Dose-dependent expression of claudin-5 is a modifying factor in schizophrenia methods
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Injection of AAV for claudin-5 suppression in the hippocampus and medial prefrontal cortex
C57/BL6J mice (8-12 weeks old) were anaesthetized using a ketamine/metadomidine mixture administered via intraperitoneal injection and placed in a stereotaxic frame. An incision was made to expose the skull, and burr holes were made using a surgical drill either above the dorsal hippocampus or the medial prefrontal cortex (mPFC). A Hamilton syringe was loaded with an AAV expressing either a shRNA against claudin-5 or a non-targeting (NT) control, and the needle was slowly lowered into the dorsal hippocampus: (co-ordinates: A/P=-1.9 mm; M/L=±1.55 mm; D/V=1.75 mm, or mPFC: (co-ordinates: A/P=+1.9 mm; M/l=±0.4 mm; D/V=2.5 mm). AAV solution of 2.0 µl was then injected at a rate of 0.5 µl per min, and once complete, the needle was left in place for 5 min before repeating the procedure in the other hemisph...
Immunocytochemistry and immunohistochemistry
HEK293 cells and Bend.3 cells were seeded on 1% fibronectin-coated Nunc Lab-Tek II Chamber Slides (Thermo Scientific, Dublin, Ireland) in Dulbecco's modified Eagle's medium. After plasmid or shRNA transfection, cells were fixed for 10 min at room temperature with ice-cold methanol, washed twice with PBS and incubated with 5% normal goat serum before overnight incubation with polyclonal rabbit anti-claudin-5 (1:100). Cells were then washed twice with PBS and incubated with Cy3-conjugated goat anti-rabbit IgG secondary antibody (1:500; Abcam, Cambridge, UK) for 2 h at room temperature and counterstained with Hoechst 33258 to visualize nuclei.
Immunocytochemistry and immunohistochemistry
Mice were killed and the brains quickly removed. One hemisphere was embedded in optimal cutting temperature compound (VWR, Dublin, Ireland), snap-frozen in liquid nitrogen and stored at -20 °C prior to slicing on a cryostat. Mouse brain cryosections (12 µm thick) were post-fixed in ice-cold methanol for 10 min at room temperature and washed three times in PBS. Sections were then incubated with 5% normal goat serum before overnight incubation with primary antibodies (rabbit anti-claudin-5 1:100, rabbit anti-ZO-1 1:100 and rabbit anti-occludin 1:100, Life Technologies). Sections were double-stained with isolectin-IB4-Alexa Fluor 488 1:300, Life Technologies, to label vessels. For permeability experiments, sections were incubated with Fluorescein isothiocyanate-conjugated polyclonal rabbit anti-human fibrinogen 1:100, DAKO, or Cy3-conjugated streptavidin...
Primary mouse brain microvascular endothelial cell isolation
Microvessels were isolated from cortical grey matter of C57BL/6J mice by collagenase/dispase (Roche) digestion and bovine serum albumin density gradient centrifugation. Purified vessels were seeded onto collagen IV/fibronectin-coated tissue-culture plates or Corning (Corning, NY, USA) HTS 24-well Transwell polyester inserts (0.4 µm pore size, vessels from five mouse brains per 3 ml) at high density. Cells were grown in EGM2-MV (Lonza, Cambridge, UK) (with 5 µg/ml puromycin during the first 3 days for endothelial cell selection) for 2-3 weeks until their transendothelial electrical resistance values plateaued.
Drug treatments
Haloperidol, lithium chloride and chlorpromazine were purchased from Sigma-Aldrich. Confluent primary mBMEC cells were treated with 0.2, 2 and 20 µ m chlorpromazine, or haloperidol diluted in culture medium containing 0.1% DMSO, and 0.1, 1 and 10 m m lithium diluted in culture medium. For in vivo injections, haloperidol and chlorpromazine were diluted in 2.5% polyethylene glycol 400 in saline and 200 µl was injected via tail vein at a dose of 1 mg/kg body weight. Lithium was diluted in saline and injected at a dose of 100 mg/kg body weight. Animals were killed 24 h following injection, and tissues were processed for protein and RNA analysis.
