Experiments were carried out in accordance with protocols approved by the Institutional Animal Care and Use Committee at Yale University School of Medicine. Adult CD1 mice (older than 3 months) were purchased from Charles River Laboratories (Wilmington, MA). Mouse colonies were maintained at Yale University, in compliance with National Institutes of Health guidelines and with the approval of Yale University Institutional Animal Care and Use Committee. For timed pregnancies, midday of the day of vaginal plug discovery was considered embryonic (E) day 0.5. Mice at embryonic and postnatal stages through adulthood were used. For embryonic stages, pregnant females were anesthetized, and pups at appropriate stages were extracted from the uterus. Embryonic brains were dissected and fixed by immersion in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS), pH 7.4. Mouse pups [postnatal (P) day 0 through P2] were deeply anesthetized on ice, and then the brains were removed and fixed in 4% PFA. At all other stages, mice were anesthetized with injectable anesthetics (100 mg/kg ketamine and 10 mg/kg xylazine) and intracardially perfused with 4% PFA.
Human and monkey brain specimens
Post-mortem human brain specimens were collected following guidelines on the research use of human brain tissue from the New York State-licensed Human Fetal Tissue Repository at Albert Einstein College of Medicine and the Croatian Institute for Brain Research, University of Zagreb Medical School. Tissues from the Zagreb Neuroembryological Collection were obtained with approval from the Medical Ethical Committee at the University of Zagreb Medical School. For each tissue donation, appropriate written, informed consent and approval were obtained. The study was approved by the Human Investigation Committee at Yale University School of Medicine. Monkey brain specimens were collected from one embryonic (E140) and two adult macaque monkeys (Macaca mulatta
; rhesus monkey). To obtain the embryo, a caesarean section was performed as previously described (Rakic and Goldman-Rakic, 1985
). In short, the pregnant monkey was first sedated with ketamine (3 mg/kg) and atropine sulfate (Lilly; 0.2 mg/kg). A catheter was introduced into the saphenous vein for continuous administration of fluids. Surgery was performed aseptically under halothane-nitrous oxide-oxygen anesthesia. Postoperatively, the animal was administered painkillers, and the wound was periodically cleaned. The animal was monitored until full recovery. The adult monkeys had been retired from a breeding colony in the Department of Neurobiology, in compliance with National Institutes of Health guidelines and with the approval of Yale University Institutional Animal Care and Use Committee.
In situ hybridization
Mouse brains were removed, postfixed overnight in 30% sucrose/4% PFA, and sectioned in the coronal plane on a Leica sledge cryomicrotome at 36 µm (Leica Microsystems, Wetzlar, Germany). Sections were mounted on slides and processed for nonradioactive in situ hybridization as described previously, with minor modifications (Tole and Patterson, 1995
; Grove et al., 1998
). An RNA probe complementary to mouse Slitrk1
(bases 322–1383 of the mouse Slitrk1
cDNA, NM_199065) was prepared and labeled with digoxigenin-11-UTP. Sections were analyzed with a Zeiss Stemi dissecting microscope or a Zeiss AxioImager (Zeiss, Oberkochen, Germany) fitted with an AxioCam MRc5 digital camera. Images were captured using AxioVision AC software (Zeiss) and assembled in Adobe Photoshop.
All antibodies used in this study were individually examined following immunostaining of mouse brain sections by confocal microscopy to ensure that the signal appeared specific and was concordant with known information about the protein. Additional information about each antibody is listed in .
