Generation of conditional RIM1αβ knock-out (KO) mice
Conditional RIM1αβ KO mice were generated according to standard procedures (Rosahl et al., 1993
; Ho et al., 2006
) using homologous recombination in embryonic stem cells that targeted exon 6 of the RIM1 gene. The original targeting vector contained a serine 413 to alanine point mutation which was introduced to probe the effect of serine 413 phosphorylation of RIM1α (Kaeser et al., 2008
). This point mutation was spontaneously repaired during homologous recombination in a subset of embryonic stem cells (Steeg et al., 1990
; Maximov et al., 2008
). By Southern blotting, PCR screening, and sequencing, we isolated these embryonic stem cells in which the RIM1 gene was homologously recombined, but the serine 413 to alanine point mutation was repaired (see result
section and supplemental materials
). We used them for blastocyst injection to produce a conditional RIM1αβ KO mouse. The male chimeric offsprings were bred to C57BL/6 females, and tail DNA of the offsprings was used for genotyping by PCR and Southern blotting to indentify germ line transmission. The absence of the serine 413 to alanine point mutation in the mice was confirmed by BglI digest and DNA sequencing. Flp recombinase transgenic mice (Dymecki, 1996
) and cre transgenic mice (O'Gorman et al., 1997
) were used to remove the double-neomycin resistance cassette and to disrupt the RIM1 gene, respectively. For a detailed description of the genomic clone, the targeting construct and homologous recombination with a targeting construct that contained a serine 413 to alanine point mutation see (Kaeser et al., 2008
), and Supplementary Figure 3
. The floxed, flp recombined allele was genotyped by PCR with oligonucleotide primers PK05163 (GACCGCTGTGCCAGGCGCACCTGC) and PK05164 (CCACAGTCTGCATTCCTACCCG). This reaction results in a 300 base pair (bp) wild type band and a 400 bp floxed band. The KO allele was genotyped with PK04159 (GCAACGTTTGCTGCTGTAAGC 3) and PK04160 (CATTCTTGTCTCAACATTCAAGCC) to identify a 340 bp wild type band and with PK04159 and PK05165 (CATCTTCACCTGCATCTCTGACC) to detect a 240 bp mutant band. All analyses were performed on littermate KO and wild-type offspring from heterozygous matings, and the genotypes were unknown to the experimenter.
5’RACE amplification and reverse transcriptase PCR analysis
Total RNA was purified from the frontal cortex of 17 day old RIM1α KO mice using a TRIzol® Plus RNA Purification Kit (Invitrogen, Carlsbad, CA, USA) according to the protocol delivered with the kit. 5’RACE amplification was performed with a 5’RACE amplification system form Invitrogen (Version 2.0) following the manufacturer’s protocol. We used antisense oligonucleotides annealing to exon 6 of the RIM1 mRNA for reverse transcription (PK 06210, GGTTGCACCACAGACTTG) and two consecutive cycles of amplification with the oligonucleotide primers against the 5’ cap supplied with the kit and nested RIM1 oligonucleotide primers (PK 06211, GGGACACGTTTGCGCTC; PK 06212, CTCCCTTGCCATTCTGCTC). The product containing the new 5’ exon 1” was sequenced and used for database analysis and RT-PCR oligonucleotide primer design. One-step RT-PCR was performed on RNA purified form the frontal cortex of 17 day old RIM1α KO and RIM1αβ KO and control littermate mice with Superscript™ One-Step RT-PCR with Platinum® Taq purchased from Invitrogen using oligonucleotide primers against RIM1α (PK 06206, CTTCACCGGGTAGCGAGCCAGG; PK 06216, ATCCGAAAGGTGAGAGCCAGAGC), RIM1β (PK 06217, CAAAGAACCACGCTCCAGATTTCG; PK 06218, GGGACATGTCACATGAGAGGAGAG) and control oligos for mouse Neuroligin2/4* (MB30, GGAATTCCTACTGGACCAACTTCGCCAAGAC; MB31, GGAATTCGTCACGCTCAGCTCCGTCGAGTAG). The RT-PCR products were purified form an agarose gel and subjected to DNA sequencing.
Protein quantitations in S2 and P2 fractions of brain homogenates
Fractionation in a particulate (P2) and soluble (S2) fraction was essentially performed as previously described (Wang et al., 2002
). In brief, brains were harvested from 3 pairs of RIM1αβ KO and wild-type littermate control mice at 8–9 weeks of age. Each brain was homogenized (in a detergent free buffer containing 25 mM Hepes pH 7.2, 0.32 M sucrose, 5 mM EDTA, 1 mM PMSF, 1 µg/ml Leupeptin and Pepstatin, 2 µg/ml Aprotinin) using a motorized glass-teflon homogenizer. After spinning for 10 min at 1500 × g, the post-nuclear supernatant was centrifuged for 1 h at 162000 × g to separate P2 from S2. Protein contents were adjusted with a BCA protein assay kit (Pierce Biotechnology, Rockford, IL, USA). 20 µg of proteins were loaded per lane on standard SDS/Page gels for immunoblotting. Protein quantitations were done with 125
I-labeled secondary antibodies as previously described (Ho et al., 2006
). We measured the levels of proteins in P2 and S2 with a Storm phosphoimager and normalized them to internal standards (valosin-containing protein, VCP, GDP dissociation inhibitor, GDI, or β-actin). % solubility was calculated by expressing the amount in S2 as a fraction of the total amount (P2+S2) without normalizing.