Generation of inducible claudin-5 knockdown mice
For behavioural experiments, all mice (Cre-positive and Cre-negative) were given doxycycline in their drinking water (2 mg/ml in 2% sucrose solution) and kept on doxycycline for the length of the behavioural experiments. Behavioural testing began 14 days after the introduction of doxycycline to drinking water to ensure maximal suppression of claudin-5. Behavioural testing was then performed for ~4 weeks before animals were killed for histological and molecular analysis.
Magnetic resonance imaging
BBB integrity was assessed in vivo via magnetic resonance imaging (MRI), using a dedicated small rodent 7 T MRI system located at TCD ( www.neuroscience.tcd.ie/technologies/mri.php ). Anaesthetized mice were physiologically monitored (electrocardiogram, respiration and temperature) and placed on an MRI-compatible support cradle, with a built-in system for maintaining the animal's body temperature at 37 °C. The cradle was then positioned within the MRI scanner. Accurate positioning was ensured by acquiring an initial rapid pilot image, which was then used to ensure the correct geometry was scanned in all subsequent MRI experiments. Upon insertion into the MRI scanner, high-resolution anatomical images of the brain were acquired (100 µm in-plane and 500 µm through-plane spatial resolution). To visualize brain damage and lesion volumes, high-resolut...
Acoustic prepulse inhibition
Sensorimotor gating was assessed by prepulse inhibition (PPI) of the acoustic startle response. The PPI apparatus consisted of a soundproof PPI chamber with a weighing scale positioned in the centre of the chamber beside a loudspeaker. Mice were maintained in a holding chamber placed on the scale. Each mouse was given 2 days to habituate to the holding chamber and PPI chamber with a constant background white noise (65 dB). Before the beginning of the experiment, all instruments were calibrated and startle stimulus (71, 77, 83, 100, 110, 120 dB) were set. PPI was divided into three stages: Two minutes habituation with constant background noise of 65 dB.
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C57/BL6J mice (8-12 weeks old) were anaesthetized using a ketamine/metadomidine mixture administered via intraperitoneal injection and placed in a stereotaxic frame. An incision was made to expose the skull, and burr holes were made using a surgical drill either above the dorsal hippocampus or the medial prefrontal cortex (mPFC). A Hamilton syringe was loaded with an AAV expressing either a shRNA against claudin-5 or a non-targeting (NT) control, and the needle was slowly lowered into the dorsal hippocampus: (co-ordinates: A/P=-1.9 mm; M/L=±1.55 mm; D/V=1.75 mm, or mPFC: (co-ordinates: A/P=+1.9 mm; M/l=±0.4 mm; D/V=2.5 mm). AAV solution of 2.0 µl was then injected at a rate of 0.5 µl per min, and once complete, the needle was left in place for 5 min before repeating the procedure in the other hemisphere. Anaesthesia was reversed with an intraperitoneal injection of atipamezole and placed in an incubator until recovered. All mice were given 7 days of recovery before introducing doxycycline into their drinking water (2 mg/ml in 2% sucrose solution). Doxycycline treatment was continued for t...
HEK293 cells and Bend.3 cells were seeded on 1% fibronectin-coated Nunc Lab-Tek II Chamber Slides (Thermo Scientific, Dublin, Ireland) in Dulbecco's modified Eagle's medium. After plasmid or shRNA transfection, cells were fixed for 10 min at room temperature with ice-cold methanol, washed twice with PBS and incubated with 5% normal goat serum before overnight incubation with polyclonal rabbit anti-claudin-5 (1:100). Cells were then washed twice with PBS and incubated with Cy3-conjugated goat anti-rabbit IgG secondary antibody (1:500; Abcam, Cambridge, UK) for 2 h at room temperature and counterstained with Hoechst 33258 to visualize nuclei.