The B-cell CLL/lymphoma 11B (BCL11B) antibody detected two bands of ~120 kD molecular weight corresponding to the two splice variants of BCL11B on Western blots of Jurkat cell lysates (manufacturer’s technical information) and detected the same two bands on Western blots of lysates of HEK293T cells transiently transfected with an expression vector encoding FLAG-CTIP2 and immunoprecipitated with an anti-FLAG antibody (Topark-Ngarm et al., 2006
). This antibody stained a pattern of cellular morphology and distribution in mouse cortical sections identical to that in previous reports (Rašin et al., 2007; Britanova et al., 2008
The calbindin 2 (CALB2; calretinin-22k) antibody recognized a common epitope for both calretinin and calretinin-22k (a region between EF-hand domains III and IV) on Western blots of mouse and rat brain extracts and dot blots (Zimmermann and Schwaller, 2002
) and stained a pattern of cellular morphology and distribution in mouse brain sections identical to that in previous reports (Brandt et al., 2003
) and to the authors’ unpublished observations.
The choline acetyltransferase (ChAT) antiserum specifically recognized a band of 68 kD m.w. in the ammonium sulfate fraction of placental proteins supplemented with affinity-purified placental ChAT but not in that fraction alone (Bruce et al., 1985
) and immunoprecipitated a 68-kD protein that displayed ChAT enzymatic activity from brain and placenta protein extracts; it also precipitated a 27-kD protein that is coexpressed with ChAT but lacks ChAT activity in some cholinergic neurons of the central nervous system (manufacturer’s technical information). This antiserum stained cholinergic neurons in rat cerebral cortex (Lysakowski et al., 1989
). The authors independently assessed the staining pattern in mouse basal ganglia sections and found it identical to previous reports (Gabriel and Witkovsky, 1998
); furthermore, preincubation with recombinant rat ChAT protein abolished staining of large striatal neurons with morphology characteristic of cholinergic interneurons.
dopamine receptor antiserum specifically recognized a band of ~50 kD m.w. on Western blots of membrane protein extracts from rat striatum and cerebellum (manufacturer’s technical information); the authors independently assessed the staining pattern in mouse brain sections and found it identical to that in previous reports (Levey et al., 1993
; Yung et al., 1995
The enkephalin antiserum was tested for specificity by immunohistochemistry; staining was eliminated by preincubation with the immunizing peptide. The antiserum stained a pattern of cellular morphology and distribution in mouse brain sections that is identical to that in previous reports (Holt and Newman, 2004
The forkhead box P2 (FOXP2) antiserum recognized a band of 80 kD m.w. on Western blots of human 293T cell lysates (manufacturer’s technical information); in chromatin immunoprecipitation assays, this antiserum identified DNA binding sites also identified with a different FOXP2 antiserum raised in rabbits immunized with the synthetic peptide EDLNGSLDHIDSNG (C-terminal region of FOXP2) conjugated to KLH (Spiteri et al., 2007
). The antiserum stained a pattern of cellular morphology and distribution in mouse brain sections identical to that in previous reports (Rašin et al., 2007; Fujita et al., 2008
The γ-aminobutyric acid (GABA) antiserum showed positive binding with GABA and GABA-KLH in dot blot assays and negative binding with bovine serum albumin (BSA; manufacturer’s technical information) and stained a pattern of cellular morphology and distribution in mouse brain sections identical to that in previous reports (Ang et al., 2003
; Lee et al., 2006
The glial fibriallary acidic protein (GFAP) antiserum detected a band of 43–45 kD m.w. on Western blots of human brain and spinal cord lysates, corresponding to the predicted molecular weight of GFAP (manufacturer’s technical information) and stained a pattern of cellular morphology and distribution in mouse brain sections identical to that in previous reports in rat (Capani et al., 2001
; Lai et al., 2003
The giantin antiserum detected a band of ~400 kD m.w. on Western blots of mammalian cell lines (including human intestine, lung fibroblasts, and liver and rat and mouse fibroblasts) and by immunostaining (manufacturer’s technical information) and stained a pattern of cellular morphology and distribution in mouse brain sections identical to that in previous reports (Ren et al., 2003
; Zheng et al., 2007
The MAP2 antibody reacted with all known forms of MAP2 (MAP2a–c) on Western blots of combined newborn and adult rat brain lysates, in which it detected two bands of 280 kD m.w. (corresponding to the similarly sized MAP2a and MAP2b) and 70 kD m.w. (MAP2c) and on immunohistochemistry with no cross-reactivity with other MAPs or tubulin (manufacturer’s technical information) and stained a pattern of cellular morphology and distribution in mouse brain sections identical to that in previous reports on rat telencephalon (Fujimori et al., 2002
The μ-opioid receptor antiserum detected a band of about 60 kD m.w. on Western blots, corresponding to the predicted molecular weight of μ-opioid receptor, and provided specific staining in trigeminal ganglion neurons (Berg et al., 2007
). Preabsorption of the antibody with the immunizing peptide blocked signal in immunolabeling (manufacturer’s technical information).
