GAD mRNA (both isoforms) (n=4) or VGluT2 mRNA (n=4). During sectioning, sections were collected in single wells. Eight to 10 free-floating sections encompassing an entire 1:6 series were combined in a single well for processing. Rostral–caudal order was re-established for mounting after processing by inspection of dopaminergic groups. The sections were washed several times in phosphate-buffered saline containing 0.1% Tween 20 (PBS-T) before transfer to hybridization buffer (HB, 50% formamide, 5× SSC, 100 μg/ml denatured sonicated salmon sperm DNA, 50 μg/ml heparin, 0.1% Tween 20) for preincubation (1 h at 65 °C). Denatured digoxigenin-labeled riboprobes (diluted in HB) were added to sections, with hybridization for 12 h at 65 °C. After a series of high to low stringency washes, sections were transferred to PBS for detection of DIG-labeled riboprobes using a mouse monoclonal anti-DIG antibody conjugated to alkaline phosphatase (Roche Diagnostics, UK; 1:2000 in PBS applied overnight at 4 °C). Sections were washed in PBS and transferred to a high pH buffer (HP, 100 mM NaCl, 100 mM Tris pH 9.5, 0.1% Tween 20, 50 mM MgCl2). A staining solution made up from NBT/BCIP (nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate) phosphatase substrate (Roche) diluted in HP was applied for 2.5 h. The phosphatase reaction was terminated by washing in PBS.
Optimal concentrations and incubation times were determined for each riboprobe in preliminary dilution series and time course experiments (results not shown). An incubation time of 2.5 h at room temperature was found to produce specific labeling of cell bodies in known GABAergic or glutamatergic cell regions, without significant background. In situ hybridization procedures were also carried out using sense DIG-labeled riboprobes to control for the occurrence of non-specific signals. No cellular labeling was observed in sections hybridized with sense riboprobes (results not shown).
Following in situ
hybridization, sections were further processed to reveal TH immunoreactivity. The primary antibody was a sheep anti-TH from Chemicon (1:5000 in PBS-T with 3% normal rabbit serum, overnight at 4 °C), followed by a biotinylated secondary antibody (diluted 1:200, biotinylated rabbit anti-sheep, Vector Laboratories; Burlingame, USA) overnight at 4 °C. An avidin–biotin peroxidase enzyme complex was prepared and applied according to manufacturer’s instructions (Vectastain Elite ABC kit). Finally, sections were incubated for 5 min in a DAB/hydrogen peroxide substrate solution (prepared according to manufacturer’s instructions, Vectastain DAB substrate kit, Vector Laboratories). Sections were mounted in an aqueous mountant (Vectashield, Vector Laboratories) and coverslipped. Coverslips were fixed in place using clear nail polish. This TH antibody is widely used (e.g. Albéri et al., 2004; Schober et al., 2007
) and only labels cells in dopaminergic regions. In control experiments where the primary or secondary antibody was omitted no labeling was observed. In addition, we have observed complete overlap between TH immunolabeling and in situ
hybridization for TH mRNA (data not shown).
Optical fractionator sampling was carried out on a Leica DMLB microscope equipped with a motorized stage and Lucivid attachment (40× objective). Sampling was implemented using the Stereoinvestigator software package (MicroBrightField Inc; Williston, USA). Midbrain dopaminergic groups were outlined on the basis of TH immunolabeling, with reference to a coronal atlas of the rat brain (Paxinos and Watson, 2005
). Boundaries separating nuclei and subdivisions were identified based on the location of anatomical landmarks (e.g. fiber tracts), the neurochemical content of cells and regional variations in cell density, orientation and morphology (in line with descriptions in previous cytoarchitectonic studies, e.g. Phillipson, 1979a,b
). For example, within the VTA the boundary separating dorsal PN (paranigral nucleus) from ventral PBP (parabrachial nucleus) was established based on visible changes in the density of dopaminergic cell bodies and their dendritic orientation. Cell bodies in PN tended to be more densely packed and with dendrites oriented in a ventromedial direction, in contrast with dopaminergic neurons in PBP, which were more sparsely distributed and appeared to lack any consistent dendritic orientation.
Total estimates of the number of GABAergic and dopaminergic neurons were obtained from four brains processed for GAD in situ hybridization and TH immunohistochemistry. Estimates of glutamatergic neurons were obtained from a further four brains processed for VGluT2 in situ hybridization and TH immunohistochemistry. In order for our counting to encompass the full rostro-caudal extent of the relevant midbrain dopamine nuclei, eight to 10 sections from a 1:6 series were analyzed for each brain. A random start was ensured by using a different well for the beginning of each series combined with the fact that the beginning of the collection of sections from the vibratome varied from brain to brain. Damaged/lost sections, of which there were few, were accounted for by the Stereoinvestigator software. Prior to beginning counting a section, average thickness was measured on a number of sections from different brains. There was no significant shrinkage from the original 38–40 μm thickness, as the sections were mounted in aqueous medium rather than dehydrated. A guard height of 8 μm was used with a sampling brick depth of 20 μm. Pilot studies were used to determine suitable counting frame and sampling grid dimensions prior to counting. Counting frame information: counting frame width (X) was 68.2 μm, height (Y) was 75 μm, depth was 20 μm. Guard regions of 8 μm thick were used for counting frame depth. Counting frame size was set so roughly 5 to 10 neurons were counted per frame and was maintained across all regions sampled. Sampling grid sizes were determined for different major regions (e.g. SN, VTA, and RRF) during pilot studies to allow approximately 15–20 sampling sites per region per section. Sampling grid size was equal for subnuclei within each major region. The sizes were: SN ((X) 366 μm, (Y) 276 μm); VTA ((X) 342 μm, (Y) 192 μm); RRF ((X) 351 μm, (Y) 251 μm). The software carried out these computations. Systematic random sampling was implemented by the Stereoinvestigator software until the designated region of interest was covered.
NBT-BCIP-positive in situ–labeled cells (blue) and DAB-positive immunolabeled cells (brown) were counted simultaneously within each counting frame position. A cell was only marked and counted if a nucleus surrounded by cytoplasm filled with a colored precipitate was clearly visible. Cells were counted only if they came into focus when racking the focus down through the sampling brick.
Image capture and processing
Color digital images were acquired using a Leitz Dialux 22 microscope equipped with a Coolsnap color camera (Photometrics; Tucson, USA) and Openlab software (Improvision; Coventry, UK). Contrast and color balance were adjusted using Adobe Photoshop CS in order to include information-containing pixels and to reflect the true appearance of the tissue as far as possible. Line drawings illustrating the distribution VGluT2-positive neurons in individual sections were prepared from images exported from Stereoinvestigator, using Adobe Illustrator drawing software.