N-terminal polyclonal antibody against DISC1 was a generous gift from Dr Gogos (Columbia univ.)49
. Additional DISC1, Kal-7 specific, and Kal-spectrin antibodies were previously described25, 27
. Antibodies to myc (Santa Cruz Biotechnology, Santa Cruz, CA), PSD95 (NeuroMab, Davis, CA), synaptophysin (Sigma-Aldrich Corp, St. Louis, MO), KIF5 (Millipore, Bedford, MA), GFP (Invitrogen, Eugene, OR), HA(Covances), phospho-Pak1 (Cell Signaling technology, Danvers, MA), Pak1 (Cell Signaling technology), Tiam1 (Santa Cruz), GluR1(Millipore), βPIX (Millipore), andAlexa 405-, 488-, 568- or 633-conjugated secondary antibodies (Invitrogen) were used.
Plasmid construction and transfection
Cloning, mutagenesis, and deletion of DISC1 and Kal-7 were conducted as described in the prior publication25
. Wild-type, constitutively-active (Rac1-CA, G12V), and dominant-negative form of Rac1 (Rac1-DN, T17N) were from Missouri University of Science and Technology (Rolla, MO). H1-RNA polymerase III promoter-driven shRNA against DISC1 (#1 with strong effect and #2 with weaker effect), which also carry EGFP under the downstream of CMV promoter, were previously described25
. Cultured cells were transfected with plasmids by the use of LipofectAMINE2000 (Invitrogen). In primary neuron culture, 2μg of pSuper-Venus RNAi were transfected into 1.5 × 105
cells. For 293 cells, 2 μg of pRK/DISC1-HA expression construct and 2 μg of pSuper-Venus RNAi construct were transfected (at 90 % confluence in 18 mm dish). In standard analyses, primary neurons were transfected after 23 DIV and maintained for 1–6 days after transfection.
Recombinant proteins tagged with GST or MBP
The full-length mouse DISC1 cDNA was cloned in pMAL vector for DISC1-MBP. Likewise, the full-length PSD95 and Kal-7 (amino acid 588–1571) were cloned in pGEX for PSD95-GST and Kal-7-GST, respectively. The expression plasmids were introduced into E. coli BL21, grown at 23 °C with 0.1 mM IPTG. Recombinant proteins were purified from E. coli with glutathione sepharose or amylose beads. Further purification was conducted with a centrifugal filter device, Amicon Ultra-100k (Millipore), by which degraded protein fragments at less than 100 kDa were removed.
Viral production and infection
Lentivirus was produced by co-transfection of RNAi-containing FUGW lentiviral constructs and helper constructs VSVG and Δ8.9 into 293FT packaging cells. Media were collected 48 h after transfection and virus was concentrated by ultracentrifugation at 25,000 × g for 90 min. For synthesis of Sindbis virus, recombinant RNAs from Sindbis vector pSinRep5 (nsP2) and packaging construct DH(26)S were generated by in vitro transcription with use of the mMESSAGE mMACHINE kit (Ambion). These RNAs were transferred into BHK cells with GenePulser XCell system (BioRad), followed by collection of media after 24 h. Viral titers were calculated by counting EGFP-positive cells after infecting with serial dilutions of the viral solution. For infection into a primary neuron, virus solution was added to the medium (3.5 × 105 unit/1.5 × 105 cells), which was changed 24 h after infection, followed by incubation for 3–7 days to allow gene knockdown. For infection of slice cultures, 1 μl of 1.8 × 105 unit/μl solution was added directly to the slice. The morphology of infected cells was depicted by the fluorescence of EGFP carried in the viral vector.