Mice were killed and the brains quickly removed. One hemisphere was embedded in optimal cutting temperature compound (VWR, Dublin, Ireland), snap-frozen in liquid nitrogen and stored at -20 °C prior to slicing on a cryostat. Mouse brain cryosections (12 µm thick) were post-fixed in ice-cold methanol for 10 min at room temperature and washed three times in PBS. Sections were then incubated with 5% normal goat serum before overnight incubation with primary antibodies (rabbit anti-claudin-5 1:100, rabbit anti-ZO-1 1:100 and rabbit anti-occludin 1:100, Life Technologies). Sections were double-stained with isolectin-IB4-Alexa Fluor 488 1:300, Life Technologies, to label vessels. For permeability experiments, sections were incubated with Fluorescein isothiocyanate-conjugated polyclonal rabbit anti-human fibrinogen 1:100, DAKO, or Cy3-conjugated streptavidin 1:100, Sigma-Aldrich, overnight at 4 °C. Following three washes in PBS, sections were incubated with Cy3-conjugated goat anti-rabbit IgG secondary antibody (1:500; Abcam) for 2 h at room temperature, washed three times with PBS and counterstained with Hoechst 33258 for 30 s a...
Microvessels were isolated from cortical grey matter of C57BL/6J mice by collagenase/dispase (Roche) digestion and bovine serum albumin density gradient centrifugation. Purified vessels were seeded onto collagen IV/fibronectin-coated tissue-culture plates or Corning (Corning, NY, USA) HTS 24-well Transwell polyester inserts (0.4 µm pore size, vessels from five mouse brains per 3 ml) at high density. Cells were grown in EGM2-MV (Lonza, Cambridge, UK) (with 5 µg/ml puromycin during the first 3 days for endothelial cell selection) for 2-3 weeks until their transendothelial electrical resistance values plateaued.
Haloperidol, lithium chloride and chlorpromazine were purchased from Sigma-Aldrich. Confluent primary mBMEC cells were treated with 0.2, 2 and 20 µ m chlorpromazine, or haloperidol diluted in culture medium containing 0.1% DMSO, and 0.1, 1 and 10 m m lithium diluted in culture medium. For in vivo injections, haloperidol and chlorpromazine were diluted in 2.5% polyethylene glycol 400 in saline and 200 µl was injected via tail vein at a dose of 1 mg/kg body weight. Lithium was diluted in saline and injected at a dose of 100 mg/kg body weight. Animals were killed 24 h following injection, and tissues were processed for protein and RNA analysis.
For behavioural experiments, all mice (Cre-positive and Cre-negative) were given doxycycline in their drinking water (2 mg/ml in 2% sucrose solution) and kept on doxycycline for the length of the behavioural experiments. Behavioural testing began 14 days after the introduction of doxycycline to drinking water to ensure maximal suppression of claudin-5. Behavioural testing was then performed for ~4 weeks before animals were killed for histological and molecular analysis.
BBB integrity was assessed in vivo via magnetic resonance imaging (MRI), using a dedicated small rodent 7 T MRI system located at TCD ( www.neuroscience.tcd.ie/technologies/mri.php ). Anaesthetized mice were physiologically monitored (electrocardiogram, respiration and temperature) and placed on an MRI-compatible support cradle, with a built-in system for maintaining the animal's body temperature at 37 °C. The cradle was then positioned within the MRI scanner. Accurate positioning was ensured by acquiring an initial rapid pilot image, which was then used to ensure the correct geometry was scanned in all subsequent MRI experiments. Upon insertion into the MRI scanner, high-resolution anatomical images of the brain were acquired (100 µm in-plane and 500 µm through-plane spatial resolution). To visualize brain damage and lesion volumes, high-resolution images were acquired using Rapid Acquisition with Relaxation Enhancement (RARE) 2-D sequence with a RARE factor of 8 and an echo time resulting in an effective time of 42.2 ms (with a flip angle of 180). With an acquisition matrix of 128 × 128 and a field of view of 1.8 × 1.8R...
Sensorimotor gating was assessed by prepulse inhibition (PPI) of the acoustic startle response. The PPI apparatus consisted of a soundproof PPI chamber with a weighing scale positioned in the centre of the chamber beside a loudspeaker. Mice were maintained in a holding chamber placed on the scale. Each mouse was given 2 days to habituate to the holding chamber and PPI chamber with a constant background white noise (65 dB). Before the beginning of the experiment, all instruments were calibrated and startle stimulus (71, 77, 83, 100, 110, 120 dB) were set. PPI was divided into three stages: Two minutes habituation with constant background noise of 65 dB.
Machine-readable layer
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