All experiments were carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. For experiments involving CGNs and cerebellar slice cultures, ΔCR-A+/−
mice (Li et al., 2007b
) were used. ΔCR-A+/−
(Fischer et al., 1996
) and Prn-p+/−
(Büeler et al., 1992
) littermates served as controls. C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, Maine). Mice were genotyped by PCR analysis of tail DNA, prepared using the Puregene DNA Isolation Kit (Gentra Systems, Minneapolis, MN). In order to obtain neural stem cells (NSCs) of different genotypes, ΔCR-A+/−
mice were mated to Prn-p−/−
mice on the C57BL/6 background, as described previously (Massignan et al., 2010
; Biasini et al., 2012b
). E13.5 mouse embryos were genotyped by PCR analysis of limb DNA.
Cerebellar granule neurons (CGNs)
CGNs were cultured from P5 mice as previously described (Li et al., 2007b
). Cerebella were removed from Prn-p−/−
mice or Tg(ΔCR)-A mice, and neurons were isolated by mechanical disruption and trypsinization. Neurons (350,000 cells/cm2
) were plated on poly-L-lysine coated coverslips in Basal Media Eagle (BME) with Earle’s salts supplemented with 10% dialyzed fetal calf serum, 2 mM glutamine, 25 mM KCl, and 0.02 mg/mL gentamycin. Aphidicholin (3.3 μg/mL) was added to cultures one day after plating. Two days after plating, PrP−/−
CGNs were transduced with recombinant lentiviruses.
Lentiviruses were constructed according to published procedures (White et al., 2008
). cDNAs encoding either enhanced GFP alone or WT or ΔCR PrP followed by an internal ribosomal entry site and enhanced GFP were cloned into the transfer vector pRRLsinCMV. 293T packaging cells were co-transfected with the resulting transfer plasmid, along with the plasmids pMD-Lg, pCMV-G, and RSV-REV. Virus was collected from the medium and concentrated.
Viral transductions were performed by incubating cells with purified virus at a multiplicity of infection of 0.1–2.0. Two days after lentiviral transduction, CGNs were collected for Western blotting or stained by immunofluorescence. Western blots were probed with anti-PrP antibody 6D11 followed by goat anti-mouse IgG (Pierce) and developed with ECL (GE Healthcare). Surface immunofluorescence staining was performed by incubating cells on ice with 6D11, after which they were fixed in 4% paraformaldehyde in PBS and labeled with Alexa-488 goat anti-mouse IgG (Molecular Probes). Cells were viewed with a 40X objective on a Nikon TE-2000E inverted fluorescence microscope, and images were captured with METAMORPH software (Molecular Devices, Sunnyvale, CA).
Neural Stem Cells
NSCs were obtained and cultured following a procedure described previously, with minor modifications (Massignan et al., 2010
; Biasini et al., 2012b
). Brains dissected from E13.5 mouse embryos were triturated in 5 ml of NeuroCult NSC basal medium containing NeuroCult NSC proliferation supplement (StemCell Technologies, Vancouver, BC) along with 20 ng/ml EGF. Once formed, neurospheres were differentiated by pipetting a 0.1–1 ml suspension (containing approximately 30–40 mature neurospheres) into each well of an 8-well chamber slide (Ibidi GmbH, München, Germany) containing NeuroCult NSC basal medium with NeuroCult NSC differentiation supplement (StemCell Technologies) along with 10 μg/ml retinoic acid. NSCs differentiated for 10 days were treated with 500 μM glutamate for 24 hrs, stained with propidium iodide (PI; Sigma-Aldrich, St. Louis, MO, USA) to estimate the number of dead cells, and with DAPI to detect cell nuclei.
Ca2+ measurement in differentiated NSCs
Cells were washed with Krebs-Ringer-Hepes (KRH) buffer (128 mM NaCl, 5 mM KCl, 1.2 mM MgSO4, 2 mM CaCl2, 10 mM glucose, 25 mM Hepes, pH 7.4) and incubated with 10 μM Fura-2-acetoxymethyl ester (Fura-2AM) in KRH buffer plus 1% BSA, for 30 min at 37 °C. After washing once with KRH buffer, chamber slides were transferred to the recording chamber of an Olympus IX81 inverted microscope equipped with a Ca2+ imaging unit (Cell R, Olympus). Fluorescence was measured at 37°C in 5% CO2/95% air by recording 669-ms frames for 100 cycles, each cycle alternating excitation at 340 nm and 380 nm and monitoring emission at 510 nm. Ca2+ response was measured after exposure to 0.5 mM glutamate beginning at cycle 20. The fluorescence ratio F340:380 was measured for each cycle and kinetic analysis of F340/380 was done by CellR software (Olympus). Data are reported as ΔF340/380, the difference between F340/380 before and after the stimulus, which is proportional to the glutamate-induced Ca2+ influx.