Neuron cultures and treatment
All procedures related to animals were performed according to the Johns Hopkins animal care and use guidelines. Dissociated cortical neuron cultures from Sprague-Dawley (SD) rat were prepared as described previously25
. Withdrawal of DL-aminophosphonovalerate (APV) (Ascent Scientific, Weston-super-Mare, UK) was used for chemical activation of NMDA receptor. Briefly, 200 μM APV was added to the culture dish 5 days after plating, and was maintained in the medium with APV. For treatment of APV withdrawal, cells were preincubated in artificial cerebrospinal fluid (ACSF) (in mM: 125 NaCl, 2.5 KCl, 26.2 NaHCO3
, 1 NaH2
, 11 glucose, 5 HEPES, 2.5 CaCl2,
and 1.25 MgCl2
) with 200 μM APV for 30 min at 37°C. Coverslips were then washed in withdrawal medium (ACSF plus 30 μM D
-serine, 100 μM picrotoxin, and 1 μM strychnine) and transferred into new withdrawal medium for 20 min. Cells were then immediately fixed for immunofluorescence or lysed with lysis buffer for biochemical analysis such as Rac1 assay and co-immunoprecipitation (see below). Organotypic cortical culture was prepared from cerebral cortex of postnatal day 3 (P3) pups by using a McIlwain-type tissue chopper. A slice (400 μm thickness) was maintained on the Organotypic cell culture insert (Millipore), which are porous and transparent membranes, with neurobasal-N1 medium [Neurobasal medium (Invitrogen) supplemented with 2 mM L-glutamine, 5% horse serum, and N1 supplement (Sigma)].
Recording for miniature excitatory postsynaptic currents (mEPSC) in cultured neurons (n
= 6 to 12 in each group) were prepared as described previously50
. Patch electrodes (3–5 MΩ) were filled with the following internal solution [(130 mM Cs-methanesulfate, 10 mM CsCl, 4 mM NaCl, 1 mM MgCl2
, 5 mM EGTA, 10 mM HEPES, 5 mM MgATP, 0.5 mM Na2
GTP, 12 mM phosphocreatine, 20 mM leupeptin, and 1 mM QX314), pH 7.2–7.3, 265–270 mosm/l]. The external solution [127 mM NaCl, 5 mM KCl, 2 mM MgCl2
, 2 mM CaCl2
, 12 mM glucose, 10 mM HEPES, and 0.5 mM TTX), pH 7.4, 300 mosm/l]. Bicuculline (10 μM) was added to the external solution to block GABAA
receptors. Recordings were obtained with an Axon Instruments 200B patch clamp amplifier that was controlled and monitored with an IBM PC running pCLAMP with a DigiData 1320 series interface (Axon instruments, Foster City, CA). Electrode resistances in the bath was typically 2–4 MΩ. After seal rupture, series resistance (4–10 MΩ) was compensated (70–90%) and periodically monitored. The cell membrane potential was held at −70 mV. The Mini Analysis Program (Synaptosoft, Leonia, NJ) was used to analyze synaptic activity. For each different condition, mEPSC recordings of 8 min were used for analysis.
Immunofluorescence cell staining
Cells were fixed with 4% formaldehyde for 30 min at room temperature (RT). Experimental details for cell staining were as described previously25
. For staining of surface GluR1 on the plasma membrane, fixed cells were incubated with a primary antibody against an extracellular region of GluR1, prior to permeabilization with Perm/Blocking buffer (2.5 % normal goat serum in PBS with 0.1 % Triton X-100). Then, permeabilized cells were stained for synaptophysin with the protocol for cell staining. For free-floating immunofluorescence staining of organotypic slice, the slices were fixed with 4% formaldehyde for 1 h at RT, followed by permeabilization with Perm/Blocking buffer (2.5 % normal goat serum in PBS with 0.3 % Triton X-100) for 1 h at RT. The slices were incubated for 48 h at 4°C with a primary antibody in Perm/Blocking buffer. After rinsing with PBS (8 times, 5 min each), sections were stained with corresponding secondary antibody, followed by mounting. Cell staining was examined with a confocal microscope (LSM 510-Meta, Zeiss, Minneapolis, MN).
Quantitative morphological analysis of spine
Staining with anti-GFP antibody was used to circumvent potential unevenness of GFP diffusion in spines. For co-transfection experiments, the neurons that were clearly transfected with both GFP and gene of interest were captured as images. Images were taken by a Zeiss LSM510-Meta with the 63 x oil-immersion objective as z-series of 6 to 12 images taken at 0.3-μm intervals (scan averaged, four times; 1024 × 1024 pixel resolution; a scan speed of 1.6 ms/pixel). The acquisition parameters were kept the same for all scans in the same experiment. Only first dendrites that were arborized from a typical apical dendrite of the authentic pyramidal neuron with triangle cell body were subjected to morphological analysis according to a protocol previously described27
. Two-dimensional projection reconstructions of z-series of images, morphometric analysis, and quantification were done using MetaMorph software version 7.1 (MDS Analytical Technologies, Sunnyvale, CA). Individual spines on dendrites were manually traced, and the area (Size), maximum length (Length), and head width (Breadth) of each spine was measured. Based on the sensitivity of this quantitative morphological analysis, which has been already established27
, we did not consider structures that had an area of less than 0.25 μm2
as the spine, because these are approaching the limit of resolution of our microscope. In each experiment, about 500 to 1000 spines from 30 to 45 dendrites derived from 10 to 15 neurons were analyzed per condition. At least two independent analyses, mostly three, were carried out while blinded to transfection condition until statistical analysis had been done.