Electrophysiology in acute brain slices
Acute transverse hippocampal slices (400 µm thick) were prepared from 3- to 7-week old mice. All recordings were performed at room temperature. The external solution for all experiments contained: 124 mM NaCl, 2.5 mM KCl, 26 mM NaHCO3, 1 mM NaH2PO4, 2.5 mM CaCl2, 1.3 mM MgSO4 and 10 mM glucose. Extracellular field potentials were recorded either in CA1 stratum radiatum (to examine Schaffer collateral to CA1 pyramidal cell synapses), or in CA3 stratum lucidum, (to examine mossy fiber to CA3 pyramidal cell synapses). Patch pipettes were filled with 1 M NaCl for extracellular recordings or with external solution for extracellular stimulation. Synaptic depression was examined using bursts of 25 stimuli at 14 Hz (90 s inter-burst intervals), and 5 responses were averaged for each experiment. Mossy fiber long term potentiation (mf-LTP) was induced with 125 stimuli at 25 Hz in the presence of 50 µM D-APV. For the MK-801 experiments, excitatory postsynaptic currents (EPSCs) mediated by NMDARs were measured in whole cell voltage clamp from CA1 pyramidal neurons (+30mV holding potential) in the presence of 10 µM NBQX and 100 µM Picrotoxin. The rate of MK-801 blockade of NMDAR-mediated EPSCs was assessed in ~2:1 Ca2+:Mg2+ external solution at 0.1 Hz stimulation. After 10 min stable baseline synaptic responses, MK-801 (25 µM) was bath applied without stimulation for 8 min. Stimulation at 0.1 Hz was then resumed and the rate of blockade was determined. Inhibitory postsynaptic currents (IPSCs) in CA1 pyramidal cells (+10 mV holding potential) were evoked by stimulating GABAergic fibers in the middle third of stratum radiatum (0.05 or 0.1 Hz basal stimulation), and were monitored in the presence of 10 µM NBQX, 3 µM CGP 55845 and 25 µM D-APV. Recordings of miniature IPSCs (mIPSCs) were obtained under similar conditions, except that 1 µM TTX was also included in the bath to block action potentials. The intracellular solution contained: 131 mM Cs-Gluconate, 8 mM NaCl, 1 mM CaCl2, 10 mM EGTA, 10 mM glucose, and 10 mM HEPES-CsOH pH 7.2 (osmolarity 293 mmol/kg). I-LTD was elicited by theta-burst stimulation (TBS) consisting of a series of 10 bursts repeated 4 times. Each burst was comprised of five 100 Hz stimuli and the inter-burst interval was 200 ms. Extracellular and whole-cell patch clamp recordings were performed using a Multiclamp 700B amplifier (Axon Instruments, Union City, CA, USA). Stimulation and acquisition were controlled by custom written software in Igor Pro 4.09A (Wavemetrics, Inc., Lake Oswego, OR, USA). The paired-pulse ratio (PPR) is defined as the ratio of the amplitude of the second synaptic response to the amplitude of the first synaptic response. The magnitude of mfLTP/I-LTD is calculated as the percentage change between baseline (averaged for 10 minutes before induction) and post-induction responses (50 to 60 minutes post induction for mf-LTP; 20 to 30 minutes for I-LTD).
Electrophysiology in dissociated neuronal cultures
Hippocampi were isolated from new born mice (at postnatal day 1) and were digested using 0.5% trypsin-EDTA for 10 min at 37°C, and then separated by trituration. The neurons were plated in high density (around 5000 cells/cm2) onto coverslips pre-treated with 2% matrigel (Collaborative Biomedical, Bedford, MA, USA). For RIM1αβ conditional KO cultures, neurons from several mice were pooled and lentiviral infections were performed at 3–4 days in vitro (DIV) using a lentivirus encoding either a cre-EGFP fusion protein or a recombination deficient deletion mutant control. For the constitutive RIM1α KO cultures, mice were cultured individually, genotyped, and KO neurons and heterozygote control neurons were chosen for analysis. Whole-cell recordings were performed at DIV 13 to 16 using a multiclamp 700B amplifier (Axon Instruments, Union City, CA, USA) and an inhibitory postsynaptic current (IPSC) was elicited by a single or a paired electrical stimulation in the presence of 10 µM CNQX and 50 µM D-APV. The recording chamber was continuously perfused with bath solution (containing 150 mM NaCl, 4 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM HEPES-NaOH pH 7.3, and 10 mM Glucose). Patch pipettes were pulled from borosilicate glass and back filled with 145 mM CsCl, 5 mM NaCl, 10 mM HEPES-CsOH pH 7.3, 10 mM EGTA, 4 mM MgATP and 0.3 mM Na2GTP. Data were collected with pClamp 9 software (Molecular Device, Sunnyvale, CA, USA) sampled at 10 Hz and filtered at 1 Hz. Off-line measurements of IPSC amplitude and charge transfer were conducted using Clampfit software (Molecular Devices, Sunnyvale, CA, USA).
PCR genotyping, Southern blotting, SDS/Page gels and immunoblotting were done according to standard methods (Ho et al., 2006
). Mouse husbandry was performed according to institutional guidelines.
All data are shown as means ± SEMs unless otherwise stated. Statistical significance was determined by the Student’s t test (two tailed distribution, paired) for quantitative protein analysis, by χ-test comparing the obtained ratio with an expected monogenic Mendelian ratio for mouse survival and recordings from cultured neurons. For electrophysiological recordings in acute hippocampal slices, statistical analysis was performed using Student’s t test or one-way ANOVA at the p < 0.05 significance level in OriginPro 7.0 software (OriginLab Corporation, Northampton, MA).