The nitric oxide synthase 1, neuronal (NOS1; nNOS) antiserum recognized a band of 155 kD m.w. on Western blots of rat brain lysates, and immunolabeling was abolished with preabsorption of the corresponding immunizing peptide (manufacturer’s technical information). The antiserum stained a pattern of cellular morphology and distribution in mouse brain identical to that in previous reports (Pla et al., 2006
; Fuentes-Santamaria et al., 2008
The parvalbumin antibody specifically recognized a spot of 12 kD m.w. on two-dimensional gels of rat cerebellar protein extracts, which is identical to purified parvalbumin; the same spot binds Ca2+
, and, furthermore, the antibody stained Purkinje cells in the cerebellum (Celio et al., 1988
) and stained a pattern of cellular morphology and distribution in mouse brain sections identical to that in previous reports (Fazzari et al., 2007
The Rab5 antiserum recognized a band of 26 kD m.w. (and occasionally a second, potentially nonspecific band) on Western blots of rat and mouse brain lysates and transfected Rab5 protein in cells by immunocytochemistry (manufacturer’s technical information) and stained a pattern of cellular distribution in mouse brain identical to that in previous reports (Cataldo et al., 2003
) and to the authors’ unpublished observations.
The Rab11 antiserum detected a single band of about 24 kD m.w. on Western blots of human platelet and mouse liver extracts (manufacturer’s technical information; Becker and Hannun, 2003
) and stained a pattern of cellular morphology and distribution in mouse brain identical to that in previous reports (Rašin et al., 2007).
The Slitrk1 antiserum recognized human SLITRK1 in direct ELISA assays and Western blots (manufacturer’s technical information). Its specificity was further examined on Western blots of mouse and human brain protein extracts in the absence or presence of the immunizing peptide (recombinant human SLITRK1 extracellular domain; R&D Systems, Minneapolis, MN; No. 3009-SK) and of protein lysates from Cos7 cells transfected with a FLAG-tagged human SLITRK1 expression plasmid (Supp. Info. Fig. 1
). In situ hybridization results presented herein, as well as our unpublished data, identified mRNA expression patterns identical to those revealed by immunostaining with this antibody.
The SMI-32 antibody recognized two bands (200 and 180 kD) that merge into a single NFH line on two-dimensional blots of rat brain homogenates and found to be specific for nonphosphorylated neurofilament H (Goldstein et al., 1987
) and stained a pattern of cellular morphology and distribution in mouse brain identical to that in previous reports (Fernandez-Chacon et al., 2004
The substance P antiserum specifically stained neurons in substantia nigra and spinal cord; staining was abolished by preincubation with the immunizing peptide (manufacturer’s technical information). The antiserum stained a pattern of cellular morphology and distribution in mouse brain identical to that in previous reports (Morgado-Valle and Feldman, 2004
; Sun and Chen, 2007
Embryonic, postnatal, and adult mouse brains were collected and fixed as described above, cryoprotected in 30% sucrose in PBS, sectioned at 40 µm using a sledge microtome in the coronal plane, and processed freely floating. Fetal and adult monkey and human brains were fixed in 4% PFA, cryoprotected in 30% sucrose, frozen by immersion in 2-methylbutane, and stored at −80°C. Blocks were sectioned frozen at 60 µm with a cryostat and processed freely floating.