Preparation of synaptosomes and postsynaptic densities
Cerebral cortex dissected from adult SD rat was homogenized in 10 volumes of buffer A (0.32 M sucrose, 1 mM NaHCO3
, 1mM MgCl2
, and 0.5 mM CaCl2
). Crude synaptosomal and postsynaptic densities were purified using the classical sucrose gradient protocol previously described27
Cells or tissue were lysed in lysis buffer [150 mM NaCl, 50 mM Tris·HCl, pH 7.4, 0.1% NP-40, and protein inhibitor cocktail (Complete; Roche Diagnostics, Indianapolis, IN)]. Lysates were sonicated, cell debris was cleared by centrifugation, and the soluble fraction was subjected to immunoprecipitation (IP), followed by immunoblotting (IB). Normal IgG (IgG) or Myc antibody was used as negative control for immunoprecipitation. The signal intensity of each band (the net signal is after subtraction of background signal obtained from the region adjacent to the band) was measured by ImageJ software (NIH, MD).
Rac activation assay
Activation of Rac1 was measured with the Rac1 activity assay kit (Upstate Biotechnology, Waltham, MA) according to manufacturer’s instructions. Briefly, fusion-protein corresponding to the p21-binding domain of Pak1, which specifically binds to the active form of Rac1 (Rac-GTP) but not to the inactive form of Rac1 (Rac-GDP), is used for precipitation for Rac-GTP. GTP-γs and GDP were used for positive and negative control for assay, respectively. Phosphorylation of Pak1, a substrate for Rac1, was also used as an indicator for Rac1 activity because the binding of Rac1-GTP to Pak1 causes autophosphorylation and activation of Pak1.
Intracerebral injections of lentiviral vectors
SD rats, aged at P21–24, were anesthetized with ketamine and xylazine and placed in a stereotaxic frame to secure the cranium. An incision was made in the scalp and a hole was drilled in the skull at the desired site of injection. The location of the injection site in the prefrontal cortex (PFC) was standardized among animals by using stereotaxic coordinates (AP = +2.2; ML = +1.0; DV = +1.0) from the skull. A total of 8 rats (4 for DISC1 RNAi, 4 for scrambled RNAi) were bilaterally injected with 3 μl of the lentiviral preparations (1.8 × 105 unit/μl) into the medial prefrontal cortex (by using a Hamilton syringe, 0.15μl/min). Eight days after injection, rats were euthanized, and the brains were perfusion-fixed in 4% paraformaldehyde for subsequent immunohistochemical analysis. The morphology of infected cells was depicted by the fluorescence of EGFP carried in the viral vector. All procedures related to animals were performed according to the Johns Hopkins animal care and use guidelines.
Electroconvulsive treatment (ECT)
ECT were induced in an unanesthetized SD rat at P21 by delivering AC current (Current, 90 mA; Frequency, 100 Hz; Pulse width, 0.5 ms; Shock duration, 1.0 s) via ear clip electrodes as described36
. For biochemical analysis, animals were euthanized by cervical fracture, followed by dissection of cerebral cortex 3 min after electroconvulsion.
For determination of the statistical significance between two groups, either the Student’s t-test (equal variances) or Mann Whitney U-tests (unequal variances) were employed. The result of the F-test was used to decide which test was appropriate. To compare three or more groups, one-way ANOVAs were used, followed by Dunnett-T3 post hoc for multiple comparisons. Statistical analyses were performed using SPSS 11.0 software (*P < 0.05; †P < 0.01; #P < 0.001). Error bar indicates s.e.m. Cross bar across a scattered plot indicates the mean of all values.