Neuronal and non-neuronal cells were distinguished based on expression of microtubule associated protein 2 (MAP2). After acquiring data for Ca2+ influx, the position of the microscope stage was recorded in order to allow exact re-alignment of each chamber slide. Cells were immediately fixed with 4% paraformaldehyde in 200 mM Hepes/NaOH pH 7.4 for 30 minutes at RT, washed in PSB and incubated in blocking solution (1% bovine serum albumin, 50 mM NH4Cl, 0.1% saponin in PBS pH 7.4) containing 10% normal goat serum (NGS) for 30 minutes at RT. Cells were then incubated with primary antibody anti-MAP2 (Sigma) diluted 1:1000 in blocking solution overnight at 4°C. After washing in PBS, cells were incubated for 1 h at RT with AlexaFluor 594-conjugated secondary antibody (1:500), then reacted with 300 nM DAPI (4′,6 -diamidino-2-phenylindole, Invitrogen) in PBS for 10 min at RT to stain nuclei. Finally, the chamber slide was re-aligned under the microscope and Ca2+ measurements were assigned to MAP2-positive or -negative cells.
Organotypic slice cultures of mouse cerebellum
After decapitation, brains were removed quickly and the cerebellum was transferred to a container with liquid agarose [low-melting point, prepared freshly each day at 2% in Hanks’ Balanced Salt Solution (HBSS); both reagents obtained from Invitrogen/Life Technologies, Carlsbad, CA, USA]. After the agarose was cooled on ice, agarose blocks were glued onto a vibratome disc, which was then placed in ice-cold HBSS in the buffer reservoir of a Vibratome 1000Plus Sectioning system (Vibratome Company, St. Louis, MO, USA). Several 300 μm sagittal slices were cut at medium speed and amplitude. Slices were collected in a Petri dish containing ice-cold HBSS with 0.5% glucose and released from the agarose under a stereomicroscope using fine forceps. All subsequent steps were carried out under sterile conditions in a cell culture hood.
Each slice was placed in a Millicell Cell Culture insert (Millipore, Billerica, MA, USA) in a 24-well cell culture plate. Culture wells contained 200 μl of slice culture medium consisting of 50% minimum essential medium (MEM; with Earle’s salts, 25 mM Hepes, without L-glutamine; Invitrogen), 22.5% HBSS, 25% horse serum (heat-inactivated; Gibco/Life Technologies), 50 U/ml penicillin/50 μg/ml streptomycin (Invitrogen), 0.5% glucose, and 2 mM L-glutamine (Invitrogen). Slice cultures were kept under standard cell culture conditions (37°C, 5% CO2) in a humidified atmosphere with the medium being changed every other day.
Toxicity assays were performed by adding to the culture media for 24 hours: L-glutamic acid (500 μM), kainic acid, N-Methyl-D-aspartic acid (NMDA), and 2-amino-3-(5-methyl-3-oxo-1,2- oxazol-4-yl)propanoic acid (AMPA) (all at 10 μM; purchased from Sigma-Aldrich); or Zeocin™ (500 μg/ml; Invitrogen). To assess cell death, cerebellar slices were kept in culture for varying time spans, after which they were incubated with 2 μM PI in culture medium for 30 minutes in a cell culture incubator. Red fluorescence was imaged on a Nikon TE2000E2 microscope with a 20x long-working range objective. In some case, nuclear labeling was performed by staining with Hoechst 34580 (2 μg/ml; Invitrogen). Several images of the cerebellar granule layer of each slice were obtained using the auto-expose function of the camera. Positive nuclei were counted with the same settings for brightness and diameter for each image using MetaMorph software (Molecular Devices, Sunnyvale, CA).
For characterization and documentation of the quality of each slice culture, some of the slices were fixed in 4% buffered formalin for 1 hour at 4°C, then washed with phosphate buffered saline (PBS) several times, subjected to paraffin embedding and hematoxylin and eosin (H&E) staining, or immunofluorescence staining. For paraffin embedding, slices were carefully removed from the membranes, placed between two biopsy foam pads in an embedding cassette, and processed according to standard histology procedures. Four μm sections were stained with H&E and assessed using a conventional light microscope.