For diaminobenzidine (DAB) staining with the goat anti-SLITRK1 antibody, sections of embryonic, postnatal, or adult mouse and of fetal or adult monkey and human brain were treated with 1% H2O2; washed in PBS; and preincubated in blocking solution (BS) containing 5% normal donkey serum (Jackson Immunoresearch Laboratories, West Grove, PA), 1% BSA, 0.1% glycine, 0.1% L-lysine, and 0.03% Triton X-100. The sections were then incubated with anti-SLITRK1 antibody for 36 hours at 4°C. After washes with PBS, the sections were incubated with secondary antibody [donkey anti-goat Biotin-SP (Jackson Immunoresearch Laboratories; 1:250 dilution)] for 2 hours at room temperature, washed in PBS, and then incubated with Vectastain ABC Elite solution (Vector, Burlingame, CA) for 2 hours. Sections were developed with 0.05% DAB (pH 7.4), 0.2% glucose, 0.01% nickel ammonium sulfate, 0.04% ammonium chloride, and 8 µg/ml glucose oxidase and then rinsed, mounted onto glass slides, allowed to dry, dehydrated, and coverslipped. For double staining with the goat anti-SLITRK1 and mouse anti-ChAT antibodies, adult mouse, monkey, and human brain sections were first incubated with the anti-SLITRK1 antibody and processed for DAB staining as described, followed by incubation with the anti-ChAT antibody for 36 hours at 4°C, washes in PBS, incubation with secondary antibody [horserasdish peroxidase (HRP)-conjugated donkey anti-mouse IgG (Vector, 1:200 dilution)] for 2 hours, washes in PBS, and then incubation with Vectastain ABC peroxidase solution (Vector) for 2 hours. Sections were developed using Nova Red peroxidase substrate (Vector) and then rinsed, mounted onto glass slides, allowed to dry, dehydrated, and coverslipped.
For immunofluorescence, mouse brain sections were placed in BS without H2O2 treatment and then incubated in primary antibodies for 36 hours at 4°C. Secondary antibodies were all highly cross-absorbed, raised in donkey, conjugated to cyanine 2 (Cy2) or Cy3 (Jackson Immunoresearch Laboratories), and used at 1:250 dilution in BS without Triton X-100 for 2 hours at room temperature. The sections were mounted, coverslipped with DAPI-containing Vectashield (Vector), and analyzed with a confocal microscope (Zeiss).
Image capture and analysis
Tissue sections were photographed by using a Zeiss Axio-Imager fitted with a Zeiss AxioCam MRc5 digital camera. Images were captured by using AxioVision AC software (Zeiss) and assembled in Adobe Photoshop. Some images were modified to adjust contrast (see ). For confocal microscopy, single optical and z-stack images were collected on a Zeiss LSM 510 laser-scanning microscope and assembled in Adobe Photoshop.
Figure 9 SLITRK1 protein expression is maintained in cholinergic interneurons of adult striatum. Sections of adult mouse, monkey, and human striatum immunolabeled with anti-SLITRK1 (black) and anti-ChAT (red). A–C: Representative SLITRK1-positive neurons (more ...)
Adult human brain protein extracts (Human Brain Protein Medley) were obtained from Clontech Laboratories (Mountain View, CA). Adult mouse brains were dissected and homogenized in lysis buffer [10 mM Tris, 200 mM NaCl, 1 mM EDTA (pH 7.4), 2% SDS] containing protease inhibitors (Roche Applied Science, Indianapolis, IN). Fifty-microgram protein samples were analyzed via SDS-PAGE under reducing conditions with a 4–15% gradient polyacrylamide gel. Western blotting was performed with anti-SLITRK1 antibody (dilution 1:1,000) and HRP-conjugated donkey anti-goat secondary antibody (dilution 1:5,000); the signal was detected by using a chemilluminescence system (ECL Western Blotting Detection system; GE Healthcare Bio-Sciences Corp., Piscataway, NJ). The blots were stripped with 2% SDS and β-mercaptoethanol and reprobed with HRP-conjugated anti-β-actin (1:5,000; Sigma, St. Louis, MO) for loading control. For peptide competition analyses, the primary goat anti-SLITRK1 antibody was preabsorbed at a 1:5 ratio with the corresponding immunizing peptide (R&D Systems) at room temperature for 2 hours prior to application for both immunostaining and Western blot assays.
Immunogold labeling and electron microscopy
Two protocols were used. For the first, adult mouse and rhesus monkey brain tissue was fixed in 4% PFA with 0.3% glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA) and sectioned with a vibratome at 70 µm. Sections were treated with 0.3% H2O2 in PBS, washed in PBS, preincubated in BS (see above), and then incubated with goat anti-SLITRK1 antibody (1:200) for 36 hours. After washing, the sections were incubated in BS with 0.1% gelatin (GE Healthcare Bio-Sciences Corp.) for 1 hour. Secondary antibody conjugated to 6- or 12-nm gold particles (1:40; Jackson Immunoresearch Laboratories) was applied overnight at 4°C. After washing and a 10-minute fixation in 2% glutaraldehyde and further washes in PBS and dH2O, silver intensification was applied as recommended by the manufacturer (Aurion). After gold immunostaining, sections were washed for 4 hours in phosphate buffer (PB) and then dehydrated as follows: 2 × 1 minutes in 50% EtOH, 45 minutes in 1% uranyl acetate in 70% EtOH, 2 × 1 minutes each in 70%, 90%, and 100% EtOH and in propylene oxide. Sections were then embedded overnight in Durcupan (Sigma-Aldrich), mounted on microscopy slides, and kept at 60°C overnight. SLITRK1-immunopositive regions were identified by light microscopy, reembedded, sectioned ultrathin (60 nm), collected on single whole copper grids (Electron Microscopy Sciences), and contrasted with lead citrate for 2–4 minutes before EM analysis. EM images were captured with a Multiscan 792 digital camera (Gatan, Pleasanton, CA). Representative immunoelectron micrographs are shown in . For the second protocol, adult mouse brain tissue was fixed in 4% PFA/0.2% glutaraldehyde in 0.25 M Hepes, pH 7.4, and postfixed overnight in the same solution. Small blocks of tissue were then embedded in gelatin, cryoprotected in sucrose, rapidly frozen in liquid N2, and sectioned ultrathin on a Leica cryomicrotome (Leica Microsystems) at 60–80 nm. Grids were immunolabeled with goat anti-SLITRK1 antibody (1:20 and 1:100) for 30 minutes at room temperature, washed in PBS, labeled with secondary antibody, washed in PBS, labeled with Protein A-gold for 30 minutes at room temperature, washed in PBS, postfixed in 1% glutaraldehyde for 5 minutes, washed in distilled water, stained with 2% neutral uranyl acetate for 10 minutes, washed briefly in water, and rinsed with 1.8% methylcellulose/0.5% uranyl acetate. Samples were examined on a Philips Tecnai 12 BioTWIN electron microscope (FEI Co.). Images were captured digitally with a CCD camera (Morada; Soft Imaging Systems). Representative immunoelectron micrographs are shown in .
Figure 7 Subcellular localization of SLITRK1 in cortical neurons. A–D: Fluorescence confocal analysis of P7 mouse neocortical sections immunolabeled with SLITRK1 (red) and appropriate subcellular compartment markers (green); cell nuclei are stained with (more ...)
Adult mouse brains were stereotaxically injected with the retrograde tracers (Retro Beads; Lumafluor, Naples, FL) or BDA-3k (dextran-3000 MW conjugated to tetramethylrhodamine; Molecular Probes, Eugene, OR) into the contralateral neocortex and ipsilateral caudate-putamen (i.e., striatum) and thalamus (lateral posterior nucleus). After 7 days, the animals were killed, and brains were processed for immunofluorescence with anti-SLITRK1 antibody.