All incubation steps for immunofluorescence staining were performed with the slices still sitting on the membranes. First, slices were immersed in a blocking solution containing 0.3% Triton X-100 and 10% fetal calf serum (FCS; heat-inactivated; Gibco/Life Technologies) for 24 hours at 4°C. On the following day, an antibody against neuronal nuclei (mouse monoclonal anti-NeuN; clone A60, Chemicon/Millipore) was added at a 1:1000 dilution in blocking solution and left on for 48 hours at 4°C. After 3 washes in PBS/0.3% Triton X-100, an Alexa Fluor 488 rabbit anti-mouse antibody (Molecular Probes/Life Technologies) was added at a 1:200 dilution in PBS/0.3% Triton X-100 for 4 hours at room temperature in the dark. Finally, slices were washed again three times in PBS and imaged using a Nikon TE2000E2 inverted fluorescence microscope equipped with a CCD camera.
All reagents for electron microscopic analysis of organotypic cellebellar slices were purchased from Ted Pella, Inc. (Redding, CA, USA). Slices were washed twice with cacodylic acid buffer and fixed for 24 hours at 4°C in 4% glutaraldehyde in cacodylic acid buffer. After another two washes in cacodylic acid buffer, the specimens were dehydrated through an ascending alcohol series. An epoxy resin consisting of Dodecenyl Succinic Anhydride (DDSA) and Araldite 502 in a 1:1 relation was added to the membrane inserts, and the inserts left on a shaker at room temperature for 2 days. Membrane patches with slices were punched out and transferred to glass vials containing epoxy resin with 2% N,N-Dimethylbenzylamin Benzyldimethylamin (BDMA), which were left on a rotating shaker for 1 hour. Membranes were then placed in electron microscopy molds with resin and left to solidify in a vacuum cabinet. Ultrathin sections were prepared from each block and stained with lead citrate according to standard electron microscopy protocols.
Whole-cell patch clamp recordings were made from CGNs after 4 days in culture, or 4 days after lentiviral transduction. Pipettes were pulled from borosilicate glass, coated with Sylgard, and polished to an open resistance of 1–10 megaohms. Experiments were conducted at room temperature with the following solutions in the patch pipette and extracellular medium: internal, 140 mM Cs-glucuronate, 5 mM CsCl, 4 mM MgATP, 1 mM Na2GTP, 10 mM EGTA, and 10 mM HEPES (pH 7.4 with CsOH); external, 150mM NaCl, 4mM KCl, 2 mM CaCl2, 2 mM MgCl2, 10mM glucose, and 10 mM HEPES (pH 7.4 with NaOH). Current signals were collected from an Axopatch 200B amplifier and digitized with a Digidata 1330 interface (Axon Instruments) or with an EPC-10 amplifier controlled by PatchMaster acquisition software (HEKA Elektronik) and were saved to disc for analysis with PClamp 9 software. Current activity was plotted as the proportion of total recording time that a cell exhibited inward current ≥ 450 pA.
To prepare cerebellar slices for acute recordings, Prn-p+/− ΔCR-A−/− and Prn-p+/− ΔCR-A+/− mice at P10 were sacrificed by decapitation, and their brains were immediately submerged in oxygenated (95% O2; 5% CO2) ice-cold Ringer’s solution [25 mM NaHCO3, 124 mM NaCl, 1 mM KCl, 2 mM KH2PO4, 10 mM Glucose, 2.5 mM CaCl2, and 1.3 mM MgCl2 (pH 7.4)]. Cerebella were affixed to an agar slab with cyanoacrylate glue and placed in a tissue holder for cutting. Four to 6 slices (300 μm thick) were cut into ice-cold Ringer’s solution with a vibrating microtome, and then equilibrated for at least 1 h at room temperature in oxygenated Ringer’s solution. Individual slices were positioned in a submersion-type recording chamber (Harvard Apparatus, Holliston, MA) on the stage of a Nikon E600 infrared-differential interference contrast microscope (IR-DIC; Micro Video Instruments, Avon, MA), and were continuously perfused with room temperature with oxygenated Ringer’s solution (2– 2.5 ml/min). Whole-cell patch clamp recordings were obtained from cerebellar granule neurons at a potential of −80 mV. Patch electrode pipettes were pulled from capillary tubes on a Flaming and Brown horizontal pipette puller (Model P87, Sutter Instrument, Novato, CA) to a resistance of 3–6 MΩ, and were filled with potassium glucuronate (KGlu) internal solution [122 mM KGlu, 2 mM MgCl2, 5 mM EGTA, 10 mM NaHEPES, 2 mM MgATP, 0.3 mM NaGTP, and 1% biocytin, (pH 7.4)]. PatchMaster software was used for data acquisition with EPC-9 and EPC-10 amplifiers, and data were saved to disc for analysis with PClamp 9 software.